A Method for Selective Concentrating of DNA Targets by ......A method for the selective...

A Method for Selective Concentrating of DNA Targets by Capillary Affinity Gel Electrophoresis

by

Andrew Chan

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Chemistry University of Toronto

© Copyright by Andrew Chan, 2013

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A Method for Selective Concentrating of DNA Targets by Capillary

Affinity Gel Electrophoresis

Andrew Chan

Doctor of Philosophy

Department of Chemistry University of Toronto

2013

Abstract

A method for the selective concentrating of DNA targets using capillary affinity gel

electrophoresis is presented. Complementary ssDNA targets are retained through hybridization

with oligonucleotide probes immobilized within polyacrylamide gels while non-complementary

targets are removed. The captured DNA targets were concentrated by step elution, where a

localized thermal zone was applied in small steps along the capillary.

Evaluation of the selective capture of a 150 nt DNA target in a complicated mixture was carried

out by factorial analysis. Gels with a smaller average pore size were found to retain a higher

amount of complementary targets. This was thought to be due to the ssDNA target migrating

through the gel by reptation, eliminating hairpin structures, making the complementary region of

the target available for hybridization.

This method was applied to a series of DNA targets of different lengths, 19 nt, 150 nt, 250 nt and

400 nt. The recovery of the method ranged from 0.5 to 4% for the PCR targets, and 13 to 18%

for the 19 nt oligonucleotide target. The purity was calculated to be up to 44% for the PCR

targets and up to 86% for the 19 nt target. This was an improvement in purity of up to 15 times

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and 1100 times in comparison to the original samples for the PCR targets and 19 nt

oligonucleotide, respectively.

The 19 nt targets were selective concentrated and delivered into a microfluidic based DNA

biosensing platform. The purity of the sample improved from 0.01% to 50% while recovery

decreased from 100% to 20% for a sample with 0.5 nM complementary and 1 µM non-

complementary targets. An improvement in the response of the sensing platform was

demonstrated on 19 nt oligonucleotide targets delivered by selective concentration versus

concentration alone into the microfluidic biosensing system.

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Acknowledgments

Firstly, I would like to thank my supervisor, Professor Ulrich Krull for his guidance, support and

patience throughout this journey. I would not have been able to achieve this without his help. I

would also like to thank the members of my supervisory committee, Professors Aaron Wheeler

and Julie Audet for their feedback over the years.

I would also like to thank Dr. Lu Chen, Uvaraj Uddayasankar and Omair Noor for their

assistance in the work with interfacing the capillary and microfluidic biosensing platform. I

would also like to thank past and present members of the Chemical Sensors Group, especially

Drs. Russ Algar, Melissa Massey, Ying Lim and April Wong for their friendship, support and

laughs.

Finally, I would like to thank my parents and family for bearing with me throughout this pursuit.

This is dedicated to you.

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Table of Contents

Acknowledgments.......................................................................................................................... iv

Table of Contents ............................................................................................................................ v

Abbreviations...................................................................................................................................x

Publications....................................................................................................................................xii

List of Tables ............................................................................................................................... xiii

List of Figures .............................................................................................................................. xix

List of Appendices ................................................................................................................... xxxiv

Chapter 1 Introduction .................................................................................................................... 1

1.1 DNA Biosensors for Detection of Real-World Targets ....................................................... 1

1.2 Goals of Pre-treatment ......................................................................................................... 5

1.2.1 Extraction................................................................................................................... 5

1.2.2 Purification................................................................................................................. 5

1.2.3 Sample Loss and Quantification ................................................................................ 6

1.3 Methods for DNA Purification............................................................................................. 8

1.3.1 Purification Methods based on Solid Phase Extraction (SPE)................................... 8

1.3.2 Purification of DNA in Conjunction with Selective Hybridization......................... 11

1.4 Enrichment and Amplification of Sample.......................................................................... 14

1.4.1 DNA Amplification by Polymerase Chain Reaction ............................................... 15

1.4.2 Concentrating by Volume Reduction....................................................................... 16

1.5 Fragmentation and Denaturation of DNA.......................................................................... 17

1.5.1 Fragmentation .......................................................................................................... 17

1.5.2 Preparation of Single-Stranded DNA Target ........................................................... 18

1.6 Integrated Microfluidic Devices ........................................................................................ 20

1.7 Methods for Purification and Concentrating in Microfluidic Devices............................... 22

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1.7.1 Filtration................................................................................................................... 22

1.7.2 Solid-Phase Extraction............................................................................................. 23

1.8 Sample Concentrating by Electrokinetic Methods............................................................. 26

1.8.1 Field Amplified Stacking......................................................................................... 26

1.8.2 Isotachophoresis....................................................................................................... 27

1.9 Contributions of this Thesis ............................................................................................... 28

Chapter 2 Materials and Method................................................................................................... 33

2.1 Reagents ............................................................................................................................. 33

2.2 DNA Targets ...................................................................................................................... 33

2.3 Instrumentation .................................................................................................................. 35

2.3.1 Capillary Electrophoresis......................................................................................... 35

2.3.2 Instrumentation for on-line capillary electrophoresis/step elution experiments...... 36

2.3.3 Confocal Fluorescence Microscope Images ............................................................ 36

2.3.3.1 Confocal fluorescence microscope slide reader for 532nm/635nm excitation (Chipreader) ........................................................................... 37

2.3.3.2 Epifluorescence microscope for 635 nm excitation (Alpha) .................... 38

2.3.3.3 Confocal Fluorescence Microscope for 534 nm excitation (Confocal) .... 38

2.3.4 UV-VIS .................................................................................................................... 38

2.3.5 Steady-State Solution phase Fluorescence Measurements ...................................... 39

2.3.6 Other Equipment ...................................................................................................... 39

2.4 Generation of Longer lengths of DNA Targets.................................................................. 39

2.4.1 150 bp targets .......................................................................................................... 39

2.4.2 250 bp targets .......................................................................................................... 40

2.4.3 400 bp targets .......................................................................................................... 41

2.4.4 Validation of DNA targets ...................................................................................... 42

2.5 Preparation of Capillary Affinity Capture Gel................................................................... 42

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2.6 Capture and elution experiments....................................................................................... 43

2.6.1 Pre-Conditioning of Affinity Capture Gel in Capillaries......................................... 43

2.6.2 Capture and Elution Experiments ........................................................................... 43

2.6.3 Factorial Design Experiments.................................................................................. 43

2.6.4 Step Elution of Captured DNA targets .................................................................... 45

2.7 Delivery of concentrated targets into microfluidic based DNA biosensing platform........ 45

2.7.1 Construction of DNA Microfluidic Biosensing Platform........................................ 45

Chapter 3 Results and Discussion................................................................................................. 49

3.0 Capture of Oligonucleotides of 20 nt Length..................................................................... 49

3.0.1 Considerations for Imaging Fused Silica Capillaries by Confocal Fluorescence Microscopy ........................................................................................................... 49

3.0.2 Capture and Elution Experiment for a 20 nt Target................................................. 50

3.0.3 Autofluorescence and Non-Selective Adsorption.................................................... 52

3.0.4 Variation of Polymer Density .................................................................................. 55

3.0.5 Quantity of Probe that was Immobilized in the Polyacrylamide Matrix ................. 57

3.0.6 Influence of Concentration of Target on the Efficiency of Capture ........................ 59

3.0.7 Capture and Elution using a Non-Complementary Target ...................................... 60

3.0.8 Examination of Selectivity Using a Five Base Pair Mismatch Target..................... 62

3.0.9 Separations of Mixtures of Complementary and Non-complementary Targets ...... 63

3.0.10 Five Base Pair Mismatch in Mixture with Fully Complementary Target ............. 65

3.0.11 Capture of 40 nt Length Targets ............................................................................ 67

3.1 Capture of Targets of Greater Lengths than 40 nt – Moving Towards Handling of NAs From Real Samples ........................................................................................................... 68

3.1.1 DNA Targets Selected for Experiments .................................................................. 69

3.2 Compositions of Affinity Capture Gels ............................................................................ 70

3.3 Selective Capture of 150 nt Target.................................................................................... 71

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3.3.1 Comparison of Capture of 150 nt DNA Targets Using Complementary and Non-complementary Probe ................................................................................... 79

3.4 Performance of the Affinity Gel for the Capture DNA Targets......................................... 82

3.5 Examination of the Effects of Varying Gel Formulation on Performance ........................ 90

3.5.1 Affect of Gel Formulation on the Quantity of Probe that was Incorporated ........... 91

3.5.2 Effect of Radical Initiators on Oligonucleotide Sequence....................................... 95

3.5.3 Cleavage of Oligonucleotide Probe by Radicals .................................................... 98

3.5.4 Examination of Damage to Nucleobases by Radical ............................................ 103

3.5.5 Examination of Conditions that Affect Capture of Complementary Targets ........ 107

3.5.6 Affinity Capture of Complementary Targets with Probes that are Immobilized in 3D Gel Supports.................................................................................................. 109

3.5.7 Effects of Gel Formulation on the Concentration of Target Injected .................... 113

3.5.8 Effects of Gel Formulation on the Quantity of Target Captured ........................... 115

3.5.9 Effects of Polymerization of Polyacrylamide ........................................................ 117

3.5.10 Effect of Probe Availability As a Function of Gel Formulation.......................... 120

3.5.11 Effect of Gel Formulation on Migration of DNA Targets ................................... 121

3.5.12 Effect of Stringency Conditions on Percent Recovery and Purity during Washing Step ...................................................................................................... 125

3.6 Selective Concentrating of Oligonucleotide Targets by Step Elution From Affinity Gels ................................................................................................................................. 133

3.6.1 Concentrating the 150 nt, 250 nt and 400 nt Targets by Step Elution ................... 139

3.7 Delivery of Concentrated Targets into Microfluidic DNA Biosensing Platform ............ 141

3.7.1 Design Aspects for Sample Transfer from the Capillary to the Microfluidic Biosensing Platform............................................................................................ 141

3.7.2 Delivery of Oligonucleotide Targets to the Microfluidic Biosensing Platform by Direct Injection and by Selective Concentration ................................................ 146

3.7.3 Response of Microfluidic Biosensing Platform to Mixtures of Targets Delivered by Direct Injection and Following Selective Concentration............................... 150

3.7.4 Response of Microfluidic Biosensing Platform to Delivery of Concentrated Oligonucleotide Targets With and Without Purification .................................... 155

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Chapter 4 Future Directions........................................................................................................ 159

4.1 Determination of Oligonucleotide Probe Incorporated into Affinity Capture Gel .......... 159

4.2 Further Factorial Experiments on Gel Formulations ....................................................... 159

4.3 Improvements to Capillary-Microfluidic Platform .......................................................... 160

4.4 Moving Towards DNA Targets in Complex Matrices..................................................... 161

Chapter 5 Conclusions ................................................................................................................ 163

References................................................................................................................................... 165

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Abbreviations

APS - Ammonium Persulfate

ATP - Adenonsine Triphosphate

AU - Arbitrary Units

BSA - Bovine Serum Albumin

CD - Compact Disc

CE - Capillary Electrophoresis

CE-MS - Capillary Electrophoresis-Mass Spectrometry

CFU - Colony Forming Units

CGE - Capillary Gel Electrophoresis

DEAE - Diethylaminoethanol

DMF - Digital Microfluidics

DNA - Deoxyribonucleic Acid

dsDNA - Double Stranded Deoxyribonucleic Acid

DTT - Dithiothreitol

EDTA - Ethylenediaminetetraacetic Acid

EKS - Electrokinetic Supercharging

ELWD - Extra Long Working Distance

EOF - Electroosmotic Flow

EWOD - Electrowetting on Dielectric

FAS - Field Amplified Stacking

HCV - Hepatitis C Virus

HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HPV - Human Papillomavirus

ID - Inner Diameter

ITP - Isotachophoresis

LATE-PCR - Linear-After-the-Exponential Polymerase Chain Reaction

LOD - Limit of Detection

MAGIChip - Microarray of Gel-immobilized Compounds on a Chip

MPS - 3-methacryloxypropyltrimethoxysilane

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NA - Nucleic Acid

OD - Outer Diameter

PCR - Polymerase Chain Reaction

PDMS - Polydimethoxysilane

PMT - Photomultiplier Tube

PNA - Peptide Nucleic Acid

POC - Point of Care

PVA - Polyvinyl Alcohol

PVP - Polyvinylpyrrolidone

RNA - Ribonucleic Acid

rRNA - Ribosomal Ribonucleic Acid

RT - Room Temperature

SMN - Survival Motor Neuron

SPE - Solid Phase Extraction

SPR - Surface Plasmon Resonance

ssDNA - Single Stranded Deoxyribnucleic Acid

TBE - Tris-Borate-Ethylenediaminetetraacetic Acid

TEMED - N,N,N′,N′-tetramethylethane-1,2-diamine

TMOS - Tetramethyl Orthosilicate

TMSPM - 3-(trimethoxysilyl)propyl methacrylate

UV - Ultraviolet

UV-VIS - Ultraviolet-Visible

WD - Working Distance

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Publications

A. Chan and U.J. Krull, "Capillary electrophoresis for capture and concentrating of target nucleic

acids by affinity gels modified to contain single-stranded nucleic acid probes", Analytica

Chimica Acta, 578(2006), pg 31-42.

A. Chan, T. Artuso, U.J. Krull, "Sample Handling Protocols for Biosensor Applications" in

Handbook of Sample Preparation. Hoboken, N.J.:John Wiley & Sons, 2010, Chapter 21, pg 385-

418.

xiii

List of Tables

Table 1.1: Examples of biosensors used for detection of NA targets from various different

sample matrices. Steps for pre-treatment protocols prior to detection are identified, as well as the

total time needed for pre-treatment and detection. When available, detection time is also

provided in parenthesis. .................................................................................................................. 4

Table 2.1: Oligonucleotide targets that were used in the experiments. Longer PCR targets are

described in a subsequent section. Melt temperature was provided by the supplier.

*oligonucleotide probes used in capillary affinity capture gels contained the Acrydite

modification at the 5' end, while probes used for immobilization onto epoxy-modified glass

slides in the microfluidic device contained a primary amino group with a C12 spacer on the 5'

end. Cy3 fluorescent label was attached to the 3' end when used.**fluorophores on these

oligonucleotide sequences were attached at 5' end when used. .................................................... 34

Table 2.2: Design matrix for the quarter 2-level fractional factorial analysis for the examination

of gel formulation on the performance of the capillary affinity capture gels. .............................. 44

Table 2.3: Experimental conditions for the two levels used for the fractional factorial design

matrix. ........................................................................................................................................... 44

Table 2.4: Design matrix for three level factorial experiment to explore capture efficiency and

selectivity of the affinity capture gel. Factors A and B are defined in Table 2.5. ....................... 45

Table 2.5: Experimental conditions for each level tested in the design matrix of Table 2.4...... 45

Table 3.1: Tabulated results for extent of probe incorporation and performance in capture for

three different probe concentrations. Affinity gel: Varying concentrations of Cy3-dT20 probe

(182 nM, 454 nM and 727 nM) in a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL

of a sample containing 1 µM Cy5-dT20 at 267 Vcm-1 for 60 s. Electrophoresis condition: 133

Vcm-1 with 1x TBE/0.1% PVP for 20 minutes. Elution condition: 267 Vcm-1 with 1x TBE/0.5 M

NaSCN/0.1%PVP for 15 minutes at 60 °C. Error bars are propagated error following correlation

of average fluorescence intensity to concentration using a calibration curve. ............................. 58

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Table 3.2: Tabulated results for effect of concentration of sample on loading of affinity gel.

Amount of target in loading of a sample was calculated based on a 10 µL of the target

concentration solution used for loading. Amount of probe and target were calculated using the

geometric volume of 0.59 µL for the capillary. Affinity gel: 0.45 µM Cy3-dT20 probe in a

12.5%T linear polyacrylamide gel. Injection condition: 10 µL sample containing Cy5-dA20 at

533 Vcm-1 for 60 seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP for 10

minutes. Elution condition: 267 Vcm-1 with 1xTBE/0.5 M NaSCN/0.1%PVP for 5 minutes at

60°C. Error bars are propagated error following correlation of average fluorescence intensity to

concentration using a calibration curve. ....................................................................................... 59

Table 3.3: Summary results for Recovery and Purity for mixture containing 150 nt and 1.5 pmol

non-complementary targets by affinity capture gel. Recovery and purity of the original solution

and by selective capture are presented. The recovery and purity were calculated based on

removal of material from the affinity capture gel by elution of the entire capillary (no

concentrating). Experimental conditions shown in Figure 3.29. .................................................. 88











Table 3.6: Summary of the migration times of the peaks observed from the CGE experiment.

The sequence of the oligonucleotide target used in these experiments: 5’ Cy5 - ACA GGG TTT

CAG ACA AAA T 3’. Error represents 1 standard from three trials. ........................................ 102

Table 3.7: Summary of melt temperatures of the oligonucleotide duplex following reaction with

the different radical initiator ratio. Error represent 1 standard deviation of three trials. ............ 107

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Table 3.8: Summary factors which were identified as significant from factorial analysis (95%

confidence). The (+) and (-) after each factor denotes whether the effect was positive or

negative. ...................................................................................................................................... 109

Table 3.9: Determination of pore size from the Ogston plot presented in Figure 3.46. The range

of DNA fragments where log(µ/µo) deviates from linearity is assumed to be the size range where

DNA transitions from Ogston to reptation. The radius of gyration of the dsDNA fragments was

calculated by Eq (11), where persistence length for dsDNA was 50 nm, and contour length of

DNA was 0.34 nm/base. ............................................................................................................. 123

Table 3.10: Probe density based on 0.5 µM probe in the original monomer solution. Values

were calculated based on the number of pores that could fit in a 1000x1000x1000 nm cube at the

lowest gel formulation (7.5 %T/1 %C). The calculation assumes that the pore volume for the

remaining gel formulation was the same as the lowest monomer concentration and number of

pores for the remaining gel formulations were calculated as such. The amount of probe was

determined based on previous experiments examining the percentage of probe incorporated, and

the accessibility of the probe from experiments performed with 19 nt targets in Figure 3.45. .. 125

Table 3.11: Summary of results of the concentrating effect of step elution of complementary

target as a function of elution length. Volume of the eluting targets was calculated based on the

peak width and mobility of the oligonucleotide, which was 86 µms-1 at 96 Vcm-1. Error

represent 1 standard deviation of three trials. Affinity capture gel: 50 nM SMN probe, 10%

LAAm affinity capture gel. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150

Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer. Step

elution for concentrating effect: coverage length: 12.5, 25 and 37.5 mm; step size: 250 µm; step

rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain 400

mV. Sampling rate: 10 Hz ......................................................................................................... 136

Table 3.12: Summary of results from step elution of complementary target as determined from

data obtained from the experiment depicted in Figure 3.51b. Results for integrated Peak Area,

Width, Height were calculated using Origin Pro 8.0 Volume of the eluting targets was calculated

based on the peak width and mobility of the oligonucleotide, which was 86 µms-1 at 96 Vcm-1.

Error represent 1 standard deviation of three trials. Affinity capture gel: 50 nM SMN probe,

10% LAAm affinity capture gel. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at

xvi

150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer.

Step elution for concentrating effect: coverage length: 12.5, 25 and 37.5 mm; step size: 250 µm;

step rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain

400 mV. Sampling rate: 10 Hz .................................................................................................. 138

Table 3.13: Summary results for Recovery and Purity from affinity capture gel for mixtures

containing varying amounts of 150 nt complementary and 1.5 pmol non-complementary targets.

The Recovery and Purity were calculated from quantitative concentration data for the eluting

peak by use of calibration curves. Errors represent propagated error following correlation of

average fluorescence intensity to concentration using a calibration curve. Affinity capture gel: 3

µM β-actin probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic injection for 20 min at

133 Vcm-1. Incubation time 5 min. Wash step: electrophoresis for 25 min at 133 Vcm-1, 45 °C,

with 25% v/v formamide/1X TBE/PVP. Concentrating step: coverage length: 12.5 mm; step

size: 250 µm; step rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition settings (Cy5): PMT gain 500

mV, translation speed: 50 µms-1, scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110

mV, Pinhole: 60 µm, 1 FPS ........................................................................................................ 139

Table 3.14: Summary results for Recovery and Purity for mixture containing varying amounts of

250 nt of complementary and 1.5 pmol non-complementary targets by affinity capture gel. Errors

represent propagated error following correlation of average fluorescence intensity to

concentration using a calibration curve. Affinity capture gel: 3 µM β-actin probe, 12.5 %T, 1

%C. Capture conditions: electrokinetic injection for 30 min at 133 Vcm-1. Incubation time 5

min. Wash step: electrophoresis for 40 min at 133 Vcm-1, 45 °C, with 25% v/v formamide/1X

TBE/PVP. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 66 µms-1;

Voltage: 96 Vcm-1; Acquisition settings (Cy5): PMT gain 500 mV, translation speed: 50 µms-1,

scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS . 140

Table 3.15: Summary results for Recovery and Purity for mixture containing 400 nt and 1.5

pmol non-complementary targets by affinity capture gel. Errors represent propagated error

following correlation of average fluorescence intensity to concentration using a calibration curve.

Affinity capture gel: 3 µM uidA probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic

injection for 40 min at 133 Vcm-1. Incubation time 5 min. Wash step: electrophoresis for 50 min

at 133 Vcm-1, 45 °C, with 25% v/v formamide/1X TBE/PVP. Concentrating step: coverage

xvii

length: 12.5 mm; step size: 250 µm; step rate: 52 µms-1; Voltage: 96 Vcm-1; Acquisition settings

(Cy5): PMT gain 500 mV, translation speed: 50 µms-1, scan rate, 50 Hz. (Cy3): Image

resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS...................................................... 140

Table 3.16: Summary of data for the response of the microfluidic DNA biosensing platform to

delivery of complementary targets by selective concentrating using the affinity capture gel. The

amount of target injected into the affinity capture gel by electrokinetic injection from the original

target solution is shown in parenthesis. The equivalent quantity was determined based on

correlation to the concentration-response curve of Figure 3.55. The enhancement factor was

calculated based on the ratio of the equivalent quantity and quantity of material injected. Errors

represent 1 standard of three trials expect for Equivalent Quantity Determined from Calibration

Curve, which is propagated error from correlation with calibration curve. Affinity capture gel:

100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL A647 SMN target, electrokinetic

injection for 1 min at 150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x

TBE/PVP running buffer. Concentrating Step: coverage length: 12.5 mm; step size: 250 µm; step

rate: 86 µms-1; Voltage: 96 Vcm-1; Delivery of purified and concentrated targets into

microfluidic biosensing platform: 500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition

settings: PMT gain 700 mV, translation speed: 50 µms-1 scan rate: 50 Hz. .............................. 149

Table 3.17: Summary of the performance of the two delivery methods. Percent recovery is

based on the proportion of the amount of target delivered to the microfluidic biosensing platform

from of the original starting sample. The values for delivery by direct injection were calculated

based on the concentration of the targets in the original sample. The values used for the delivery

selective concentrating were calculated based on the response of the biosensing platform. The

enrichment factor is the ratio of the percent complementary target with and without selective

concentrating. Errors represent propagated error resulting from calculating derived values. ... 154

Table 3.18: Position and peak width of the eluted targets from the concentrating of the two

captured targets along the capillary (from the injection end). Error represent 1 standard deviation

of three trials. Affinity capture gel: 10% LAAm, 100 nM SMN and 5 µM β-actin probes. Target

injection: 10 µL of 1 nM A647-SMN and 1 µM Cy3-β-actin targets, electrokinetic injection for 1

min at 150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running

buffer. Concentration step: coverage length: 12.5 mm; Step rate: 86 µms-1; Voltage: 96 Vcm-1;

xviii

Acquisition settings (Alexa647): PMT gain 700 mV, translation speed: 50 µms-1, scan rate, 50

Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS ...................... 156

Table 3.19: Quantitative information of the eluted A647-SMN target during stacking from gels

which containing only SMN probe (selective concentrating) and containing both β-actin and

SMN probe (concentrating only). Errors represent 1 standard deviation of three trials. Affinity

capture gel: Selective concentrating: 10% LAAm with 100 nM SMN probe. Concentrating only:

10% LAAm with 100 nM SMN and 5 µM β-actin probes. Target injection: 10 µL of 1 nM

A647-SMN and 1 µM Cy3-β-actin targets, electrokinetic injection for 1 min at 150 Vcm-1.

Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer. Concentrating

Step: coverage length: 12.5 mm; Step rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition settings

(Alexa647): PMT gain 700 mV, translation speed: 50 µms-1, scan rate, 50 Hz. ....................... 156

xix

List of Figures

Figure 1.1: Radical polymerization reaction of acrylamide in the presence of an Acrydite™

modified oligonucleotide. ............................................................................................................. 13

Figure 1.2: Schematic of differences between (a) conventional denaturation by heat and (b)

denaturation by heat with ancillary blocking oligonucleotides. With permission from Analytica

Chimica Acta. Copyright 2004, Elsevier [131]. .......................................................................... 19

Figure 1.3: (a) Schematic diagram of the microchip layout for pre-concentration, (b) image of

the pre-concentrator channel, and (c) schematic of how the filtration membrane is placed in

between the microchip and the coverplate. With permission from Analytical Chemistry.

Copyright 2004, American Chemical Society [153]..................................................................... 23

Figure 1.4: Fluorescence images of fluorescein-labeled ricin injected a) without pre-

concentration, and b) with pre-concentration for 1 minute. With permission from Analytical

Chemistry. Copyright 2004, American Chemical Society [153]. ................................................ 23

Figure 1.5: Schematic and SEM images of the microfabricated silica pillars for SPE of DNA.

With permission from Biosensors and Bioelectronics. Copyright 2003, Elsevier [158]............. 25

Figure 1.6: Schematic representation of selective concentrating as done in the work of this

thesis. First, the target was captured onto the affinity capture gel column. Elution took place in a

localized area of the capillary by means of application of heating to a narrow zone such that only

targets captured in that region were denatured. This process took place during electrophoresis,

and the denatured targets moved along in the electric field. The heated zone was then physically

moved along the column. This allowed for the continual release of targets into a stacked zone of

significantly smaller volume than the original sample volume. ................................................... 30

Figure 2.1: Schematic of the capillary electrophoresis set-up .................................................... 35

Figure 2.2: Set-up for online capillary electrophoresis and step elution experiments................ 36

Figure 2.3: Schematic of the instrumental setup for how confocal fluorescence microscope

images were obtained.................................................................................................................... 37

xx

Figure 2.4: Schematic for the construction of the microfluidic DNA sensing platform with a

capillary interface. Left: original microfluidics template and position of the template capillary.

The capillary (100 µm I.D., 375 µm O.D.) was positioned over the microfluidic channel such that

the inner diameter was within the width of the channel. PDMS was poured over the template and

cured on a hotplate. The template capillary and microfluidic template were removed and the

PDMS chip had the channel structure and capillary port. Right: schematic of the microfluidic

DNA sensor platform. The PDMS template was trimmed such that only the straight channel

remained, and this was positioned over the two oligonucleotide probe spots on the epoxy

modified slides. ............................................................................................................................. 46

Figure 2.5: Schematic of the electrophoresis setup for the capillary to microfluidic DNA

biosensor. ...................................................................................................................................... 47

Figure 3.1: Confocal fluorescence microscope images of affinity capture capillaries from a

capture and elution run using a complementary target showing: (a) the affinity gel material

inside the capillary prior to loading of target oligonucleotide sequence, (b) running for 35

minutes following electrokinetic injection of target Cy5 – dA20 and (c) after elution for 25

minutes at 60 °C. Affinity gel: 0.45 µM Cy3-dT20 probe in a 12.5%T linear polyacrylamide

gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dA20 at 267 Vcm-1 for 60

seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP for 35 minutes. Elution

condition: 267 Vcm-1 with 1x TBE/0.5 M NaSCN/0.1% PVP for 25 minutes at 60 °C. Images

were obtained using the Chipreader.............................................................................................. 51

Figure 3.2: Confocal fluorescence microscope images of affinity capture capillaries showing the

Cy3 (left) and Cy5 (right) channels from a portion of the capillary shown in Figure 3.1(a), which

shows the autofluorescence signal of the system before the loading of any fluorescently labelled

materials. Images were enhanced in ImageJ using the Window/Level function for better clarity.

....................................................................................................................................................... 52

Figure 3.3: Confocal fluorescence microscope images of affinity capture capillaries showing the

Cy5 channel (a) before loading of complementary target and (b) following elution. A

fluorescence signal was apparent following elution, indicating retention of 13.6 nM or 8 fmol of

target. Injection condition: 5 µL of a sample containing 1 µM Cy5-dT20 at 267 Vcm-1 for 60

seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1 %PVP for 35 minutes. Elution

xxi

condition: 267 Vcm-1 with 1x TBE/0.5 M NaSCN/0.1 %PVP for 25 minutes at 60 °C. Images

were enhanced in ImageJ using the Window/Level function for better clarity. ........................... 54

Figure 3.4: (a) Change in the average Cy5 fluorescence intensity over time, measuring the loss

of any adsorbed materials used in the pre-treatment of the columns. Pre-treatment protocol: a 5

µL sample of 2 µM Cy5-dC20 at 267 Vcm-1 for 2 minutes, followed by electrophoresis at 133

Vcm-1 in 1xTBE/0.1% PVP for 5 minutes, and a second injection of a 5 µL 2 µM Cy5-dC20 at

267 Vcm-1 for 2 minutes and electrophoresis at 133 Vcm-1 in 1xTBE/0.1% PVP for 15 minutes.

(b) The original fluorescence intensity of the capillary channel prior to the loading of any

material (baseline). Error bars represent 1 standard deviation of three trials. ............................. 54

Figure 3.5: Confocal fluorescence microscopy images of capillary affinity capture gel of the

Cy3 channel showing the elution of 0.45 µM Cy3-dT20 probe immobilized in a 7.5%T linear

polyacrylamide gel. The image was taken of the capillary in (a), following pre-conditioning and

pre-treatment and (b), following loading 1 µM Cy5-dA20 and running for 35 minutes. Injection

condition: 5 µL of a sample containing 1 µM Cy5-dA20 at 267 Vcm-1 for 60 seconds.

Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP for 35 minutes. Images were

acquired using the Chipreader. ..................................................................................................... 56

Figure 3.6: Effect of amount of target on the efficiency of capture using a 454 nM Cy3-dT20

probe affinity gel. The ratio of captured target versus available probe was plotted against

varying concentrations of Cy5-dA20 targets. Error bars are propagated error following

correlation of average fluorescence intensity to concentration using a calibration curve. ........... 60

Figure 3.7: Confocal microscope images of capillaries examining the use of a non-

complementary target in the affinity gel from (a) prior to loading the non-complementary target,

(b) after loading and running for 25 minutes and (c) after the elution step was applied. Affinity

gel: 0.45 µM Cy3-dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection condition:

5 µL of a sample containing 1 µM Cy5-dC20 at 267 Vcm-1 for 60 seconds. Electrophoresis

condition: 133 Vcm-1 with 1x TBE/0.1% PVP for 35 minutes. Elution condition: 267 Vcm-1

with 1xTBE/0.5 M NaSCN/0.1%PVP for 25 minutes at 60°C. Images were obtained using the

Chipreader..................................................................................................................................... 61

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Figure 3.8: Confocal microscope images of capillaries showing non-complementary target

(Cy5-dC20) as it moved electrophoretically through the capillary after (a) 60 seconds, (b) 120

seconds, (c) 240 seconds and (d) 420 seconds. Only the images of the Cy5 channel are shown.

Affinity gel: 0.45 µM Cy3-dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection

condition: 5 µL of a sample containing 1 µM Cy5-dC20 at 267 Vcm-1 for 60 seconds.

Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP. Images were obtained using the

Chipreader..................................................................................................................................... 61

Figure 3.9: Confocal microscope images of capillaries examining the loading of Cy3-dA8C5A8

(5 base pair mismatch) target through an affinity capture gel. Images were taken after (a) 5

minutes and (b) 30 minutes. Affinity gel: 1.8 µM dT20 probe immobilized in a 12.5%T

polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy3-dA8C5A8 at

267 Vcm-1 for 60 seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP.

Images were obtained using the Chipreader. The images in (b) was enhanced in ImageJ using

the Window/Level function for better clarity. .............................................................................. 62

Figure 3.10: Confocal microscope images of capillaries tracking a time course experiment for

loading a mixture of dT20-Cy3 and dC20-Cy5. Images shown were taken after (a) 120 seconds,

(b) 240 seconds, (c) 540 seconds and (d) 840 seconds. Affinity gel: 1.8 µM dA20 probe

immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition: 5 µL of a sample

containing 0.5 µM Cy3-dT20, 0.5 µM Cy5-dC20 at 267 Vcm-1 for 60 seconds. Electrophoresis

condition: 133 Vcm-1 with 1x TBE/0.1% PVP. Images were obtained using the Chipreader. ... 63

Figure 3.11: Profile plot taken from the inlet to outlet end of the capillary from confocal

microscope images of the Cy5 channel after a 4 minute run of the (a) fully complementary

(dT20-Cy3) target with (b) non-complementary (dC20-Cy5) targets in a dA20-probe affinity gel.

Images were acquired using the Chipreader. ................................................................................ 64

Figure 3.12: Profile plot taken from the inlet to outlet end of the capillary from confocal

microscope images of the Cy5 channel. Profile plots following migration of (a) Cy3-dT20 and

(b) Cy5-dC20 through an unmodified polyacrylamide gel after five minutes. The profile of the

entire capillary length is not shown. The distance is shown from the injection end to the elution

end. Images acquired using the Chipreader. ................................................................................. 65

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Figure 3.13: Confocal microscope images taken from an affinity gel column containing

immobilized dT20-probe after loading a 1:1 mixture of Cy3-dA8C5A8 and Cy5-dA20. Images

taken after (a) 120 seconds, (b) 240 seconds, (c) 360 seconds, (d) 660 seconds and (e) 960

seconds. Affinity gel: 1.8 µM dT20 probe immobilized 12.5%T linear polyacrylamide affinity

gel. Injection condition: 5 µL of a sample containing 0.5 µM Cy3-dA8C5A8 and 0.5 µM Cy5-

dA20 at 267 Vcm-1 for 60 seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1%

PVP. Images acquired using the Chipreader. The Cy3 channel image in (e) was enhanced using

the Window/Level function in ImageJ.......................................................................................... 66

Figure 3.14: Confocal microscope images taken from an affinity gel column containing

immobilized dT20-probe after loading a 9:1 mixture of Cy3-dA8C5A8 Cy5-dA20 target. Images

were taken after (a) 240 s, (b) 540 s and (c) 840s. Affinity gel: 0.9 µM dT20 probe immobilized

12.5%T linear polyacrylamide affinity gel. Injection condition: 5 µL of a sample containing 1.8

µM Cy3-dA8C5A8 and 0.1 µM Cy5-dA20 at 267 Vcm-1 for 60 seconds. Electrophoresis

condition: 133 Vcm-1 with 1x TBE/0.1% PVP. Images acquired using the Chipreader. ............ 67

Figure 3.15: Confocal microscope images of capillaries for the Cy5 channel demonstrating the

loading and capture of a 1 µM 40 nt target sequence, Cy5-dC10T20C10 through the affinity gel.

Images were taken after electrophoresis following electrokinetic injection for (a) 300 s and (b) 25

min. Affinity gel: 1.8 µM dA20 probe immobilized 12.5%T linear polyacrylamide affinity gel.

Injection condition: 5 µL of a sample containing 1 µM Cy5-dC10T20C10at 267 Vcm-1 for 60

seconds. Electrophoresis condition: 133 Vcm-1 with 1x TBE/0.1% PVP. Images were acquired

using the Chipreader. .................................................................................................................... 68

Figure 3.16: Fluorescence intensity values from the Cy5 channel as measured from confocal

microscope images of the capillary taken at various times during the capture and elution

experiment. Values were obtained by taking the average of the values generated from the profile

plot of the confocal image. The capillary containing the affinity capture matrix was first imaged

to establish the background fluorescence signal (‘before’). A 10 µL, 1.67 µM solution of the

Cy5 labeled 150 nt complementary target was then loaded into the affinity capture gel

(7.5%T/6%C, 1.8 µM β-actin probe) electrokinetically for 20 minutes at 167 Vcm-1 (‘load’)

(The fluorescence intensity following this step saturated the detector and the actual value is not

shown). The fluorescence intensity for the (‘wash’) step was taken after the entire capillary was

xxiv

heated to 95 °C for 5 minutes, allowed to sit for 20 minutes at 20 °C, and following the

application of a voltage of 167 Vcm-1 for 20 minutes at 20 °C. Finally the captured targets were

eluted by the application of a voltage of 167 Vcm-1 for 15 minutes at 65 °C. Images were

acquired using the Chipreader. ..................................................................................................... 72

Figure 3.17: Profile plots from the outlet end to inlet end of the capillary from confocal

microscope images obtained for the Cy5 channel of the capillary. a) Fluorescence intensity

profile of the Cy5 channel of the affinity gel following the capture of the (0.14 µM) Cy5 labeled

150 nt target. Affinity capture gel: 7.5%T, 6 %C, 3 µM β-actin probe. b) Profile of the Cy5

channel of the affinity gel following the capture of the (1 µM) Cy5 labeled 19 nt target (SMN).

Affinity capture gel: 10%T, 5%C, 0.5 µM affinity capture probe (SMN). Images were acquired

using the Chipreader. .................................................................................................................... 73

Figure 3.18: Profile plots from the outlet end to inlet end of the capillary from confocal

microscope images obtained for the Cy5 channel of the capillary. Profile plots for the capture of

a (0.14 µM) 150 nt target using a 20 nt length probe and a 10 nt length probe. Affinity capture

gel: 7.5%T, 6 %C, 3 µM affinity capture probe (β-actin). Images acquired using the Chipreader.

....................................................................................................................................................... 76

Figure 3.19: Examples of hairpin structures as calculated by OligoAnalyzer software. Settings

used for calculations were 25 °C, 50 mM Na+ concentration, suboptimality 50% and maximum

foldings 20. Probe region is highlighted in the drawn box. ......................................................... 77

Figure 3.20: A histogram of the number of partial interactions possible between the target, its

complementary strand and the probe as calculated by OligoAnalyzer software. The number of

interactions was binned by the number of base pairs forming the interactions. Calculation

conditions were oligonucleotide concentration = 0.25 µM, [Na+] = 50 mM................................ 78

Figure 3.21: Data for comparison of the amount of 150 nt target (100 nM) captured by the

affinity capture gel after the capillary was heated to 95 °C for 5 minutes following the injection

relative to the amount captured by an unheated column. Affinity capture gel: 7.5%T, 6 %C, 3

µM β-actin probe. Error bars are propagated error following correlation of average fluorescence

intensity to concentration using a calibration curve. .................................................................... 79

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Figure 3.22: Difference in the amount of target retained after the ‘wash’ step from capture and

elution experiments between affinity capture gels that were complementary (3 µM β-actin) and

non-complementary (3 µM non-β-Actin) to a 150 nt length DNA target (20 nM). The data was

obtained from confocal fluorescence images (Chipreader) of the capillaries and values were

obtained from the profile plot function. Error bars are propagated error following correlation of

average fluorescence intensity to concentration using a calibration curve. .................................. 80


elution experiments between affinity capture gels that were complementary (3 µM β-actin) and

non-complementary probe (3 µM non-β-Actin) to a Cy5 labelled 250 nt DNA target (10 nM).

The experimental conditions are the same as in Figure 3.17 except the last step in the ‘wash’ step

was applied for 40 minutes rather than 20 minutes. The data was obtained from confocal

fluorescence images (Chipreader) of the capillaries and values were obtained from the profile

plot function. Error bars are propagated error following correlation of average fluorescence

intensity to concentration using a calibration curve. .................................................................... 81


elution experiments between affinity capture gels that contained complementary (3 µM uidA)

and non-complementary (3 µM SMN) to a Cy5 labelled 400 nt DNA target (130 nM). The

experimental conditions are the same as in Figure 3.17 except the last step in the ‘wash’ step was

applied for 50 minutes. The data was obtained from confocal fluorescence images (Chipreader)

of the capillaries and values were obtained from the profile plot function. Error bars are

propagated error following correlation of average fluorescence intensity to concentration using a

calibration curve............................................................................................................................ 81

Figure 3.25: Amount of 150 nt target captured onto affinity capture gel as a function of

concentration. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of

150 nt target in 1xTBE/PVP, 20 minute electrokinetic injection at 133 Vcm-1. Incubation time: 5

min. Wash Step: electrophoresis at 133 Vcm-1 for 25 min at 25 °C with 1xTBE/PVP buffer. The

data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values

were obtained from the profile plot function. Error bars are propagated error following

correlation of average fluorescence intensity to concentration using a calibration curve. ........... 82

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Figure 3.26: Amount of 150 nt target captured from samples in mixtures of complementary and

non-complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection

step: 10 µL of 150 nt target and non-complementary target in 1XTBE/PVP, 20 minute

electrokinetic injection at 133 Vcm-1. Incubation time: 5 min. Wash Step: electrophoresis at 133

Vcm-1 for 25 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP buffer. The data was

obtained from confocal fluorescence images (Chipreader) of the capillaries and values were


average fluorescence intensity to concentration using a calibration curve. .................................. 83

Figure 3.27: Comparison of amount of material retained by the 150 nt complementary target

when treating with 100 nt non-complementary target. Affinity capture gel: 12.5%T/1%C, 3 µM

β-actin probe. Injection step: 10 µL of 40 nM complementary (150 nt Cy5-β-actin) and 45 nM

non-complementary targets (100 nt Cy3-LAMA3) in 1xTBE/PVP, 20 minute electrokinetic

injection at 133 Vcm-1. Incubation time 5 min. Wash Step: electrophoresis at 133 Vcm-1 for 25

minutes at 40 °C with 1xTBE/PVP with different concentrations of formamide. Data was derived

from images obtained from epifluorescence images of Cy3 channel (Alpha). Error bars represent

1 standard deviation of three trials................................................................................................ 84

Figure 3.28: 1% Agarose gel electrophoresis for non-complementary target used in efficiency

experiments. Lane 1: DNA Ladder. Lanes 2 and 3: non-complementary target used in factorial

analysis. Run conditions: 100 V, 1 hour, 1x TBE buffer............................................................ 85

Figure 3.29: Amount of material captured for 150 nt target in mixture of constant concentration

of non-complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe.

Injection step: 10 µL of 150 nt target and 1.5 pmol of non-complementary target in 1XTBE/PVP,

20 minute electrokinetic injection at 133 Vcm-1. Incubation time: 5 min. Wash Step:

electrophoresis at 133 Vcm-1 for 25 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP

buffer. The data was obtained from epifluorescence microscope images (Alpha) of the capillaries

and values were obtained from the profile plot function. Error bars are propagated error


....................................................................................................................................................... 86

Figure 3.30: Amount of material captured for 250 nt target in mixture of constant concentration

of non-complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe.

xxvii

Injection step: 10 µL of 250 nt target and 1.5 pmol of non-complementary target in 1XTBE/PVP,

30 minutes electrokinetic injection at 133 Vcm-1. Incubation time: 5 min. Wash Step:

electrophoresis at 133 Vcm-1 for 40 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP




....................................................................................................................................................... 87

Figure 3.31: Amount of 400 nt target captured in mixture of non-complementary targets onto

affinity capture gel as a function of concentration. Affinity Capture Gel: 12.5%T/1%C, 3 µM

uidA probe. Injection step: 10 µL of 400 nt target and 1.5 pmol of non-complementary target in

1XTBE/PVP, 34 minutes electrokinetic injection at 133 Vcm-1. Incubation time: 5 min. Wash

Step: electrophoresis at 133 Vcm-1 for 50 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP




....................................................................................................................................................... 87

Figure 3.32: The relative fluorescence intensity following reaction between a 1 µM Cy3 labeled

oligonucleotide with different ratios of TEMED/APS radical initiator system after a period of 20

minutes. The loss was calculated relative to the in initial fluorescence intensity measured of the

solution immediately following the addition of the radical initiator. Error bars represent 1

standard deviation of three trials................................................................................................... 92

Figure 3.33: UV-VIS spectra of bulk polyacrylamide gels before and after polymerization for

20 minutes as a function of different gel formulations. a) 12.5%T/5%C, b) 12.5%T/1%C, c)

7.5%T/5%C, d) 7.5%T/1%C ........................................................................................................ 94

Figure 3.34: Reaction products between the sulfate radical anion and (a) adenine, (b) guanine,

(c) cytosine and (d) thymine. Adapted from [207]. ..................................................................... 97

Figure 3.35: Scheme of the reaction between the radical and the nucleotide base that can lead to

either removal of the nucleoside base or strand cleavage. Adapted from [213]........................... 98

xxviii

Figure 3.36: Electrophoretogram for a solution containing 2 µM 12nt, 0.5 µM 19 nt and 1.0 µM

20 nt oligonucleotides. Cy5 labeled targets. Injection: 10 µL sample volume, 142 Vcm-1, 4 s.

Run condition: 142 Vcm-1 in 1xTBE/PVP buffer........................................................................ 99

Figure 3.37: Electrophoretograms of the reaction products between a 19 nt Cy5 labelled target

and different amounts of TEMED/APS. Injection: 10 µL sample volume, 142 Vcm-1, 4 s. Run

condition: 142 Vcm-1 in 1xTBE/PVP buffer. a) Control b) TEMED/APS in 10%/10%. c)

TEMED/APS 10%/4%, d) TEMED/APS 4%/10%, e) TEMED/APS 4%/4% ........................... 102

Figure 3.38: Representative melt curve for a sample of 0.3 µM 19 bp duplex (SMN) in 1 x TBE.

Error bars represent 1 standard deviation of three trials. ............................................................ 104

Figure 3.39: Melt curves for a sample of 0.3 µM 19 bp duplex in 1 x TBE where the probe was

reacted with different amounts of TEMED and APS for 20 minutes prior to addition of the

complementary target. a) TEMED/APS 10%/10%, b) TEMED/APS 10%/4%, c) TEMED/APS,

4%/10%, d) TEMED/APS 4%/4%. Error bars represent 1 standard deviation of three trials. ... 106

Figure 3.40: Decrease in average fluorescence intensity as labelled DNA targets wash out of the

capillary following injection of the DNA target into affinity capture gels containing

complementary (�) and non-complementary (x) probe. Fluorescence intensity is normalized

against the initial fluorescence intensity. Experimental conditions: Affinity capture gel: 10%T,

5%C, 2 µM β-actin probe, 2 µM non-β-actin probe. Injection conditions: 10 µL, 250 nM Cy5-

50 nt target, electrokinetic injection at 133 Vcm-1 for 20 minutes. Incubation Time: 5 mins.

Wash conditions: 133 Vcm-1 at 25 °C with 1xTBE/PVP. The data was obtained from confocal


plot function. Error bars represent 1 standard deviation of three trials....................................... 111

Figure 3.41: Concentration of Target Injected. Values corrected for differences in fluorescence

intensity as a function of different gel formulations. Affinity capture gel: Conditions as

prescribed in factorial design. Injection: 10 µL of 170 nM (low probe) 500 nM (high probe)

target. Electrokinetic injection at 133 Vcm-1 for 20 minutes. The data was obtained from confocal


plot function. Error bars are propagated error following correlation of average fluorescence

intensity to concentration using a calibration curve. .................................................................. 113

xxix

Figure 3.42: Electrophoretic mobility of 150 bp target using different gel filled capillaries with

different gel formulations. Gel formulations used were as previously prescribed, with radical

initiator concentrations of TEMED/APS of 10%/10%. Mobility calculated based on the time

required to travel 2.6 cm along the capillary. Injection conditions, 10 µL, 0.5 µM Cy5-150 bp

target, 86 Vcm-1, 15 s. Run conditions, 143 Vcm-1, 1x TBE/PVP buffer. PMT gain 400 mV.

Error bars represent 1 standard deviation of three trials. ............................................................ 114

Figure 3.43: Amount of material captured by affinity capture gel. Values were corrected for

differences in fluorescence intensity as a function of different gel formulations. Affinity capture

gel: Conditions as prescribed in factorial experiment. Injection: 10 µL of 170 nM (low probe)

or 500 nM (high probe) Cy5-150 nt target. Electrokinetic injection at 133 Vcm-1 for 20 minutes.

Incubation time: 5 mins at 10 °C. Wash Step: electrophoresis at 133 Vcm-1 for 25 mins at 10 °C

with 1xTBE/PVP. The data was obtained from confocal fluorescence images (Chipreader) of the

capillaries and values were obtained from the profile plot function. Error bars are propagated

error following correlation of average fluorescence intensity to concentration using a calibration

curve............................................................................................................................................ 116

Figure 3.44: Schematic representation of polyacrylamide gel and how oligonucleotide probes

are incorporated into the gel. a) indicates how gels with large pores are formed and b) indicates

how gels with small pores are formed. ssDNA with hairpin structures are presented in the center

and illustrates the difference between how DNA might move through the different gel structures;

a) unhindered via Ogston, and b) stretched by reptation. ........................................................... 118

Figure 3.45: Amount of target captured for a 19 nt probe/target pair at different monomer and

crosslinker levels as examined in the factorial analysis. Affinity capture gel: 0.5 µM SMN

probe. TEMED and APS used were at 10% w/v and v/v, respectively. Injection: 10 µL, 0.5 µM

Cy5 complementary target. Electrokinetic injection at 133 Vcm-1 for 10 min. Incubation time: 5

mins at 10 °C. Wash step: electrophoresis at 133 Vcm-1 for 15 min at 10 °C with 1xTBE/PVP.

The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and

values were obtained from the profile plot function. Error bars are propagated error following

correlation of average fluorescence intensity to concentration using a calibration curve. ......... 120

Figure 3.46: log(µ/µo) versus number of bases for DNA fragments from a Low Range DNA Gel

ladder (Fast Ladder) using TOPRO3 in different gel formulations. Capillary Gel: Different gel

xxx

formulations as described in factorial analysis with 1 µM TOPRO-3. Injection: 10 µL of ladder

incubated with 10 µM TOPRO3 (1:1), 5 seconds, 570 Vcm-1. Run: 93 Vcm-1 in

1xTBE/PVP/1µM TOPRO3. Acquisition settings: PMT Gain 400 mV, sampling rate 1Hz. .. 122

Figure 3.47: Percent recovery and percent purity of sample following washing of the affinity

capture gel. Data represents the average from the duplicates defined in the factorial design.

Affinity capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt

target and 12 nM of non-complementary target. Electrokinetic injection at 181 Vcm-1 for 20

minutes. Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm-1 for 25

minutes at 10 °C, 25 °C, 40 °C using 0%, 10% and 25% v/v formamide of 1xTBE/PVP. The data

was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were


average fluorescence intensity to concentration using a calibration curve. ................................ 128

Figure 3.48: Fluorescence profile plots of capillaries taken from the outlet to inlet end generated

from confocal microscope (Chipreader) images tracking the non-complementary target following

affinity capture and a subsequent wash step using two different stringency conditions. Affinity

capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and 12

nM of non-complementary target. Electrokinetic injection at 181 Vcm-1 for 20 minutes.

Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm-1 for 25

minutes, at (a) 10 °C with 1xTBE/PVP and (b) 40 °C with 25%v/v formamide/1x TBE/PVP. 131

Figure 3.49: Profile plots of capillaries from the outlet to inlet end from confocal microscope

(Chipreader) images of the complementary target following affinity capture the wash step in the

affinity capture and a subsequent wash step using two different stringency conditions. Affinity

capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and 12

nM of non-complementary target. Electrokinetic injection at 181 Vcm-1 for 20 minutes.

Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm-1 for 25

minutes, at (a) 10 °C with 1xTBE/PVP and (b) 40 °C with 25%v/v formamide/1x TBE/PVP. 132

Figure 3.50: Electrophoretograms comparing the relative fluorescence intensities

(concentrations) of short oligonucleotide target by step elution from complementary and non-

complementary probes. a) Affinity capture gel: 50 nM SMN probe, 10% LAAm. Target

injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150 Vcm-1. b) Affinity capture gel: 50 nM

xxxi

β-actin probe, 10% LAAm. Target injection: 10 µL 5 µM Cy5-SMN target for 1 min at 150

Vcm-1. Capture step: 10 min, 1xTBE/PVP running buffer, 150 Vcm-1. Concentrating step:

coverage length: 25 mm; step size: 250 µm; step rate: 86 µms-1; Voltage: 96 Vcm-1; Acquisition

settings: ND 4, 8 and 16 filters, PMT gain 400 mV. Sampling rate: 10 Hz. ............................ 135

Figure 3.51: Schematic diagram of different permutations of the step elution sweeps where a)

the resistive heating element was started at the same point at the injection end of the capillary,

and the terminal position was varied. In b), the step elution again swept through different

distances, but stopped at the same position along the capillary.................................................. 137

Figure 3.52: Schematic diagram illustrating two possible orientations for creating an

interconnect between the capillary column and the microfluidic channel. The interconnect can

be created by orienting the capillary (a) orthogonal to the microfluidic channel and (b) in-plane

with the microfluidic channel. The area that is shaded in blue represents the filled area of the

capillary and microfluidic channel.............................................................................................. 143

Figure 3.53: Line scans of the microfluidic channel of the DNA biosensing platform following

delivery of fluorescently-labelled complementary target by selective concentration. Affinity

capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL 5 nM A647 SMN

target, electrokinetic injection for 1 min at 150 Vcm-1. Capture: electrophoresis for 10 min at

150 Vcm-1 with 1x TBE/PVP running buffer. Concentration step: coverage length: 12.5 mm;

step size: 250 µm; step rate: 86 µms-1; Voltage: 96 Vcm-1; Delivery of concentrated targets into

microfluidic biosensing platform: 500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition

settings: PMT gain 700 mV, translation speed: 50 µms-1 scan rate: 50 Hz. .............................. 144

Figure 3.54: Line scans of the fluorescence intensity along a microfluidic channel of the DNA

biosensing platform following delivery of complementary target by selective concentration. a)

both pad probe spots are complementary to the SMN target sequence, and b) one probe pad is

complementary (SMN) and the second is non-complementary (β-actin probe). Affinity capture

gel: 100 nM SMN probe, 10% LAAm gel. Target injection: a) 10 µL 1 nM A647 SMN target,

b) 10 µL 1nM A647 SMN target, 1 µM Cy3 β-actin target, electrokinetic injection for 1 min at

150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer.

Concentration step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms-1; Voltage:

96 Vcm-1; Delivery of purified and concentrated targets into microfluidic biosensing platform:

xxxii

500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV,

translation speed: 50 µms-1 scan rate: 50 Hz............................................................................... 147

Figure 3.55: Response of the microfluidic based DNA biosensing platform to quantities of

complementary target a) average fluorescence intensity signal level and b) integrated

fluorescence intensity. Different concentrations of DNA were mixed in 10% LAAm gel and

injected into an empty fused silica capillary using a syringe to CE adapter. Delivery of

complementary oligonucleotide into microfluidic biosensor: 500 V, 10 minutes, 1xTB/PVP/20

mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms-1, scan rate: 50

Hz. Error bars represent 1 standard deviation of three trials. ..................................................... 148

Figure 3.56: The response of the microfluidic biosensing platform for samples containing

complementary and non-complementary target comparing delivery with and without selective

concentrating. Direct Injection: mixture of A647 SMN and Cy3 β-actin target in 10% LAAm

gel. Selective concentrating: Affinity capture gel: 100 nM SMN probe, 10% LAAm gel.

Target injection: 10 µL of A647 SMN and Cy3 β-actin target, electrokinetic injection for 1 min

at 150 Vcm-1. Capture: electrophoresis for 10 min at 150 Vcm-1 in 1x TBE/PVP running buffer.

Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms-1; Voltage:

96 Vcm-1; Delivery of oligonucleotide targets into microfluidic biosensing platform: 500 V, 10

minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed:

50 µms-1 scan rate: 50 Hz. Error bars represent 1 standard deviation of three trials. ................. 151

Figure 3.57: Effect of the probe concentration on the amount of target introduced into the

affinity gel by electrokinetic injection. Affinity gel: Varying concentrations of dA20 probe (1.8

µM, 0.45 µM, no probe) in a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL

sample containing 0.5 µM Cy3-dT20 at 267 Vcm-1 for 60 seconds. The amount of target in the

original sample was 2.5 pmol. Error bars represent 1 standard deviation of three trials............ 154

Figure 3.58: Comparison of the response of the microfluidic biosensing platform (containing

probe for SMN) for a sample containing A647-SMN and Cy3-β-actin targets as prepared by

selective concentrating and concentrating of the all oligonucleotide targets. Delivery of

oligonucleotide targets by selective concentrating was done as previously described. Delivery of

oligonucleotide targets by non-selective pre-concentration was done using 10% LAAm affinity

xxxiii

capture gels which contained 100 nM SMN probe and 5 µM β-actin probe. Error bars represent 1

standard deviation of three trials................................................................................................. 158

xxxiv

List of Appendices

A. Factorial Design Experiment ................................................................................................. 182

A1. Fractional Factorial Designs............................................................................................ 183

A2. Design Matrix for Quarter Fractional Factorial Design .................................................. 184

A3. Choice of Factors and Levels .......................................................................................... 185

B. Synthesis of DNA Targets...................................................................................................... 186

B1. Construction of 250 bp Target......................................................................................... 186

B2. Construction of 400 bp Target......................................................................................... 189

B3. Confirmation of DNA Targets......................................................................................... 190

B4. Examination of Sequence for the 150 nt Target .............................................................. 195

C. Generation of Single Stranded DNA Targets......................................................................... 199

D. Suppression of Electroosmotic Flow (EOF) in Capillary Affinity Capture Gels ................. 203

E. Factorial Analysis for Probe Incorporated into Affinity Capture Gel .................................... 207

E1. Table of Results ............................................................................................................... 207

E2. Factorial Analysis for Percentage of Probe Incorporated................................................ 207

E2.1 Analysis of Results ................................................................................................. 207

E2.2 Pareto Effects Plot .................................................................................................. 208

E2.3 Magnitude of Effects .............................................................................................. 208

E2.4 ANOVA Table........................................................................................................ 210

E2.5 Normal Probability Plot.......................................................................................... 211

E2.6 Examination of Model Adequacy........................................................................... 212

E3. Amount of Probe Incorporated ........................................................................................ 213

F. Amount of Target Captured.................................................................................................... 216

F1. Data Reflecting the Quantity of Target Captured ............................................................ 216

F2. Concentration of Target Injected ..................................................................................... 216

xxxv

F3. Amount of Target Captured ............................................................................................. 219

G. Evaluation of Hybridization and Stringency Conditions ...................................................... 222

G1. Effectiveness of Gels for Purification of Target ............................................................. 222

G2. Hybridization Time and Wash Voltage for Samples Containing Only Complementary Targets............................................................................................................................. 222

G3. Stringency Conditions for Samples Containing Complementary and Non-complementary Targets................................................................................................... 223

H. Effect of Step Size in the Step Elution Process ..................................................................... 226

1

Chapter 1 Introduction

Nucleic acid (NA) sequences can be used for species identification, to identify the presence of

pathogens, and to detect genes and gene mutations that are associated with risk of various

dysfunctions. NAs typically represent a relatively small component of the biological make up of

cells, and are in mixture with a wide variety of other molecules when real samples from

biological sources are considered. The intention of the work in this thesis is the investigation and

development of a method for the selective concentration of one or more targeted NAs within a

mixture, for delivery to a microfluidic based NA biosensor. The DNA targets of interest were

captured selectively onto an affinity gel inside a capillary column. This affinity capture gel

consisted of oligonucleotide probes incorporated into a polyacrylamide gel matrix.

Concentrating of the captured targets was accomplished by eluting the targets into a smaller

volume than the original sample solution. This was achieved by using a localized thermal elution

zone that was swept across the capillary to stack the targets as they dehybridized from the

capture gel. Selective concentrating of the DNA target prior to detection was intended to

improve the limit of detection of the biosensor by increasing the concentration of the target DNA

while removing non-complementary NA sequences from the original sample solution. An

overview of the importance of sample pre-treatment protocols that are used prior to the detection

of DNA from real world samples, as well as some of the most common methods for DNA

purification will first be considered.

1.1 DNA Biosensors for Detection of Real-World Targets

A DNA biosensor is a self-contained device that is designed to provide quantitative information

about the presence of a specific target. It typically uses single stranded oligonucleotide probe

sequences to interact with a specific region of the target. Typically these probes are immobilized

onto a surface near the transduction element to integrate the biological recognition element with

the transducer [1, 2]. When the target DNA hybridizes with the immobilized probe sequences,

the extent of binding is interrogated to produce a signal by the transduction strategy, ultimately

resulting in production of a quantifiable electrical signal [3, 4].

2

Biosensors are intended to operate as highly sensitive and selective devices with the ability to

achieve detection of low quantities of targets, and excellent discrimination within mixtures of

closely related compounds [3, 5]. Biosensors are defined as reusable devices, and it is desirable

that the sensor can quickly be regenerated to be capable of analysis of a series of samples [1, 3,

5, 6]. DNA based biosensors have been of interest for the rapid and sensitive detection of targets

such as pathogens in environmental samples, meat, water and soil; the presence of an infectious

disease in various clinical samples such as blood, urine or saliva; and for the identification of

genetic markers which differ by as little as one base pair [1, 2, 5, 7, 8]. Such applications require

rapid detection of very low concentrations of nucleic acids in complex sample matrices [9].

Early detection of diseases may allow for early treatment which may provide a better outcomes

[10–12]. Small amounts of foodborne pathogens can result in illness. For pathogens such as

Listeria monocytogenes and Campylobactor spp, infectious doses starting from 400 Colony

Forming Units (CFU) have been reported [3]. Ten bacterial cells of E. coli O157:H7 can result in

infection [13]. Inhalation of more than 104 spores of Bacillus anthracis requires medical

attention within 24-48 hours. However, symptoms can take up to 60 days to appear in humans,

delaying necessary treatment [14].

In addition to the need to detect low concentrations of target, it must be recognized that the target

is also present in a large amount of background that can interfere with analysis. For example, E.

coli O15:H7 in stool samples require detection of below 105 CFU/mL, and are contained in a

complex mixture of non-pathogenic strains [15]. For blood samples, the amount of genomic

DNA present from the blood may be 1014 times higher than that DNA from pathogenic species

[9]. Cells in whole blood are predominantly erythrocytes and account for 99% of total cells,

while the DNA containing leukocytes make up less than 1% [16]. Detection of B. anthracis from

soil samples requires differentiation with other closely related non-pathogenic Bacillus species

such as B. cereus, B. mycoides and B. thuringiensis [17]. The presence of a high background in a

sample matrix may hinder detection and can compromise selectivity [9, 15]. If sample analyte is

in very low concentration, detection may become an issue if the signal generated by background

is sizeable [18]. Components in the sample matrix may also affect the long-term stability of the

recognition element and transducer [9]. These can include nucleases which can degrade the DNA

target and immobilized probes, compounds which can inhibit target amplification by Polymerase

3

Chain Reaction (PCR), and aggregation factors or other cells or cell fragments that can clog

downstream processing [13, 15].

Currently, tests for the identification of cell-based pathogens are usually performed with

inclusion of a culturing step. The cells are allowed to grow and multiply in selective media to

enrich the concentration, and this is followed by biochemical tests selective towards that strain's

phenotypes. This can take 20 hours to several days to complete [8, 9, 19, 20]. There is a need for

technology that can offer more rapid detection of targets with high sensitivity in complex

matrices at the point of care level [9, 15, 21, 22]. DNA biosensors have demonstrated

quantitative function within several hours, depending on the type of sample preparation required

[9, 20, 23, 24].

Table 1.1 provides some examples of biosensors that have been applied for the detection of DNA

targets in real samples. Listed are the transduction method, type of sample pre-treatment

protocol as well as the limit of detection that was reported. Additionally, the total analysis time

from the point of obtaining the sample to the actual quantitative detection step is provided. This

information was compiled based on the time for each pre-treatment step as listed in the

experimental method, as well as time that was indicated for detection. The time required for

only the detection step is also shown when available. Some of the pre-treatment protocols listed

will be discussed further.

As can be seen in Table 1.1, detection of targets from real samples often require multiple pre-

treatment steps for purification and to also concentrate the targets prior to detection. Some of

these pre-treatment steps can become quite involved, and in some cases can take more time than

the actual analysis.

Sample purification and concentrating are typically necessary for detection of NA targets from

real samples. In addition to removal of background matrix and amplification of the target, the

NA targets must be released from inside cells, and must be in a form in terms of length and

folding that it is amenable for detection using hybridization [18]. Concentrating the NA targets

can occur by reduction of volume in which the analyte is contained and can alleviate sensitivity

demands placed on a detector [18, 25]. For NA analysis, a more common strategy for

improvement of detection sensitivity is amplification of NAs during sample pre-treatment by

Polymerase Chain Reaction (PCR).

4

Table 1.1: Examples of biosensors used for detection of NA targets from various different sample matrices. Steps for pre-treatment protocols prior to detection are identified, as well as the total time

needed for pre-treatment and detection. When available, detection time is also provided in parenthesis.

Transduction Mode

Target Sample Pre-treatment

Protocol LOD

Total Analysis

Time Ref

Spectroscopic E. coli In buffer

Culture enrichment,

centrifugation, lysis, chloroform-

phenol extraction,

ultrasonication

1 pg/mL

> 12 hours (20

second detection)

[26]

Visual Vibrio cholerae Clinical

Samples

PCR, concentration by

lateral flow

5 ng in 10 µL

~1 hour (10 min

detection) [27]

SPR Tumor necrosis

factor (TNF-alpha)

In buffer PCR,

denaturation by heat

0.677 pM

~120 min [23]

Piezoelectric HPV Clinical cervical

scrapings

Centrifugation, DNA Extraction

(Commercial Kit), PCR

50 nM ~10 hrs (20 min

detection) [28]

Piezoelectric E. coli Water

Culture enrichment, lysis,

phenol-chloroform extraction,

centrifugation, PCR,

denaturation

1 µg/mL >12 hrs (10 min

detection) [29]

Piezoelectric E. coli O157:H7 In buffer

Culture enrichment,

centrifugation, extraction,

Asymmetric PCR

2.67x102

CFU/mL 22 hrs [30]

Piezoelectric Aspergillus

flavus and A. parasiticus

Flour/feed Extraction, PCR 0.03 µM 2.5 hours (20 min

detection) [31]

Piezoelectric HPV Clinical samples

Plasmid extraction

1.21 pg/L

3 hours [32]

Piezoelectric CaMV 35S GMO -

Tabacco Plants

Extraction, ultrasonication

0.25 ng/µL

30 minutes

[33]

Electrochemical Phosphinothricin acetyltransferase

GMO soybean

PCR 2.7x

10-14

M

2 hours (40 min

detection) [34]

Electrochemical E. coli Water Magnetic beads,

heat lysis/denaturation

0.5ng/µL

40 minutes

(30 minute

detection)

[35]

Electrochemical E. coli O157:H7 Cultured samples

Centrifugation, cell lysis,

concentration using magnetic

2.5 aM , 0.01

CFU/mL 6 hours [8, 20]

5

beads, asymmetric PCR

Electrochemical Human

Interleukin 2

From cultured samples

Centrifugation, Liquid N2 lysis,

genomic extraction,

purification, PCR

69 pM 3 hours (15 min

detection) [24]

1.2 Goals of Pre-treatment

The major goals of the pre-treatment of a sample for detection by a biosensor are: extraction of

the target material from the sample matrix, the purification of the target, sample enrichment

through amplification or volume reduction and conversion of the NA targets into short, single

stranded fragments.

1.2.1 Extraction

Extraction may be required to isolate the analyte of interest from the background matrix. For the

extraction of NA, the cells of interest must be lysed and the NA separated from the intracellular

material. Common methods for isolation of NAs from the sample include filtration and

centrifugation. Cell lysis can be accomplished by chemical or mechanical means. Such

techniques have been reported and extensively reviewed [36–43].

1.2.2 Purification

The removal of background components may be necessary to avoid inhibition or competition

with the bio-recognition between the probe and its target [44, 45], or if contaminants can also

generate a signal [37, 46]. Since biological materials are generally used as the selective reagents

to develop biosensors, issues related to the stability of the probes in the background matrix may

arise [37].

Purification may also be required in order to differentiate between viable and dead cells prior to

the extraction of DNA. This is important where only viable cells are of interest; for example in

the detection of a pathogen in consumable products where only live cells will adversely affect

human health. One challenge is that extraction methods will extract NAs from both viable and

dead cells [44], making it very difficult to differentiate whether there is any actual danger

associated with biologicals in a consumable product [44, 47, 48].

6

Purification of samples can also be important when considering the quantitative determination of

a target. Variations in the components of the matrix between samples may pose a problem for

quantitative analysis [1]. Calibration curves are often constructed using solutions of known

concentrations of the target in water or buffer. Unless steps are taken to ensure that the

background matrix from a “real” sample would not interfere with the quantitative determination,

purification is necessary to ensure that the background matrix of the sample was compatible with

that used with the calibration curve [1]. Alternatively, a standard addition approach might be

considered, but this is of little interest if the matrix interferes with signal development.

The majority of purification protocols done to extract DNA are intended to remove compounds

that might inhibit amplification of NA targets by PCR. PCR is a common step for amplification

of NAs prior to detection by a biosensor to improve sensitivity, and can also serve in itself as a

detection strategy, confirming the presence or absence of the analyte [8]. As can be seen from

the examples listed in Table 1.1, PCR amplification is commonly used to amplify the target

sequence.

Environmental samples, such as those from water, soil and food materials are known to contain

compounds which can inhibit PCR or that can interfere with detection [49–51]. Such

compounds include humic acids, polyphenolic compounds, polysaccharides, urea, soot, dust and

pollen, silt, clay, metal ions, chelators, milk products, fat in foods, hemoglobin, iron, heparin,

acidic polysaccharides, as well as NAs from non-target microorganisms [38, 45, 49, 50, 52–54].

Humic acids and polysaccharides can bind to DNA polymerase, and to chelating agents which

may be co-factors for the enzyme. It has been shown that as little as 1 ng of humic acid can

inhibit PCR [45]. Haemachrom in erythrocytes is also known to be an inhibitor of PCR.

Removal of red blood cells from white blood cells is required for NA purification [16].

1.2.3 Sample Loss and Quantification

The sample pre-treatment protocols outlined in examples in Table 1.1 often involve multiple

steps before the target molecules can be detected by a biosensor. Therefore, consideration must

be given that with each purification step taken, there is the possibility of loss of the analyte by

partial transfer and non-selective adsorption. An extensive multi-step protocol may lead to

significant loss of target, which can be disproportionately detrimental in cases where the quantity

7

of target is low [55]. Oftentimes each sample pre-treatment protocol is optimized to suit each

application and sample type, resulting in variations in the pre-treatment protocols [44, 56, 57].

The loss induced by the purification protocol must be accounted for when quantitative

information provided by a biosensor is to be meaningful. However, achievement of reproducible

efficiency of a purification protocol is not always a simple matter. For example, differences in

cell wall structures may also result in differences of adhesion behaviour of cells with particulate

matter in the sample, resulting in variation of loss due to adsorption [36, 58]. Differences

between bacterial cells will also alter the effectiveness of typical lysis protocols when attempting

to release intracellular materials for assay [36, 56, 58]. While gram-negative organisms were

successfully lysed using an alkaline agent, gram-positive organisms, having a thicker cell walls,

required the action of a surfactant (TritonX-100) and an enzyme (lysozyme) in order to

efficiently lyse the cells [59, 60]. It was reported by Honoré-Bouakline et al. and Kotlowski et

al. that some commercially available kits do not allow for the complete lysis of mycobacterial

cells such as Mycobacterium tuberculosis [59, 61].

The recovery (quantity recovered) and purity (relative amount of target within sample) of

extracted NA often varies depending on the protocol that is being used, as well as the

composition and type of sample [36, 44, 54, 62, 63]. The recovery of DNA from standard

phenol-chloroform extraction was demonstrated to be lower when using a sample such as canned

tuna in comparison to DNA extracted from raw, fresh tuna [64]. Purification methods such as

gradient centrifugation, glass bead extraction, chromatography and spin columns may also result

in a significant loss of extracted NA [50, 54, 65]. As a general rule, the more extensive the

purification protocol, the lower will be the recovery of NA [54, 66]. Additionally, since many

biological molecules are charged, loss due to adsorption by electrostatic interaction with the

materials used for purification is common.

Many of the established purification protocols can only provide semi-quantitative assays unless

steps are taken to account for the loss of material during purification [67]. Quantification at each

stage of a multi-step extraction procedure is considered important to account for loss during

purification [68].

8

1.3 Methods for DNA Purification

The following presents an overview of some of the most common methods used for DNA

purification. Most are based on the capture of the DNA onto a solid support surface, removal of

any non-DNA material by washing, and elution from the support surface. Many of the methods

are not selective towards specific DNA sequences, while a few methods exist where the target of

interest is purified selectively.

1.3.1 Purification Methods based on Solid Phase Extraction (SPE)

Solid phase extraction is a commonly used technique for the purification of DNA from a

complex matrix following cell lysis [69, 70]. DNA will bind to silica or glass particles by

hydrogen bonding or electrostatic interactions in the presence of a chaotropic agents such as NaI,

NaClO4, guanidine hydrochloride or guanidine thiocyanate [59, 71–73]. The captured DNA can

be eluted by application of a low salt buffer [72].

The use of solid phase extraction eliminates the need for organic solvents associated with

conventional extraction and purification techniques such as phenol-chloroform extraction [70].

Many commercially available kits for extraction and purification of DNA also use a solid phase

extraction step [69, 70]. Solid phase extraction has been used for the purification of DNA from a

wide variety of different types of samples including plants, fungi, micro-organisms as well as

human hair, teeth, bone and blood [70]. Concentrating the sample can be accomplished by

eluting the DNA into a smaller volume than the original sample [72, 74].

Common solid phase extraction materials include hydrophobic surfaces such as alkyl-bonded

silicas (C18, C20), and copolymers such as cross-linked polystyrene and divinylbenzene [2, 75].

Normal phase materials such as those containing diol, aminopropyl or cyanopropyl functional

groups have also been used [75].

Proteins that are present in a biological sample can also bind to solid phase extraction material,

limiting the binding capacity of DNA on the columns [76]. Capture is non-selective, and any

compounds with similar properties to NAs may also be retained by the resin, decreasing the

purity of the extracted material [77].

9

Landers’ group has demonstrated a SPE purification system for DNA by using monolithic

extraction columns made from 3-(trimethoxysilyl)propyl methacrylate (TMSPM) modified with

85% v/v tetramethyl orthosilicate (TMOS). The addition of TMOS has been shown to increase

DNA binding by the monolith. Elution of the captured targets into a final volume of 1 µL was

done by using a low ionic strength Tris buffer at pH 8. Solid phase capture of PCR amplified

380 bp human genomic β-globin fragments as well as human genomic DNA purified from blood

was demonstrated. Both samples still required the addition of guanidine hydrochloride prior to

binding. Extraction efficiency as quantified by a PicoGreen assay was 86% for the PCR

amplified genomic DNA and 60% for DNA from whole blood. These efficiencies were greater

than DNA purified using a commercially available solid phase extraction kit [78].

Chromatographic techniques such as size exclusion, ion-exchange and affinity chromatography

has also been demonstrated for SPE purification of DNA [36, 79]. Gel filtration is a common

method for size exclusion that can purify DNA with minimal loss of the target [36, 80].

Ion exchange columns can selectively bind or elute DNA based on the pH and ionic strength of

the buffers used. Anion-exchange has been the most prominent technique. Positively charged

diethylaminoethyl (DEAE) tertiary or quaternary amine anion exchange resins will bind to

negatively charged DNA [79]. Elution of NA from the resin can be done by adjusting the pH of

the mobile phase [77]. Recovery of 80% of NA bound on the resin has been reported [36, 79].

Polymeric monoliths can be used as an alternative to resins. Monoliths are single pieces of

porous material where the pores are interconnected, forming channels with diameters ranging

from 13 nm to 4000 nm and 60-80% porosity within the monolith material. The pore size can be

selected to increase the surface area available for binding of NAs. Monoliths tend to offer good

mass transfer between mobile and stationary phases within channels and low back pressures at

high flow rates [36, 81, 82]. Methacrylate-based monoliths have been demonstrated for the

separation and purification of varying sizes of DNA molecules. For example, DNA binding was

calculated to be 9 mg/mL for a number of different DNA targets, including plasmid DNA from

E. coli as well as 200 kbp and 50 kbp genomic DNA. These results are notable when compared

to binding of less than 200 µg/mL on anionic Sepharose resins. Recovery of the DNA was

achieved by adjusting pH and ionic strength of the elution buffer. Under optimal conditions,

recoveries of up to 80% were obtained within 10 minutes [79].

10

A DEAE monolithic anion exchange column has been used to purify genomic DNA [82].

Samples of bacterial and eukaryotic genomic DNA purified by a commercially available kit were

tested on the monolithic column. Elution of the DNA was performed by increasing the

concentration of salt in the elution buffer. The 3 mm x 12 mm monolithic column could also be

used to process large volumes of DNA samples. Binding of ~4 mg of DNA from a 30 mL

sample was demonstrated with no DNA being detected in the wash fractions. It was also shown

that RNA present in the purified samples could also be removed by the column [82].

Magnetic beads can also be used for the capture of DNA similar to solid phase extraction [77,

71, 83–85]. Modifications of the magnetic beads can include a silica surface [86], polymer

surfaces [87], polyvinyl alcohol (PVA) or hyperbranched polyamidoamine dendrimers [69]. The

latter two provide for an additional electrostatic component for the capture of charged material

[69]. Carboxy-coated magnetic beads have also been used for bioaffinity adsorption of DNA

[88].

Anion-exchange resins that contain a paramagnetic core are available commercially (Whatman

DEAE-Magarose). These beads have been used to isolate plasmid DNA from bacterial cell

lysate and genomic DNA from bacterial cells and blood samples by means of electrostatic

attraction [77]. Magnetic beads can be functionalized with a dense coating of amino groups on

the particle surface by an aminosilane reagent, 3-[2-2-aminoethy(amino)-ethylamino-

propyetrimethoxysilane]. DNA can then be captured by electrostatic interaction between the

positively charged amino-modified beads and the negatively charged DNA [84]. An automated

sample pre-treatment using magnetic beads has been introduced for the extraction of DNA from

solid biomaterials [71].

However, the different methods for non-selective purification of NA material presented may not

have the same efficacy in purifying a sample. For example, a study conducted by Trochimchuk

et al. compared the efficacy of purification of DNA from E. coli present in cattle manure by

phenol-chloroform extraction, gel filtration and SPE. The efficacy was measured by its removal

of compounds which inhibit PCR amplification. It was noted that NA samples purified by SPE

purified were successfully amplified by PCR, while samples purified by phenol/chloroform and

gel filtration did not yield successful PCR amplicons unless the original sample was diluted [80].

11

Bencina et al. also compared the efficacy of NA purification using ion-exchange columns and by

SPE. It was observed that both methods recovered comparable quantities of NA content and

removed inhibitor compounds to PCR. However, optimization of anion-exchange columns

required adjusting a number of factors such as flow rate, ionic strength and pH. Additionally,

fragments of 50kbp and larger resulted in poor recovery with the ion-exchange columns, which

might be due to the large number of charge interaction between the DNA and the anion exchange

resin [79]. The efficacy of SPE to purify NA samples for PCR amplification, its relatively ease of

use as well as its widespread and commercial availability makes SPE one of the most attractive

and commonly used methods for non-selective NA purification.

1.3.2 Purification of DNA in Conjunction with Selective Hybridization

The purification methods outlined in the previous section can be considered non-selective

methods that collect any NA material present in the sample. An issue that arises by such non-

selective concentration methods is that a large amount of non-target NA is collected concurrent

with the target NA, which may hinder detection downstream.

Methods where only the NA target of interest is retained and purified from other NA in the

sample have also been reported. These methods use oligonucleotide probes selective towards a

particular target incorporated into support materials for the selective capture and purification of

DNA from a sample. Following the selective capture of the DNA targets, the column can be

washed, and the captured DNA can be eluted as a relatively pure sample [77]. Through selective

capture of complementary material followed, it may be possible to reduce or eliminate issues that

are associated with non-selective adsorption of non-complementary target on detection elements.

Magnetic beads have been modified with oligonucleotide probes to capture a specific DNA

target [69]. DNA that is captured on the magnetic beads can be used for direct detection without

eluting the DNA [85, 89, 90]. For example, an electrochemical biosensor using magnetic beads

and PCR for E. coli. analysis demonstrated that 2.5 aM of PCR products could be detected [20].

Fuentes et al. demonstrated the detection of 10-18 g/mL (two molecules) of HCV cDNA from a 1

mL sample by PCR amplification of DNA targets that were selectively captured on magnetic

beads. The detection was not impaired by the addition to the sample solution of 2.5 million fold

excess of non-complementary DNA. Non-selective adsorption of DNA was decreased by

blocking active sites with aldehyde-aspartic-dextran. The use of the negatively charged polymer

12

reduced the positive charge of the support surface and also allowed for a strong secondary amine

bond to be formed between the oligonucleotide probe and the polymer coated magnetic beads.

However, some non-specific adsorption was still observed [85].

Even though purification using magnetic beads may offer a much simpler and faster extraction

method than conventional phenol-chloroform extraction, the recovery and purity of the extracted

DNA may not be as high as conventional methods [83]. A study by Faggi et al. comparing

purification of DNA from yeast using magnetic beads and conventional phenol-chloroform

extraction found that phenol-chloroform resulted in higher recovery of extracted DNA with

better purity than magnetic beads. The amount of DNA extracted from three different types of

fungi by phenol-chloroform was 6684, 2022 and 945 µg/mL. Purity was indicated as the ratio of

optical densities of UV absorbance at 260 nm and 280 nm, which was reported to be 1.8, 1.4 and

1.4, respectively. By comparison, the amount of DNA extracted from the same three types fungi

by magnetic beads were 443, 223 and 329 µg/mL with purities of 1.3, 1.2 and 1.4, respectively.

Additionally, the cost per test using magnetic beads was higher than phenol-chloroform

extraction [83].

Peptide nucleic acids (PNAs) have also been demonstrated for affinity capture of DNA targets.

PNAs are synthetic mimics of oligonucleotides that are stable against the action of peptidases

and nucleases [91]. Duplexes of PNA-DNA and PNA-RNA show higher thermal stability and

faster hybridization kinetics versus their DNA-DNA and DNA-RNA counterparts [92, 93]. The

improvement in hybridization kinetics is believed to be due to the neutrality of the PNA

backbone, eliminating the electrostatic repulsion observed for the hybridization of two DNA

strands [92]. PNA immobilized onto beads has been reported to offer enhanced recovery and

detection of rRNA. rRNA was captured and detected on PNA-coated Lumavidin beads from 0.1

ng of RNA within 15 minutes. By comparison, the use of DNA immobilized beads required an

overnight hybridization before a detectable signal was obtained from the same amount of RNA

[92]. Unfortunately, PNAs are not highly soluble in water and have a tendency to self-aggregate

and sediment in low salt concentrations [91].

Oligonucleotide probes can also be incorporated into chromatography support materials for the

selective capture and purification of DNA from a sample. Various research groups, including

13

those of Mathies, Olsen and ours have utilized a commercially available oligonucleotide

modification sold under the name of Acrydite™.

The acrylamide modified oliogonucleotide probe was first introduced by Rehman et al., and

allows for the oligonucleotide probes to be incorporated into polyacrylamide gels during radical

polymerization [94]. Acrydite™ represents a modification that adds an acrylamide monomer

unit to the 5’ end of an oligonucleotide sequence. When mixed with a solution of acrylamide

monomer and polymerized, the resulting polyacrylamide gel has oligonucleotide probes

incorporated into the gel matrix [95–97]. DNA targets are then driven through the modified gels

by electrophoresis [98]. Any debris or unwanted salts can be washed away, and the

complementary targets are subsequently released by denaturation [95–97]. This chemistry has

also been used for incorporation of the NA probes into a 3D gel matrix, and has been used to

clean up of PCR amplicons for CE sequencing, pyroseqeuncing and as a biosensor platform [96,

97, 99–101].

Figure 1.1: Radical polymerization reaction of acrylamide in the presence of an Acrydite™ modified

oligonucleotide.

14

Whitney et al. manufactured polyacrylamide gels that were modified with oligonucleotides.

These affinity gels were cut into 1 cm diameter disks and were used to capture DNA from a

sample volume of 2.4 mL by electrophoresis. The captured target was subsequently eluted into a

volume of 50 µL by the addition of NaOH. A 5 fold increase in the quantity of DNA was

recovered from stool samples when compared to the use of immobilized probes on magnetic

beads [98].

Polyacrylamide gels modified with capture probe have also been used inside microfluidic

channels by Olsen and Mathies [96, 97]. After the running conditions were optimized, this

method was capable of desalting and concentrating a sample of PCR amplification product in

120 seconds with a 100 fold increase in concentration based on volume reduction [96].

We have previously reported the quantitative determination of the efficiency of capture by

probe-modified gels loaded inside fused silica capillaries (100 µm I.D. X 7.5 cm length). By

measuring the fluorescence intensity of labelled oligonucleotide targets, the capture efficiency

was determined to be approximately 90% of the amount of probe molecules that were

incorporated into the gel. Elution by heating and chemical denaturation released approximately

95% of the captured target. A constant loss of 8 femtomoles of the target DNA by adsorption

onto the gels was observed after elution, and was independent of probe and target concentration.

This loss of DNA was still observed even after efforts to block active sites by pre-conditioning

the capture gels with a solution of non-complementary oligonucleotides [95]. One objective of

the work presented in this thesis will be to examine the response of a biosensing platform to

samples processed by selective versus non-selective methods of NA purification.

1.4 Enrichment and Amplification of Sample

Enrichment and amplification of the target DNA is often necessary in order to detect low target

concentrations that are present in clinical, food and environmental samples. The most common

methods increase the total amount of target present, and make use of methods such as cell

culturing prior to DNA extraction, or by PCR.

15

1.4.1 DNA Amplification by Polymerase Chain Reaction

PCR allows for a small number of copies of DNA to be amplified exponentially. This process is

often used for amplification of DNA targets for applications in trace analysis, and is still a

necessary step for detection of low concentrations of DNA by biosensors [20, 32, 102].

PCR uses the target DNA as a template that will be copied. DNA primers are used to flank the

target sequence to mark the initiation and termination locations for copying. Nucleotide

triphosphates are used to build the new DNA copies, and are stitched together by DNA

polymerase. Heat stable DNA polymerase is used. Amplification starts by first annealing the

primers to the target DNA, followed by an increase in temperature to 72°C for polymerase

activity and 94°C to denature the newly formed double-stranded DNA (dsDNA). The

temperature of the reaction vial is lowered again to anneal the primers to the target DNA and the

cycle is repeated, and the quantity of DNA is amplified exponentially [103]. Theoretically, more

than 109 copies of target DNA can be produced after 25 to 30 cycles [104]. The length of the

PCR amplicons can be controlled by designing the DNA primers to bind to different areas of the

DNA target. Fluorescently labelled-DNA targets can be generated by adding specific

fluorescence modification to the monomeric nucleotides prior to assembly by polymerase..

On average, a typical 30 – 40 cycle PCR amplification in bulk solution can take upwards of 2-3

hours to complete, but is wholly dependent on the reaction times for each step [23, 28, 29].

Amplification yields are rarely truly exponential and will plateau over the course of the PCR

amplification [105]. This is thought to be the result of the consumption of reagent at higher

cycle numbers, poor template to primer hybridization, contamination in the sample, inefficient

thermal cycling and poor temperature control [103]. Efficient amplification will depend on rapid

heat cycling so to allow for rapid temperature transitions. More rapid and efficient heat cycling

can be achieved by reducing the volume of the PCR mixture to less than 1 µL [103]. Such small

volume PCR reactions has been demonstrated by construction of small microreactors on

microfluidic devices [106–108].

Another major contributor to amplification efficiency is the purity of the template DNA.

Inhibiting compounds can deactivate the thermostable DNA polymerase or degrade the target

nucleic acids [22, 109]. For any sample pre-treatment protocol that includes amplification by

PCR, stringent purification protocols are usually implemented in order to remove most if not all

16

of the inhibitor compounds. Some recent relevant developments to overcome such limitations

include work by Kermekchiev et al., where they recently identified mutants of thermostable Taq

DNA polymerase that were found to be more resistant to inhibitor compounds in whole blood

and crude soil samples [110]. Also, the Collins group has examined a method for performing

PCR directly from lysed cells without requiring purification of the DNA from the cell lysates

[111, 112]. A sufficient dilution factor was found where the concentration of inhibitors in the

solution no longer inhibited PCR. PCR was performed successfully on a 10 fold dilution of

E. coli O157:H7 cell lysates (original concentration of 200 cells/mL) [112].

1.4.2 Concentrating by Volume Reduction

Biosensors typically require small sample volumes for detection. Volumes usually range from

nano- to microlitres. However, for trace detection of analytes in “real” samples, collection of

microlitres may not be statistically representative of the sample.

Bacteria or viruses may not be homogenously distributed within samples, necessitating that large

volumes be collected for representative sampling [113]. Additionally, the target pathogens may

be present in low numbers, again requiring large volumes to be collected so as to have

confidence in quantification [13, 47, 114, 115].

For example, Mycobacterium ulcerans, which is a human pathogen that causes chronic necrotic

skin disease, has a threshold “load of concern” that is estimated as approximately 0.5 cells per

100 mL of water [59]. The infectious dose for many foodborne pathogenic bacteria is often only

a few cells [116, 117]. For E. coli O157:H7 in ground beef, contamination levels are usually less

than 100 CFU/g of beef [52, 118].

In the detection of cancerous colon cells from stool samples, the amount of DNA obtained from

colon cells represents only 0.01 to 0.1 % of the total DNA that is recovered from the samples. Of

that small percentage, only about 1% of those cells may be cancerous [98]; the balance being

primarily from DNA associated with bacterial cells.

The problem of low abundance of the target for these cases results in the need for large sampling

volumes. The challenge is to ensure that a sufficient quantity of pathogen has been collected to

meet the limit of detection for the sensor, as well as to provide replicates for statistical

confidence measures of results. For example, assume that a RNA-based biosensor had a limit of

17

detection of 16 ng/µL of RNA for a 2 µL sample. Assuming a homogeneous sample, and that

the extraction and purification protocol recovery an average of 0.02 ng of RNA per cell, then the

sampling volume would be about 6.4 L if the solution contained 250 cells/L (i.e. 800 cells in

total) to achieve a detectable amount of rRNA [119]. Volumes taken for environmental samples

are often several orders of magnitude larger than the volumes actually required by a biosensor in

the measurement step. This requires some form of volume reduction so that the volume is

reduced without significant loss of the target. Volume reduction is usually done by capture or

sequestering of the target, purification, and subsequent elution of the target into a smaller

volume. This selective concentrating of the target results in enhancement in signal-to-noise,

improving detection sensitivity and the reliability of analysis [120, 121].

1.5 Fragmentation and Denaturation of DNA

The length of the DNA targets can impact hybridization kinetics, and this is particularly

important for hybridization with immobilized probes. DNA fragments need to be sufficiently

short to allow for fast hybridization. Lengths of DNA target commonly used are on the order of

25 to 300 base pairs. By comparison, the lengths of DNA from cells are orders of magnitude

longer. For example, the genomic DNA for most E. coli strains is on the order of 4 million base

pairs in length [122]. Short target sequences within long fragments of DNA may be sterically

hindered from interacting with immobilized probes [122, 123]. Furthermore, if the target

fragments are too long then intramolecular structures may be present that prevent hybridization.

1.5.1 Fragmentation

Various mechanical cell lysis methods used to release intercellular material have been shown to

fragment DNA to differing degrees [36, 38]. Bead beating often results in the most fragmented

DNA [52, 124, 125] while ultrasonication can be used to shear as well as denature DNA [122–

124].

Fragmentation of DNA using ultrasound occurs by mechanical damage through heating of the

solution and by chemical damage by free radicals attack on the DNA. Our group has previously

demonstrated that ultrasonication (85 W power, 20 kHz) can fragment genomic DNA extracted

from E. coli cells to produce products in the 100-400 bp range within 30 seconds. Application of

18

ultrasound will also heat the sample solution, which will also cause denaturation of the DNA

fragments [122].

Enzyme catalyzed scission of DNA involves use of restriction enzymes, and this approach has

also been used to generate shorter DNA fragments than typically obtained from short periods of

application of high power ultrasound [122, 126].

1.5.2 Preparation of Single-Stranded DNA Target

The most common method for denaturation of DNA targets is by heating the DNA solution at

95 °C for 5 minutes followed immediately by cooling to ice temperature [126–128]. A

modification to thermal denaturation has been to introduce blocking oligonucleotides directly to

the denaturation mixture. These blocking oligonucleotides are 10-30 nt length single-stranded

oligonucleotides that are complementary to a region on the DNA target. Following thermal

denaturation at 95 °C, the solution is cooled to allow for annealing of the blocking

oligonucleotides to the DNA targets. This prevents the re-annealing of the denatured strands

while still leaving the target region available for binding to the oligonucleotide probes on the

biosensor [126–128].

As one example, a SPR-based DNA biosensor was used to examine samples of bovine DNA

processed by thermal denaturation with blocking oligonucleotides. These experiments showed a

43% increase in signal in comparison to the same samples that were only thermally denatured

[126]. Similar results were also observed for samples of apolipoprotein E gene in humans and

the 35S promoter sequence that is present in most genetically modified organisms [126].

Short, single stranded DNA targets can also be produced by asymmetric PCR. In conventional

symmetric PCR, equal concentrations of forward and reverse primers are used in the reaction

mixture and the amplicons are in double stranded form. Denaturation by heat followed by rapid

cooling has been used to generate single stranded products. In asymmetric PCR, a higher

concentration of one primer is added over the other [129]. Over the course of the amplification,

the amplicons will be predominantly in a single stranded form, extended from the excess primer

[130].

19

Figure 1.2: Schematic of differences between (a) conventional denaturation by heat and (b) denaturation by heat with ancillary blocking oligonucleotides. With permission from Analytica Chimica Acta. Copyright

2004, Elsevier [131].

The use of asymmetric PCR provides an amplification yield that is significantly less than that

from conventional PCR. For asymmetric PCR, amplification occurs exponentially until the

primer in lower abundance is exhausted. Subsequently, the available number of templates

remains constant, and amplification occurs in a linear fashion as cycling continues. Asymmetric

PCR often requires extensive optimization to determine of the ratio between the primers,

amounts of starting materials and number of cycles to provide sufficient amplification of a

particular target [130].

Wangh’s group has introduced a modification to asymmetric PCR called Linear-After-The-

Exponential PCR (LATE-PCR). In LATE-PCR, the length and nucleotide base composition of

the two primers are adjusted so that the difference in melt temperatures is zero or the limiting

primer has a higher melt temperature than the excess primer.

The amplification efficiency of LATE-PCR over asymmetric PCR was demonstrated for an

application involving the CD∆508 cystic fibrosis allele and the TSD 1278 Tay-Sachs disease

allele, where more products were detected by fluorescence from LATE-PCR after 80 cycles

[130].

20

1.6 Integrated Microfluidic Devices

There is an interest to integrate the extraction, purification and concentration steps with detection

into a single package. A miniaturized device can reduce reagent use and assay cost, often

increases speed of the relevant processes, and allows a lab to become “portable”. A system that is

free of human interaction with the sample improves reproducibility and ameliorates opportunity

for contamination or sample loss [15, 102].

A fully integrated microfluidic device was recently developed by Liu et al., where cell capture

by immunomagnetic beads, purification, concentration and thermal lysis were all done within the

same device. The capture efficiency using the magnetic beads was determined to be at 40%.

Discrimination of single-nucleotide polymorphism targets was demonstrated following on-chip

PCR amplification. The detection of 103-106 E. coli K12 cells from a 1 mL whole blood sample

was shown and required 2.7 hours. It was observed that the presence of the magnetic beads in

the PCR mixture reduced the amplification efficiency by 50% [132].

Landers’ group has demonstrated a fully integrated microfluidic device based on purification by

SPE that could generate a genetic profile from whole blood. Upon loading the sample into a

glass-based microfluidic device, a chemical lysis agent was mixed with the blood. This was

followed by loading onto a silica bed for purification of genomic DNA. The eluted DNA was

amplified by PCR. The entire process took less than 30 minutes, and 1500 to 2000 CFU of

Bacillus anthrax was detected in 750 nL of whole blood [25].

Integrated microfluidic systems for DNA detection have also been demonstrated using digital

microfluidic (DMF) platforms. In contrast to conventional microfluidic where solution is

continuously moved through the microchannels in the chip, DMF moves independently

addressed droplets of solution with typical volumes of micro- to pico-litres discretely on an

electrode array by a phenomenon termed electrowetting on dielectric (EWOD) [133, 134].

Using DMF, droplets of different chemical reagents can be dispensed from a reservoir and mixed

together by moving the droplets onto the same location on the array. This allows for bench-top

protocols to be reduced in scale for implementation in DMF. DMF may provide a modular

platform for the miniaturization and integration of multiple conventional bench-top scale pre-

treatment techniques and assays into a microfluidic scale [133–135]. Sample pre-treatment

techniques such as immunomagnetic capture of cells onto magnetic beads followed by

21

concentrating [133, 136], liquid-liquid extraction of oligonucleotides from histones [137] and

PCR [134–136, 138] have been demonstrated using DMF.

Besides electrokinetics, fluids can also be manipulated in a microfluidic platform by centrifugal

forces. Centrifugal or Compact Disc (CD) based microfluidic platforms use disc-shaped devices

where a centrifugal force is generated by rotation of the disc [139–143]. Centrifugal-

microfluidic platforms have an advantage over electrokinetic based microfluidic devices in that

they can be operated using inexpensive CD drives and do not require the use of specialized

equipment such as high voltage power supplies [144, 145]. Additionally, commercially available

CDs or DVDs can be used as substrate materials, which are low cost and offer a number of

surface attachment chemistries for probe immobilization [144, 146]. Other polymer materials

such as PDMS can be made into the CD form factor for use in centrifugal microfluidics [142].

Multiple channels can also be designed into the discs, allowing for high throughput screening or

parallel processing of the sample in one device [139–142].

Fluid flow rate in the disc can be determined by the rotation speed of the motor [145]. The

system can handle a variety of fluids such as organic solvents or other aqueous buffers;

movement of sample matrices such as blood, milk and urine have been demonstrated [145].

Fluid velocity is not influenced as strongly by factors such as pH, ionic strength as compared to

electrophoretic methods, and flow rates from 10 nL/s to 100 µL/s can be achieved [139–141].

The main driving force for fluid transport is centrifugal force, but can be further controlled by

coriolis forces [139–141, 143, 145], valves [139–141] or external magnets [143]. A number of

pre-treatment techniques for biomolecules have been demonstrated in the centrifugal

microfluidic platform, including: cell culturing [139]; separation of red blood cells from plasma

in a whole blood sample by sedimentation [143]; immunomagnetic separation [147]; cell lysis

using glass beads [142]; separation by affinity [145], ion exchange [145] and size exclusion

chromatography [139–141]; DNA extraction based on solid phase extraction [145, 147]; PCR

[147]. Detection strategies in centrifugal microfluidic devices include: DNA hybridization [144,

145, 147]; immunoassays [143, 147–149] and colorimetric analysis [143].

For development of fully integrated microfluidic-based DNA biosensors, there is a need to

develop sample pre-treatment and purification methods that are amenable to channel based and

digital microfluidic devices. Sample pre-treatment for microfluidic applications is still a

22

challenge and a limiting factor in many chip designs [150]. Many of the pre-treatment and

purification technologies have aimed to miniaturize macro-scale pre-treatment techniques for use

inside microfluidic channels. Conventional techniques such as centrifugation or precipitation are

not readily adaptable into a microfluidic device [16, 69, 88, 151]. Other techniques such as

filtration, magnetic particle based separation and solid-phase extraction have been miniaturized

to operate on chips [103, 151]. Section 1.7 will review some of the purification methods as well

as concentrating methods that are available for channel based microfluidic devices, with the

focus of the work in this thesis aligning with channel-based microfluidic systems.

1.7 Methods for Purification and Concentrating in Microfluidic Devices

1.7.1 Filtration

Filtration in a microfluidic device can be accomplished either by incorporating a piece of

membrane filter material, most commonly by sandwiching it in between two pieces of the

microfluidic device, or by construction of microstructures such as micropillar arrays or

microweirs [16, 152]. Sample filtration and concentrating in a microchip was demonstrated with

porous membranes. Long et al. sandwiched a 10 nm pore diameter membrane between two

pieces of PDMS which had microchannels etched inside. Molecules small enough to pass

through the nanopores were moved across the membrane by the application of a voltage. This

allowed the collection of larger molecules at the membrane by size exclusion, resulting in

concentrating of the sample [152]. The filter was used to concentrate a sample of PCR amplified

DNA target. An 80 fold enhancement in the signal was observed after 40 seconds of filtration.

This enhancement factor could be further increased by using longer sampling times as well as an

increased applied voltage [152].

Foote et al. have demonstrated a nanoporous silica membrane that is capable of a 100 fold

enrichment of DNA after 5 minutes. As shown in Figure 1.3(c), the silica filter membrane was

placed between the silicon-based microchip and a glass cover plate, which trapped the DNA

molecules while still allowing current to flow. Figure 1.4 shows the concentration of

fluorescently labeled ovalbumin using this chip. When coupled with enrichment by field

amplified stacking, a 600 fold enhancement in the signal was observed [153].

23

Figure 1.3: (a) Schematic diagram of the microchip layout for pre-concentration, (b) image of the pre-

concentrator channel, and (c) schematic of how the filtration membrane is placed in between the microchip and the coverplate. With permission from Analytical Chemistry. Copyright 2004, American

Chemical Society [153].

Figure 1.4: Fluorescence images of fluorescein-labeled ricin injected a) without pre-concentration, and

b) with pre-concentration for 1 minute. With permission from Analytical Chemistry. Copyright 2004, American Chemical Society [153].

1.7.2 Solid-Phase Extraction

The incorporation of solid-phase extraction in microfluidic devices has been demonstrated for

purification and concentration of biological targets such as DNA [72, 73, 154]. Capture

efficiency can be improved by repetitively cycling the sample through the SPE matrix in the

microchannel [88]. Following capture, the target can be released into a smaller volume,

concentrating the sample for further downstream processing and detection [155, 156]. A method

to increase concentration has been achieved using an inline solid phase extraction using amino-

silica monoliths. Concentrating factors of 100 fold were achieved for DNA from crude E. coli

lysates. [157]

24

Methods of incorporating SPE into microfluidic devices include filling the microfluidic channels

with silica resins or beads [72, 88, 155, 156, 158, 159], as well as constructing monoliths or

microstructures inside the channels [158]. Wolfe et al. demonstrated a silica material that was

able to extract 500 bp DNA from a 25 µL sample of HindIII digested λ-phage DNA. The

extraction efficiency was reported at 80% and the material was eluted into a final volume of 5 µL

[160]. Other examples of work using SPE for purification of DNA samples in microfluidic

devices have been reported by Yu et al. [161] and Oleschuk et al. [162].

Binding capacity of DNA can be improved by use of beads of smaller diameter to increase the

surface area of capture. However, this results in an increase in back pressure and requires the

application of higher pressure to achieve reasonable flow rates [73, 74, 76]. In addition, the use

of microbeads in the channel requires consideration of a mechanism to retain microbeads inside

the microfluidic channel. This is most commonly done by placing a frit in the channels [28,

163].

Landers’ group has introduced an alternative approach based on immobilization of the silica

beads with sol-gels. The sol-gel is thought to act as an “interparticle glue” to hold the particles

in place. Extraction and purification of genomic DNA from samples of human whole blood in 25

minutes was demonstrated using a solid-phase extraction material that was based on silica gel

immobilized using a sol-gel within a microchannel [160].

Monoliths can be used for the isolation and concentrating of DNA. Monoliths offer larger

surface areas, controllable pore size, and higher mass transfer from porous structures than silica

beads [74, 76, 164] and can be cast in situ by photopolymerization. Pore size and porosity can

be adjusted by altering the concentration and type of porogenic solvent that is selected [72, 164].

Examples of the use of porous monoliths for the purification of DNA have been reported by

Bhattacharyya et al. [72] and Satterfield et al. [164]. A sol-gel based monolith has been

demonstrated for the isolation of DNA from clinical samples. The monolith was a tetramethyl

ortho-silicate based sol-gel loaded inside a microchannel. The addition of ethylene glycol as a

porogen resulted in a monolith with pores in the micrometer scale, providing a large surface area

for adsorption of DNA with little back pressure. Extraction efficiencies of 85% for λ-phage

DNA and 70% for the extraction of human genomic DNA from human blood were observed.

Using this system, a 200 µL solution containing DNA can be concentrated by eluting into a final

25

volume of 12 – 18 µL. Extraction of viral DNA from human spinal fluid using this monolithic

material has also been demonstrated. Repeated extraction using a single device showed blockage

of the pores by components in the lysed cells [165].

DNA can be captured onto silica-based microstructures such as pillars inside the microchannels

[72, 88]. The use of these pillars increases the surface area, but requires complex fabrication

procedures, increasing the cost of each chip [73]. Cady et al. demonstrated an increase in

surface area available for the capture of DNA by using silica-coated pillars (Figure 1.5).

Depending on the etch depth (20 to 50 µm), an increase in surface area from 300% to 600% was

observed. Selective binding of genomic DNA was observed in the presence of a chaotropic salt

followed by washing with ethanol and elution in a low-ionic strength buffer. Samples were

moved through the extraction device by application of positive pressure, eliminating the need for

centrifugation. The binding capacity for DNA in the device was calculated to be approximately

82 ng/cm2 [158]. Polycarbonate based microchips with microposts inside the polycarbonate

channels have also been used for the capture of DNA [88].

Figure 1.5: Schematic and SEM images of the microfabricated silica pillars for SPE of DNA. With

permission from Biosensors and Bioelectronics. Copyright 2003, Elsevier [158].

Yeung et al. developed a gel-based method for concentrating DNA following PCR that was

accomplished on a microfluidic chip. One of the primers used in the PCR process was modified

with a biotin label while the other primer was fluorescently labeled. The capture gel consisted of

immobilized strepavidin moeities. The biotin labeled PCR amplicons interacted with the

immobilized streptavidin to be captured as the PCR products were moved through the gel. Since

the PCR products are double stranded, application of heat to melt the target was used to release

26

the second strand that carried the fluorescent label. This allowed for the capture of all targets in

the PCR amplicon, including those that had mismatches, which would otherwise not have been

captured if specific capture strands for hybridization had been immobilized into the gel. The

method required a total of 40 minutes; half the time of conventional methods, with provision of a

10 fold increase in concentration [166].

1.8 Sample Concentrating by Electrokinetic Methods

The manipulation of solutions and targets by an electric field in microfluidic devices has

demonstrated enrichment of low concentrations of targets by manipulation of their

electrophoretic mobility inside a channel so that they are confined in a smaller volume. Such

techniques include field amplified stacking and isotachophoresis [163, 167, 168].

1.8.1 Field Amplified Stacking

Field amplified stacking (FAS) involves a concentrating effect that is achieved when the target is

introduced electrokinetically into the separation channel. The targets are first dissolved in a

buffer that has conductivity lower than that of the buffer in the channel. Upon electrokinetic

injection, the targets move at a higher velocity in the sample buffer until they reach the interface

between the sample buffer and the buffer in the separation channel. At this point the targets

experience a sharp decrease in velocity. Stacking into a smaller volume occurs, concentrating

the targets. Concentration increases of 3-100 fold have been observed, depending on the charge

of the targets and the conductivities of the buffer [163, 167].

The difference in conductivity between the two buffers should be as large as possible to achieve

the largest stacking effect. A large difference in conductivity allows the targets to stack much

more quickly while limiting the velocity of the stacked band in the high conductivity buffer.

Ideally, the use of water as the low conductivity buffer would yield the greatest enhancement.

However, real sample matrices often contain substances which could increase the conductivity of

the buffer [169–171]. A solution to this is to dilute the sample with water prior to injection to

lower the conductivity of the sample [169, 172]. Alternatively, a short plug of water can be

introduced into the injection end of a channel for sample stacking. The target is injected by

electrokinetic injection into the water and is stacked against the separation buffer. The increase

in concentration is improved since stacking occurs between the water and the separation buffer

27

rather than within the buffer of the original sample. Concentrating factors of 1000 fold have

been reported using such an approach [163].

As an example, Shim’s group has introduced a method to concentrate DNA to improve

sensitivity of downstream electrochemical detection. The system first introduces a short plug of

water into the channel where DNA is loaded by electrokinetic injection. Stacking occurs against

a buffer that contains hydroxypropyl cellulose modified with gold nanoparticles and sodium

citrate. Enhanced stacking occurs due to an increase in conductivity of the stacking buffer, as

well as a decrease of the electrophoretic mobility of the DNA by adsorption onto the large gold

nanoparticles. A concentrating factor of 25000 was observed using this method, which is 2 to 3

orders of magnitude higher than other stacking methods. The stacking, separation and detection

of a sample of 100 bp DNA ladder was completed within 435 seconds [167].

1.8.2 Isotachophoresis

In isotachophoresis (ITP), the target is concentrated and separated from other components in a

discontinuous buffer system. The target plug is positioned between two different buffers. A

leading buffer contains a large concentration of higher mobility ions (higher conductivity) than

those of the targets, and a terminating buffer contains ions with lower mobility at the injection

side (lower conductivity) [171–173]. When a potential is applied across the channel, the local

field strength across the leading buffer will be less than that of the terminating buffer to maintain

a constant velocity of all charged species across the entire channel [172]. Therefore, an ion

traveling through the low conductivity buffer will experience a higher field strength than one in a

high conductivity buffer. The velocity of the ions will decrease upon moving from the low

conductivity buffer to the high conductivity buffer [172].

During isotachophoresis the charged targets in the sample plug having faster electrophoretic

mobilities move ahead into the leading buffer. As the targets begin to separate from the other

ions in the sample plug, they create a local region of high conductivity, reducing the local field

strength. The velocity of the fast moving targets decrease until they reach a constant velocity

[172, 173].

Other targets with different mobilities that are present in the sample will also separate into

different zones until a constant velocity is achieved [172]. At equilibrium, the sample

28

components are contained in sharp zones with the same concentration as the leading electrolyte.

Therefore, the degree of concentration is determined by the composition of the leading buffer

[163]. ITP requires loading and maintaining zones of the different buffers, and knowledge of the

conductivity of the targets is needed for an efficient separation [120]. This method can be

cumbersome due to the need to use discontinuous buffer systems [174]. Selection of proper

buffers to use in ITP for sample stacking can also become complex [168].

As one example, the concentration of digested HaeIII dsDNA fragments has been demonstrated

by ITP. The ITP device was constructed from poly(methyl methacrylate). The leading edge

buffer consisted of 15 mM HCl, 36 mM imidazole (pH 7) and the terminating edge buffer was a

solution of 20 mM HEPES, 36 mM imidazole (pH 7.2). The high mobility ion in the leading

buffer was chloride and the low mobility ion in the terminating buffer was HEPES. Injection

and stacking was completed in 70 seconds. By measurement of fluorescence from an

intercalating dye, stacking was shown to increase the fluorescence intensity by 40 fold. It is

noteworthy that the high salt content used in ITP may interfere with PCR of the stacked DNA

[173–175].

Recently, electrokinetic supercharging (EKS) has been reported as a concentration technique that

uses field amplified stacking followed by transient ITP [175, 176]. It has been demonstrated to

be capable of handling very dilute samples. Here, the sample is first injected into a channel

filled with the leading electrolyte. The trailing electrolyte is then injected and stacking by ITP

occurs. Concentration of targets inside capillaries was observed to be improved by up to 100 000

fold. Inside microchannels, concentration factors from 10 to 100 fold were observed [175].

1.9 Contributions of this Thesis

The work presented in this thesis examines a method for the selective concentrating of DNA

targets for delivery into channel-based microfluidic. These studies were combined with work

done by other researchers in the Krull group who are developing biosensor technology for use in

a microfluidic system. It can be observed in the review of other purification methods that many

of the methods for extraction, purification and concentrating of DNA from cellular samples have

been based on the non-selective collection of DNA targets onto a solid support. The materials

captured on the support are subsequently washed, and then eluted into a volume smaller than the

original sample volume. As previously noted, a significant issue that arises by implementation

29

of such non-selective concentration methods is that a large amount of non-target DNA is

collected with the target DNA, which may hinder selective detection downstream. Through the

selective concentrating of the complementary material, it may be possible to reduce or eliminate

issues that are associated with non-selective adsorption of non-complementary target on

detection elements. The Acrydite capillary affinity gel electrophoresis chemistry that I have

previously reported to capture short oligonucleotide targets was further examined and developed

herein for the selective purification and concentration of longer base pair DNA targets [95, 177].

Although a number of pre-treatment techniques have been adapted and miniaturized to operate in

the microfluidic regime, an issue still present is the processing of large volume samples with

these devices. As eluded to in the introduction, large sample volumes (mL to L) are often

required where the target analyte is present in very small concentration; for example, for the

detection of pathogenic bacteria from consumable products. Such samples must be "scaled-

down" prior to detection using a microfluidic device.

Additionally, since it would be difficult to account for all the components present in the sample

matrix for any given sample, it is likely that an unforseen component in the matrix could

irreversible damage the integrated microfluidic biosensor and limit its reusability. For example,

clogging of the channels can be a possibility if the solution was not adequately filtered. A

modular approach to developing pre-treatment protocols, such as the one used in this thesis

where the selective purification chemistry is inside a capillary that can be inserted into the main

biosensing, be more practical. The chemistry inside the capillary can be replaced if damaged or

used without having to replace the biosensing platform.

As already indicated, the Mathies group has used this affinity capture gel chemistry as a method

for the purification of DNA targets [96, 178–180]. Mathies has used this chemistry for the

removal of buffers, excess primers and template PCR products for downstream sequencing

experiments in a channel-based microfluidic device. They have also reported the optimization of

binding efficiency for PCR products in the affinity gel as a function of electric field strength and

temperature [96].

The work presented in this thesis further examines the ability of Acrydite chemistry to be used

for purification of a target that is in a mixture containing non-complementary DNA. A thorough

evaluation of the selective capture of one oligonucleotide target in a complicated mixture has not

30

yet been reported. The investigation considered the effects of gel formulation, and different

loading and elution conditions on recovery and purity. This was addressed systematically and

guided by a factorial analysis (see Appendix A for background of factorial analysis). It was

identified that a higher amount of complementary target was retained in the affinity capture gels

made with higher concentrations of monomer. This was thought to be a result of such gels

having a smaller average pore size than the radius of gyration of the ssDNA target examined,

causing the ssDNA target to migrate by reptation. It was proposed that migration by reptation

stretches out the ssDNA targets, eliminating any hairpin structures present in the ssDNA. This

made the complementary region on the target available for hybridization with the gel

immobilized probe, resulting in a higher amount of capture of the complementary target by the

affinity capture gel.

The release and delivery of the DNA targets was performed by step elution for concentrating the

DNA targets. Use of a localized elution zone that was applied in small steps along the length of

a capillary column allowed for the targets to stack as they were denatured from the capture gel.

This is shown schematically in Figure 1.6. The elution was accomplished by denaturation using

a resistive heating element.

Figure 1.6: Schematic representation of selective concentrating as done in the work of this thesis. First, the target was captured onto the affinity capture gel column. Elution took place in a localized area of the

capillary by means of application of heating to a narrow zone such that only targets captured in that region were denatured. This process took place during electrophoresis, and the denatured targets moved along in the electric field. The heated zone was then physically moved along the column. This allowed for

the continual release of targets into a stacked zone of significantly smaller volume than the original sample volume.

31

Since only the target of interest was retained by the affinity capture gel, the target was

concentrated and eluted at high purity. The concentrated target was delivered into a microfluidic

channel that had been modified with immobilized probe oligonucleotides at various spots on one

channel wall. The capillary interfaced to the microfluidic device acted to bridge the disparity in

volumes between the macro-environment to the microfluidic device.

A capillary-to-microfluidic interconnect was used to deliver target analyte into the microfluidic

device. The volume of the capillary (350 nL) was larger than the microfluidic channel (19 nL),

and this offered the opportunity to use stacking to reduce the volume of the material delivered

into the microfluidic device. The use of affinity capture gel inside the capillary as a component

separate from the microfluidic channel allowed for sequential processing of samples, and the

opportunity from replacement of the gel in a manner that was independent of the sensor

component.

This method of selective concentrating of DNA targets was intended as an adjunct protocol for

the purification of samples following PCR amplification or ultrasonication, where DNA targets

would be available as shorter, single-stranded sequences. This method is not meant to replace

any particular purification method, but provides an additional method to further purify and

concentrate a sample.

The targets examined in this thesis were selected to cover a range of lengths as might be

encountered in real samples, and included 19 nt, 150 nt, 250 nt and 400 nt oligonucleotides. The

19 nt oligonucleotide was a synthetic oligonucleotide target while the 150, 250 and 400 nt targets

was obtained by PCR. Selective capture of these targets in the presence of non-complementary

material was achieved by the affinity capture gel, and this was followed by step elution to

achieve target concentration. The recovery of the method ranged from 0.5 to 4% for the PCR

targets, while it was 13 to 18% for the 20 bp oligonucleotide target. The purity was calculated to

be up to 44% for the PCR target and up to 86% for the 19 nt target. This was an improvement in

purity of 15 times and 1100 times in comparison to the original samples for the PCR targets and

19 nt oligonucleotide, respectively. The lowest concentration of the 150, 250 and 400 nt targets

that saw an advantage by selective concentration was 1 nM of complementary in 150 nM non-

complementary target. The lowest concentration of the 19 nt oligonucleotide target that could be

32

processed to see advantage in selective purification and concentration was 0.5 nM

complementary target in 1 µM non-complementary target.

Finally, the 19 nt oliogonucleotide targets were delivered by selective and non-selective

concentration into a channel based microfluidic DNA biosensing platform, and the response of

the was clearly improved when selective concentration was invoked for sample processing.

33

Chapter 2 Materials and Method

2.1 Reagents

Acrylamide, bis-acrylamide, agarose gel, APS and TEMED were all electrophoresis grade from

Sigma-Aldrich (Mississauga, ON, Canada). DNA modifying enzymes, DNA ladders, gel

loading dyes and all other molecular biology reagents were from Fermentas Inc. (Burlington,

ON, Canada). The QIAquick PCR purification kit and Genomic DNA extraction kit were from

Qiagen Inc (Mississauga, ON, Canada). Fluorescent DNA intercalating dyes were from

Invitrogen Inc. (Burlington, ON, Canada). Fused silica capillary (100 µm I.D., 375 µm O.D.)

was from Molex Corporation (Phoenix, AZ, USA). PDMS microfluidic chips were cast using

Sylgard 184 Silicone Elastomer kit (Ellsworth Adhesives, Stoney Creek, ON, Canada). Epoxy

modified glass substrates (Super Epoxy II) were from Array-it (Sunnyvale, CA, USA). All other

reagents were from Sigma-Aldrich (Mississauga, ON, Canada) unless stated otherwise.

2.2 DNA Targets

Table 2.1 lists all of the oligonucleotide sequences (probes, primers, targets) that are used in the

experimental work. Oligonucleotide sequences were from Integrated DNA Technologies

(Coralville, IW, USA), and were used as received.

34

Table 2.1: Oligonucleotide targets that were used in the experiments. Longer PCR targets are described in a subsequent section. Melt temperature was provided by the supplier. *oligonucleotide probes used in

capillary affinity capture gels contained the Acrydite modification at the 5' end, while probes used for immobilization onto epoxy-modified glass slides in the microfluidic device contained a primary amino group with a C12 spacer on the 5' end. Cy3 fluorescent label was attached to the 3' end when used.

**fluorophores on these oligonucleotide sequences were attached at 5' end when used. Oligonucleotide

Probes* Length

(bp) Sequence Tm*

(°C)

SMN 19 5' ATT TTG TCT GAA ACC CTG T 3' 49.3

β-actin 19 5’ CCC TCC CCC ATG CCA TCC T 3’ 62.3

β-actin (1 bpm) 19 5’ CCC TCC CCC ATG CCA CCC T 3’ 65.2

Non-complementary

β-actin

19 5' ACG CGG TCT GAT GCC CTG T 3' 62.3

β-actin (short) 14 5’ CCC TCC CTC ATG CC 3’ 51.8

uidA 22 5’ AGT CTT ACT TCC ATG ATT TCT T 3’ 49.3

DNA Targets**

SMN 19 5’ ACA GGG TTT CAG ACA AAA T 3’ 49.3

Β-actin 19 5' AGG GTG GCA TGG GGG AGG G 3' 62.3

uidA 22 5' AAG AAA TCA TGG AAG TAA GAC T 3' 49.3

Non-complementary

20 5' CCG CGA CGG ATT GAT TGT TT 3' 56.2

PCR Primers**

β-actin forward 20 5’ TCA CCC ACA CTG TGC CCA TC 3’ 60.0

β-actin reverse 20 5’ GTG GTG GTG AAG CTG TAG CC 3’ 58.4

LAMA3 forward 20 5’ CTG GGC TAC AGT TCA CAG CA 3’ 57.3

LAMA3 reverse 20 5’ TCC ACA TAA CTC GCT TGC AG 3’ 55.2

400 forward 24 5’ CTT GTC CAG TTG CAA CCA CCT GTT 3’ 60.0

400 reverse 24 5’ ATG CGG TCA CTC ATT ACG GCA AAG 3’ 59.8

35

2.3 Instrumentation

2.3.1 Capillary Electrophoresis

Figure 2.1: Schematic of the capillary electrophoresis set-up.

Figure 2.1 shows the capillary electrophoresis setup used for the experiments. Microcentrifuge

tubes of 0.65 mL volume were used to hold the running buffer (0.1 % PVP in 1x TBE, pH 8.0).

The capillary was inserted into the microcentrifuge tubes via holes drilled into the top of the

tubes. A Spellman CZE1000R (Hauppauge, New York, USA) high voltage power source was

used as the power supply. Electrodes were inserted through a second hole made in the side of

the microcentrifuge tube. Temperature was controlled by pumping water heated to the desired

temperature into a syringe connected to a water jacket system surrounding the capillary.

36

2.3.2 Instrumentation for on-line capillary electrophoresis/step elution experiments

Figure 2.2: Set-up for online capillary electrophoresis and step elution experiments.

Figure 2.2 shows the set-up used for online electrophoresis experiments and step elution

experiments. A Labsmith HVS448 3000V (Livermore, CA, USA) was used as the high voltage

power supply. Microcentrifuge tubes of 250 µL volume were used to hold running buffer (1x

TBE with 0.1% PVP) and were mounted onto a flat, plastic surface using epoxy glue. The level

of tubes were adjusted to ensure that the capillary was level when placed inside the setup.

Localized step elution of the captured targets on the capillary was accomplished by heating,

using a 45W soldering iron with approximately 0.8 mm metal wire coiled around the soldering

tip. The resistive heating element covered approximately 0.8 mm of the capillary at a time. The

temperature of the resistive heating element was measured to be around 85 °C. The heating

element was moved across the capillary in discrete steps at a defined rate using a Sigma-Koki

SGSP20-85 motorized Stage attached to a Shot-602 two-Axis Stage controller (Tokyo, Japan)

and controlled using custom software written in National Instruments LabVIEW 8.0 (Austin,

TX, USA).

2.3.3 Confocal Fluorescence Microscope Images

In addition to obtaining data from electrophoretograms, images of the capillaries were acquired

by confocal fluorescence microscope. Such images were obtained by placing the capillary on the

stage of the fluorescence microscope and measuring the fluorescence emission intensity as the

stage was rastered in the x and y directions. This produces a fluorescence emission map of the

37

capillary. In order to measure fluorescence emission from the inside of the capillary, a large

portion of the polyimide coating was burned off the capillary. Figure 2.3 shows a schematic of

how fluorescence images of the capillaries were acquired. Offline images of the entire fused

silica capillary were also obtained for data processing.

Figure 2.3: Schematic of the instrumental setup for how confocal fluorescence microscope images were

obtained.

The images were then processed further as detailed in the results and discussion section. The

affinity gel capillaries were imaged using the following instruments.

2.3.3.1 Confocal fluorescence microscope slide reader for 532nm/635nm excitation (Chipreader)

Affinity gel-filled capillaries were imaged off-line using a Bio-Rad VersArray ChipReader

confocal fluorescence microscope (Hercules, California, USA). The ChipReader was designed

to excite and detect fluorescence of Cy3 and Cy5 dyes. Laser excitation of the Cy3 and Cy5

dyes occurred at 532 nm and 635 nm, and the fluorescence emission was detected at 570 nm and

670 nm, respectively. The focal plane was first determined by scanning a capillary filled with a

solution of dye-labelled oligonucleotide and focusing until the maximum signal intensity of the

dye was obtained. Scanning parameters were as follows: laser power 10% (100% = 8 mW

power), detector sensitivity 800mV, 1x detector gain, image resolution 15 µm, and an image scan

speed of 25 lines per second.

38

2.3.3.2 Epifluorescence microscope for 635 nm excitation (Alpha)

Real-time monitoring of Cy5 fluorescence during electrophoresis and step elution pre-

concentration experiments was performed using a custom a epifluorescent microscope based on a

Nikon Eclipse L150 microscope platform (Melville, NY, USA). The excitation source was a

10 mW 635 nm solid-state laser (Coherent, Santa Clara, CA, USA) directed through a 40x

microscope objective (Plan Fluor, ELWD, Numerical Aperture 0.60) using a 630-650 nm band

pass excitation filter. Emission was collected through the same objective using a 660 nm long

pass dichroic filter and a 665-695 nm band pass emission filter (Chroma Technology Corp,

Bellows Falls, VT, USA) into a Hamamatsu H574-20 PMT detector. The microscope stage was

controlled by a Conix Research Inc. XYZ 4000ML Stage Manipulator (Springfield, OR, USA).

Image acquisition software for the custom microscope was written in LabVIEW.

2.3.3.3 Confocal Fluorescence Microscope for 534 nm excitation (Confocal)

A Nikon C2 confocal system mounted onto a Nikon L150 microscope setup was used for real-

time monitoring and some off-line images of Cy3 fluorescence. The excitation source was a

10 mW 534 nm laser (Research Electro-Optics, Boulder, CO, USA). Excitation radiation was

delivered through a 4x microscope objective (Plan Fluor , WD 17.2 mm, Numerical Aperture

0.13) by the Nikon C2 system. Emission was collected through the same objective and passed

into the 3-PMT detector. Filters were set up such that emission intensity collected from 560 to

610 nm were assigned to the Cy3 channel. Data obtained from off-line images from this

instrument will be noted.

All fluorescence intensity images were analyzed and processed using ImageJ (NIH).

Enhancements were made to some images by adjusting the Window/Level parameters to aid in

visualization. Such enhanced images were identified in the figure caption.

2.3.4 UV-VIS

UV-VIS absorbance measurements were collected using a HP8425A. Melt temperature

experiments were done using a HP89090A Peltier temperature controller.

39

2.3.5 Steady-State Solution phase Fluorescence Measurements

All steady state fluorescence measurements were done using a QuantaMaster PTI

Spectrofluorimeter (Photon Technology International, Lawrenceville, New Jersey, USA). A 45

µL volume ultramicro fluorescence cell was used to hold samples (path length 3 mm, Hellma

Ltd, Concord, Ontario, Canada). The excitation and emission settings were as follows: Cy3 –

Excitation 513 nm, Emission 550 – 600 nm, integration time 1 second, Cy5 – Excitation 625 nm,

Emission 645 – 680 nm, integration time 1 second. Each sample was scanned three times to

provide an average signal.

2.3.6 Other Equipment

Ultrasonication of DNA was done using a Vibra-cell Ultrasonic Processor VC-250 equipped

with a 5 mm diameter tapered microtip (Sonics & Materials, Danbury, CT, USA). PCR was

performed on an iCycler Thermal Cycler (Biorad, Hercules, CA, USA). Aqueous solutions of

PCR amplified DNA were dried using a centrifugal vacuum evaporator (DNA Plus, Jencons,

Leighton Buzzard, UK). Agarose gel electrophoresis made use of a BioRAD powerPAC power

supply and gel chamber, and the Mini-Sub Cell GT Agarose Gel Electrophoresis System. Gels

were imaged using a Biorad Gel-Doc 1000 system. PDMS microfluidic chips were plasma

oxidized using a Herrick PDC-32G Plasma Cleaner/Sterilizer.

2.4 Generation of Longer lengths of DNA Targets

2.4.1 150 bp targets

Longer DNA target sequences were synthesized from samples provided by Dr. Paul Piunno from

the Department of Chemical and Physical Sciences, UTM (Mississauga, ON, Canada). The

150 bp target provided is a target from the β-actin gene, which codes a cytoskeletal protein found

in networks of microfilaments and stress fibers that influence the cytoarchitecture of a cell [181].

It is a highly conserved housekeeping gene, and typically is used to normalize molecular

expression studies [182]. The 150 bp length DNA target was generated by PCR. The PCR used

a combination of: 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.08% Nonidet P40, 1.5 mM MgCl2,

0.5 µM of each primer, 25 nM dNTPs, 0.02 µg/mL template, 2.5 units of Taq. PCR conditions

were as follows: an initial denaturation step at 95 °C for 5 minutes, 45 cycles of denaturation: 1

min at 95 °C, annealing: 30 seconds at 61 °C, extension: 30 seconds at 72 °C, and a final

40

extension step at 72 °C for 10 minutes. Attachment of a Cy5 fluorescent label to the target

strand of the interest was accomplished by using a Cy5 labeled reverse primer in the PCR mix.


The 250 bp target was constructed by ligation of the 150 bp target prepared as described in

Section 2.4.1 with a 100 bp target supplied by Paul Piunno. The 100 bp fragment is a target for

the LAMA3 gene. This gene codes the α3A part of the Laminin-5 heterotrimer filament protein,

which is an important structural component in basement membranes. Laminins contribute to cell

proliferation, migration, differentiation and promotion of tissue survival [183].

The enzymatic reactions were carried out using the protocols provided by the supplier. The 5’

end of the 150 bp target was first phosphorylated with T4 Polynucleotide Kinase so that the

subsequent reaction with T4 DNA Ligase would occur. The reaction conditions were as follows:

20 pmol of DNA target, 1x supplied reaction buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl2,

6 mM DTT, 0.1 mM spermidine, 0.1 mM EDTA), 20 pmol of ATP, 10 units of T4

Polynucleotide Kinase. The reaction was allowed to proceed for 30 minutes at 37 °C, following

which the enzyme was inactivated by increasing the temperature to 75 °C for 10 minutes. The

reaction mixture was used in the ligation step without further purification.

The 150 and 100 bp target was then incubated with T4 DNA Ligase for 7 hours at 22 °C to allow

for ligation to occur. The reaction used equal volumes of 150 and 100 bp length DNA targets, 1x

ligation buffer as provided (40 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT, 0.5 mM

ATP), 5%v/v PEG 4000 and 2 units of T4 DNA Ligase. Following ligation, the enzyme was

inactivated by heating at 65 °C for 10 minutes, as suggested by the supplier. Following the

reaction, the reaction mixture was run on a 1% agarose gel with 1x SYBR Gold intercalating dye

in 1x TBE (100 V, 1 hour) along with a 100 bp DNA Ladder. DNA bands were visualized on a

UV trans-illuminator and the appropriate gel bands were excised and DNA recovered using the

QIAquick Gel extraction kit. Cy5 labeled targets were generated using fluorescently tagged

primer in the PCR mixture [40 cycles, denaturing at 95 °C for 1 min, annealing at 53 °C for 30 s,

extension at 72 °C, 30s].

41


400 bp length targets were obtained from genomic DNA extracted from E. coli K12 cell cultures

in a number of steps. The E. coli K12 strain was provided by Fong Ly from the Department of

Biology, UTM (Mississauga, ON, Canada).

First, genomic DNA was extracted from the cell cultures using a Qiagen genomic DNA

extraction kit (Qiagen). The protocol was followed without modification. Three volumes of a

proprietary solution which contained a chaotropic agent was added to 1 volume of the excised

agarose gel and incubated at 50 °C until the gel was dissolved. The same volume of isopropanol

was added to the solution as the volume of gel. The resulting solution was added to a silica gel

based spin column and centrifuged for 1 minute at 13 000 x g. A volume of 500 µL of the initial

binding solution was added to the spin column and centrifuged again. A volume of 750 µL of a

wash buffer that contained absolute ethanol was added to the spin column, and this was allowed

to sit for 2 minutes before centrifugation. The eluent was discarded and the spin column was

centrifuged for an additional minute. 50 µL of elution buffer (10 mM Tris-Cl, pH 8.5) was

added to the spin column and allowed to stand for 1 minute before the spin column was

centrifuged for one minute to collect the purified DNA.

Next, a 1000 bp length target was generated from the genomic DNA by a double restriction

enzyme digest. Two restriction enzymes, BfmI and Bsp119I, were used in a restriction enzyme

digest of the extracted genomic DNA to generate a 1000 bp length DNA target. Reaction

conditions were as follows: 0.5 µg of extracted genomic DNA , 1x Tango™ buffer (33 mM

Tris-acetate (pH 7.9), 10 mM magnesium acetate, 66 mM potassium acetate, 0.1 mg/mL BSA), 2

units of Bsp119I and BfmI each. The double digest was allowed to proceed at 37 °C overnight,

after which the enzymes were inactivated by heating to 80 °C for 20 min.

The 400 bp length target was generated from the 1000 bp length target by PCR. Primers were

designed using the web-based PrimerQuest tool available at www.idtdna.com. The sequence of

the 1000 bp length target was obtained from geneBLAST. The design criteria were that the

primers were 24 bp in length, with a Tm of 60 °C and a target length of 400 bp containing the

target region, which was in the middle of the sequence. PCR was used to generate Cy5 labeled

DNA targets. PCR conditions (40 cycles of 95 °C denaturation for 1 min, 55 °C annealing for 30

seconds and extension at 72 °C for 30 seconds)

42

After the PCR reaction was completed, all DNA targets were purified using a QIAquick PCR

purification kit. The protocol was as follows: Binding buffer (which is a proprietary solution

that contains guanidine HCl and isopropanol) was added in 5:1 volume of the PCR reaction

solution. The resulting mixture was added to the silica gel based spin column and centrifuged at

13 000 x g for 1 minute. A volume of 750 µL of 30% w/v Guanidine HCl was added to the spin

column following the initial binding of the DNA to the silica column, and the spin column was

then centrifuged at 13 000 x g for 1 minute. A volume of 750 µL of the wash buffer

(proprietary) with absolute ethanol was added to the spin column, followed by two rounds of

centrifugation at 13 000 x g for 1 minute. The bound DNA target was eluted by adding 50 µL of

the buffer in the kit (Tris-HCl) to the spin column, which was then centrifuged at 13 000 x g for

1 min.

2.4.4 Validation of DNA targets

The targets as synthesized by PCR were run in an agarose gel electrophoresis system (1%

agarose, 1x TBE, 25 Vcm-1) and compared with standard DNA ladders. The gels contained 0.8x

SYBR Gold to allow for visualization by use of a standard UV trans-illuminator. DNA targets

were sequenced at The Centre for Applied Genomics (Toronto, ON, Canada).

2.5 Preparation of Capillary Affinity Capture Gel

Fused silica capillaries were first cut into 5.5 cm long pieces and the polyimide coating was

removed by burning with a flame to create a 4.5 cm window in the center of the capillary to

allow for imaging by fluorescence microscopy.

The capillary was filled with a solution of 2% w/v polyvinylpyrrolidone (average molecular

weight 360,000, PVP) for 5 minutes. The affinity capture gel material, which consisted of

varying amounts of 50% w/v acrylamide, 2% bis-acrylamide, 0.1% PVP in 1X TBE solution and

acrylamide modified probe, was initiated using 0.5 µL of 4% w/v ammonium persulfate (APS)

and 0.5 µL of 4% v/v N,N,N,N-tetramethyl-ethylenediamine (TEMED). The initiated affinity

gel mixture was injected into the capillary and allowed to polymerize for one hour, and then was

stored at room temperature overnight prior to use. The capillary was filled using a vacuum

injection system similar to that described by Baba et al. [184]. A rubber septum was used to cap

a small, empty glass vial, where a capillary was inserted into the septum using a needle. A

43

vacuum line was connected to the vial through the septum, and solution was injected into the

capillary by placing the end of the capillary not inside the vial in the solution while a vacuum

was applied.

2.6 Capture and elution experiments

2.6.1 Pre-Conditioning of Affinity Capture Gel in Capillaries

The capillaries were pre-conditioned prior to use by operating the electrophoretic system at 67

Vcm-1 for 30 minutes. A 10 µL, 2 µM sample of the non-complementary oligonucleotide

sequence was electrokinetically loaded at 267 Vcm-1 for 2 minutes into the capillary to block

non-selective adsorption sites. After injection of the non-complementary oligonucleotide, the

sample was run for 5 minutes at 133 Vcm-1. The non-complementary target was loaded a second

time in the same manner, and the process was repeated for 15 minutes at 133 Vcm-1.

2.6.2 Capture and Elution Experiments

A general protocol for the loading and capture of the longer length DNA targets was followed.

Target solutions contained varying concentrations of the DNA targets in1x TBE and 0.1% PVP

in deionized water. DNA target solutions were first boiled at 95 °C for 5 minutes and

immediately put on ice for 10 minutes. A 10 µL sample of the denatured DNA target was loaded

into the affinity capture gel by electrokinetic injection (167 Vcm-1 for 15 minutes).

Electrophoresis was stopped following electrokinetic injection and the temperature surrounding

the capillary was then maintained at 20 °C for 20 minutes using a water jacket system that

surrounded the outside of the capillary. A voltage was then applied across the capillary at 167

Vcm-1 for 20 minutes while the temperature around the capillary was maintained at 20 °C for

removal of any excess, uncaptured target. Elution of the captured targets was achieved by

application of the same voltage while the temperature of the capillary was increased to 65 °C

using the water jacket.

2.6.3 Factorial Design Experiments

Fractional factorial design experiments were used to identify optimal gel formulation and capture

conditions for the DNA targets. A quarter fractional 2-level factorial design was used for the

examination of gel formulation on effect of probe incorporation and target loading. Table 2.2

and 2.3 shows the design matrix used and experimental conditions, respectively. The factors

44

examined were the monomer concentration (%T), crosslinker content (%C), the concentration of

oligonucleotide probes, and the concentrations of TEMED and APS used for initiation of the

polymerization reaction.

Table 2.2: Design matrix for the quarter 2-level fractional factorial analysis for the examination of gel formulation on the performance of the capillary affinity capture gels.

Standard Order

Run Order %T (A)

%C (B)

Probe (C)

TEMED (D)

APS (E)

1 9 3 2 - - - + +

2 10 5 8 - - + - -

3 11 6 3 - + - + -

4 12 2 1 - + + - +

5 13 8 4 + - - - +

6 14 4 6 + - + + -

7 15 1 7 + + - - -

8 16 7 5 + + + + +

Table 2.3: Experimental conditions for the two levels used for the fractional factorial design matrix.

Factor - +

A (%T) 7.5 % 12.5 % B (%C) 1 % 5 % C (DNA probe)

0.5 µM 3.0 µM

D (TEMED) 4 %v/v 10 %v/v E (APS) 4 %w/v 10 %w/v

The total monomer concentration (%T) and percentage of crosslinker (%C) is given by:

%100100

% xsolutionmL

ercrosslinkgmonomergT

+= (1)

%100% xrecrosslinkgmonomerg

rekcrosslingC

+= (2)

Examination of conditions for capture efficiency and selectivity of the affinity capture gel were

explored using a 3 level 2 factor factorial design. The amount of target captured was examined

based on the incubation time for hybridization and the wash voltage. Response of the affinity

capture gel to mixtures of targets was examined by changing the elution temperature and

formamide content as a denaturant. Tables 2.4 and 2.5 show the design matrix and experimental

conditions that were used. Statistical analysis was done using Minitab R14 (Minitab Inc, State

College, PA, USA).

45

Table 2.4: Design matrix for three level factorial experiment to explore capture efficiency and selectivity of the affinity capture gel. Factors A and B are defined in Table 2.5.

Standard Order

Run Order

A B

1 10 5 13 1 1

2 11 3 12 1 2

3 12 7 10 1 3

4 13 6 14 2 1

5 14 1 17 2 2

6 15 8 18 2 3

7 16 4 11 3 1

8 17 2 16 3 2

9 18 9 15 3 3

Table 2.5: Experimental conditions for each level tested in the design matrix of Table 2.4.

Capture Efficiency Selectivity

Level Sit Time (A)

Wash Voltage (B)

Temperature

(°C) (A)

%v/v formamide (B)

1 5 min 300 V 10 0

2 20 min 600 V 25 10

3 40 min 900 V 40 25

2.6.4 Step Elution of Captured DNA targets

Localized elution of the captured DNA targets was done using the set-up shown in Figure 2. For

these experiments, 7 cm long capillary pieces were cut and filled with the affinity capture gel. A

1.5 cm detection window was made at the elution end of the capillary. Following the capture of

the DNA targets, the resistive heating element was moved across the capillary using a step size

of 250 µm, at a step rate that was determined based on the electrophoretic velocity of the DNA

target of interest. The elution of the stacked oligonucleotide target as it moved through the

detection windows was monitored in real time by an epifluorescence microscope setup.

2.7 Delivery of concentrated targets into microfluidic based DNA biosensing platform

The stacked, eluted targets were delivered from the capillary into a microfluidic DNA biosensing

platform based on a design by Erickson et al. via a capillary-microfluidic interconnect [185].

2.7.1 Construction of DNA Microfluidic Biosensing Platform

Amine terminated oligonucleotide probes at 25 µM were covalently immobilized onto epoxy

modified glass slides by spotting them in borate buffer followed by storage in a humid chamber

46

for 48 hours. Excess probes were removed by washing the slide in 70 °C water for 1 minute.

The location of the probe spots on the glass slide is shown in Figure 2.4.

The design of the microfluidic DNA biosensor is shown in Figure 2.4. The master template was

provided courtesy of Uvaraj Uddayasankar and Omair Noor. The design was based on an H-

shaped channel. The dimension of the main microfluidic channel was 185 µm (W) x 8 µm (H).

Figure 2.4: Schematic for the construction of the microfluidic DNA sensing platform with a capillary

interface. Left: original microfluidics template and position of the template capillary. The capillary (100 µm I.D., 375 µm O.D.) was positioned over the microfluidic channel such that the inner diameter was within the width of the channel. PDMS was poured over the template and cured on a hotplate. The

template capillary and microfluidic template were removed and the PDMS chip had the channel structure and capillary port. Right: schematic of the microfluidic DNA sensor platform. The PDMS template was

trimmed such that only the straight channel remained, and this was positioned over the two oligonucleotide probe spots on the epoxy modified slides.

The capillary-to-microfluidic interconnect was made by positioning a piece of empty capillary

using an XYZ translation stage (Coherent Inc., Santa Clara, CA, USA) orthogonally over the

microfluidic channel on the template. The microfluidic channel was made by casting a 10:1

mixture of the silicone elastomer base and curing agent over the template. Curing was

accomplished by heating at 120 °C on a hotplate for 20 minutes. Removal of the capillary

47

following casting left a hole where the affinity capture capillary could be inserted. The PDMS

mold was trimmed and then plasma oxidized for 30 seconds. The side-arm channels were

removed from the mold so that only the straight channel was present in the final PDMS chip.

The length of the final channel was 1.5 cm from the interconnect to the reservoir well. The mold

was then placed over the epoxy modified glass slides so that the channel was positioned over the

immobilized probe spots. The open ends of the channel were sealed with a drop of PDMS. A

schematic of the final microfluidic system is shown in Figure 2.4.

The microfluidic chip was first pre-conditioned by running electrophoresis in buffer on a 65°C

block heater for 5 minutes, followed by injection of a 5 µM non-labelled non-complementary

sequence to block non-selective absorption sites.

Step elution of the oligonucleotide targets was done prior to interfacing with the microfluidic

chip. The affinity capture capillary was 4.5 cm in length, and was filled with 10% LAAm gel

containing 100 nM complementary (SMN) probe. Step elution of the targets was done as

outlined previously. Immediately following the elution process, the leading 2.5 cm portion of

the capillary was cut and removed, and the remaining 2 cm portion of the affinity capture

capillary was inserted into the microfluidic chip via the interconnect port. Voltage (500V) was

applied across the capillary and microfluidic channel as shown in Figure 2.5.

Figure 2.5: Schematic of the electrophoresis setup for the capillary to microfluidic DNA biosensor.

48

Delivery of material without any pre-treatment (direct injection) was done using the same

capillary-to-microfluidic interface. Solutions of 10% LAAm were made and mixed with the dye-

labeled DNA targets. The complementary target used for these experiments was Alexa647-SMN

and the non-complementary target was Cy3-β-actin. The solutions were then injected into pre-

cut capillaries using a syringe-to-CE coupler, and these were used to deliver sample into the

microfluidic channel.

Experiments examining samples where all DNA targets were concentrated (without purification)

were performed using affinity capture gels that contained a mixture of 100 nM (SMN) and 5 µM

(β-actin) probes.

49

Chapter 3 Results and Discussion

3.0 Capture of Oligonucleotides of 20 nt Length

I have previously published results which demonstrated the successful attachment of the

acrylamide modified oligonucleotide probe into polyacrylamide gel in Analytica Chimica Acta,

578(2006), pg 31-42, titled "Capillary Electrophoresis for Capture and Concentrating of Target

Nucleic Acids by Affinity Gels Modified to Contain Single-Stranded Nucleic Acid Probes".

This system was used to capture a 20 nt length oligonucleotide target, and demonstrated

selectivity of the capture gel when a mixture containing both fully complementary target and a 5

base pair mismatch was used. This work reported the quantity of probe that could be

incorporated into a polyacrylamide gel, as well as the capability of the affinity gel to capture

targets at different concentrations. A summary of the methods and highlights from this

published work is now presented.

3.0.1 Considerations for Imaging Fused Silica Capillaries by Confocal Fluorescence Microscopy

Data for the affinity capture gel experiments were obtained from images of the capillary acquired

by confocal fluorescence microscopy. A number of instruments were used to obtain

fluorescence microscope images and described in Section 2.3.3.1 to 2.3.3.3. At various steps of

the experiment (i.e., following pre-conditioning of the capillaries, following injection of the

DNA targets, following elution of the targets), the capillary was removed from the CE setup,

placed on a microscope slide and the entire capillary was imaged using the fluorescence

microscope. Data which were obtained from images taken by confocal microscopy will be

indicated as such in the caption. Quantitative analysis of signal intensity of fluorescence images

was done using ImageJ. A straight line with a width of 20 pixels was drawn along the capillary

and a profile plot was generated using the Plot Profile function. This plots the fluorescence

intensity (integrating the 20 pixel width) along the 4 cm window of capillary length. Since the

capillary may not be completely straight when being imaged, a 20 pixel wide line was used so

that the fluorescence information from the entire capillary was captured in a single straight line

during analysis. The average fluorescence intensity was then calculated from the profile plot.

50

In order to image the fluorescently labelled NAs were used in the experiments, and these were

detected by the confocal fluorescence microscope (Chipreader). Fused silica capillaries are

coated with a protective layer of polyimide. This material is fluorescent, and the intensity of

emission was sufficient to saturate the detector of the microscope. This polyimide layer was first

removed so that the affinity gel inside the capillary could be imaged. However, removal of the

polyimide coating resulted in mechanical brittleness when handling the bare fused silica.

Therefore, only 4 cm of the polyimide coating was removed from the center of the capillary to

allow for imaging while retaining some durability for physical manipulation of the capillaries.

The confocal fluorescence microscopy images represent a 4 cm window, rather than the entire

length of the capillary. It was assumed that the affinity gel was homogeneous throughout the

capillary. Confocal fluorescence microscope images of the capillaries were stored as two

separate files, one containing the fluorescence emission data from the Cy3 channel and the other

from the Cy5 channel. For experiments where both fluorophores were present, the images are

shown as a pair. The left image represents the fluorescence emission data from the Cy3 channel

and the right image the emission from the Cy5 channel. For experiments where only one

fluorophore was used, only a single image of that dye channel is presented. Some images shown

were processed using ImageJ by adjusting the Window/Level parameters to aid in visualization.

Such enhancement of images has been identified in the figure captions.

3.0.2 Capture and Elution Experiment for a 20 nt Target

Figure 3.1 shows an example of confocal fluorescence microscopy images of capillaries obtained

during a capture and elution experiment for a 20 nt length complementary target using the

affinity gel. The affinity gel consisted of a 0.45 µM dT20-Cy3 oligonucleotide probe in a 12.5

%T linear polyacrylamide gel. Briefly, the left channel represents the fluorescence image of the

Cy3 channel, which corresponds to the Cy3 labelled oligonucleotide probe (dT20-Cy3) used in

the affinity capture gel while the right channel represents the fluorescence image of the Cy5

channel, corresponding to the Cy5 labelled oligonucleotide target (dA20-Cy5). The

complementary target, a 5 µL sample of 1 µM dA20-Cy5, was introduced electrokinetically at

267 Vcm-1 for 1 minute. Figure 3.1(a) shows the image of the capillary prior to the loading of

the complementary target. Successful capture of the complementary Cy5 labelled target can be

seen by the images in Figure 3.1(b) after injection and electrophoresis for 35 minutes. Elution of

the captured target was achieved by the addition of 0.5 M NaSCN to the running buffer and

51

application of a 60 °C water jacket around the capillary for 25 minutes, as shown in Figure

3.1(c). The use of sodium thiocyanate salt has been previously demonstrated to be a potent

denaturant of double stranded DNA [186].

Figure 3.1: Confocal fluorescence microscope images of affinity capture capillaries from a capture and

elution run using a complementary target showing: (a) the affinity gel material inside the capillary prior to loading of target oligonucleotide sequence, (b) running for 35 minutes following electrokinetic injection of

target Cy5 – dA20 and (c) after elution for 25 minutes at 60 °C. Affinity gel: 0.45 µM Cy3-dT20 probe in a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dA20 at 267 Vcm

-1 for 60 seconds. Electrophoresis condition: 133 Vcm

-1

with 1x TBE/0.1% PVP for 35 minutes. Elution condition: 267 Vcm-1

with 1x TBE/0.5 M NaSCN/0.1% PVP for 25 minutes at 60 °C. Images were obtained using the Chipreader.

The probe was labelled with a Cy3 fluorophore. The presence of a Cy3 fluorescence signal inside

the capillary indicated that the Acrydite™ modified probes were successfully incorporated into

the polyacrylamide matrix. The Cy3 signal was observed throughout the entire capture and

elution experiment, showing that the EOF was adequately suppressed by the dynamic coating

with PVP; otherwise, a loss in Cy3 fluorescence as the gel was physically ejected from the

capillary would have been observed.

52

3.0.3 Autofluorescence and Non-Selective Adsorption

A low level of fluorescence intensity was observed in the Cy5 channel following the elution of

the captured target in the experiments described in Figure 3.1. An effort was made to identify

the source of this residual fluorescence signal.

Prior to the loading of any materials into the gel filled capillary, a fluorescence signal was

observed from the capillary in both the Cy3 and Cy5 channels, as shown in Figure 3.2. The

average fluorescence signals along the length of the capillary column in the Cy3 and Cy5

channels were 379 AU and 699 AU, respectively. This background fluorescence was attributed

to autofluorescence from the fused silica capillary and polyacrylamide gel. It was also important

to note that while the fluorescence emission observed in the Cy3 channel was only from the gel

region inside the capillary channel, the fluorescence emission in the Cy5 channel was from the

entire capillary.

Figure 3.2: Confocal fluorescence microscope images of affinity capture capillaries showing the Cy3 (left) and Cy5 (right) channels from a portion of the capillary shown in Figure 3.1(a), which shows the

autofluorescence signal of the system before the loading of any fluorescently labelled materials. Images were enhanced in ImageJ using the Window/Level function for better clarity.

A control experiment included introduction of both Cy3 and Cy5 labelled targets to a

polyacrylamide gel that contained no immobilized probe. There was an increase in the intensity

relative to the background fluorescence following the loading and migration of a sample through

the capillary. The increase represented retention of 5 ± 5 fmol and 10 ± 5 fmol of Cy3 and Cy5

labelled oligonucleotides, respectively (as determined using a concentration-response calibration

curve). In these experiments the affinity gels had not been pre-treated using non-labelled non-

complementary targets and the labelled targets were being non-selectively adsorbed onto the gel.

53

For applications that involve the selective capture of low concentrations of material, any non-

selective adsorption would be unfavourable. Therefore, as a pre-treatment step, non-labelled non-

complementary oligonucleotide target was pre-loaded into the capillary.

The effectiveness of the pre-treatment protocol was tested using a 5 µL sample of 1 µM dC20-

Cy5 non-complementary target that was introduced to an affinity gel containing 0.45 µM Cy3-

dT20 probe in 12.5 %T linear polyacrylamide. The amount of material left in the gel as a result

of non-selective adsorption corresponded to 10 ± 9 fmol for columns without any pre-treatment,

and 4 ± 4 fmol for columns with pre-treatment (data corrected for autofluorescence).

The effectiveness of the pre-treatment protocol was examined using complementary targets. The

affinity gels consisted of 0.45 µM Cy3-dT20 probe in a 12.5 %T linear polyacrylamide. Samples

containing 1 µM dA20-Cy5 were used as the complementary target. Following sample elution

with denaturation of hybrids, affinity gels that were not pre-treated retained approximately 19 ±

8 fmol of dye-labelled oligonucleotide while the affinity gels that were pre-treated indicated

retention of 8 ± 8 fmol of oligonucleotide target. Pre-treatment helped in reducing but did not

completely eliminate non-selective adsorption.

Figure 3.3 shows Cy5 fluorescence emission following elution of the captured target. Since

retention of material was observed with both complementary and non-complementary targets

following elution, it was concluded that the retention of material was based on non-selective

adsorption, rather than incomplete elution of the captured targets.

The pre-treatment likely resulted in a condition where adsorbed material was being leached from

the gel during the capture and release experiment. This is analogous to the process encountered

when using dynamic coatings to suppress electroosmotic flow on capillary walls, where a small

amount of the surface active agent must be included in the running buffers to replenish the

coating.

The possibility of loss of adsorbed material used in pre-treatment over time due to leaching was

examined. Here, Cy5-labelled dC20 was used in place of the non-labelled oligonucleotide

during the pre-treatment protocol. Following pre-treatment, the capillary was subjected to an

electric field in 1xTBE / 0.1% PVP running buffer and scanned for fluorescence at various times.

54

The resulting plot shows the average fluorescence intensity of the Cy5-labelled dC20 inside the

capillary over time.

Figure 3.3: Confocal fluorescence microscope images of affinity capture capillaries showing the Cy5

channel (a) before loading of complementary target and (b) following elution. A fluorescence signal was apparent following elution, indicating retention of 13.6 nM or 8 fmol of target.

Injection condition: 5 µL of a sample containing 1 µM Cy5-dT20 at 267 Vcm-1

for 60 seconds. Electrophoresis condition: 133 Vcm

-1 with 1x TBE/0.1 %PVP for 35 minutes. Elution condition: 267 Vcm

-1

with 1x TBE/0.5 M NaSCN/0.1 %PVP for 25 minutes at 60 °C. Images were enhanced in ImageJ using the Window/Level function for better clarity.

600

700

800

900

1000

1100

1200

1300

1400

15 35 55 75 95

Time (minutes)

Avera

ge F

luo

rescen

ce I

nte

nsit

y (

AU

)

(a)

(b)

Figure 3.4: (a) Change in the average Cy5 fluorescence intensity over time, measuring the loss of any

adsorbed materials used in the pre-treatment of the columns. Pre-treatment protocol: a 5 µL sample of 2 µM Cy5-dC20 at 267 Vcm

-1 for 2 minutes, followed by electrophoresis at 133 Vcm

-1 in 1xTBE/0.1% PVP

for 5 minutes, and a second injection of a 5 µL 2 µM Cy5-dC20 at 267 Vcm-1

for 2 minutes and electrophoresis at 133 Vcm

-1 in 1xTBE/0.1% PVP for 15 minutes. (b) The original fluorescence intensity

of the capillary channel prior to the loading of any material (baseline). Error bars represent 1 standard deviation of three trials.

55

The data of Figure 3.4 shows a decrease in the average fluorescence intensity over time,

indicative of loss of the non-complementary material used to pre-treat the capillary. It can be

observed that the decrease in average fluorescence intensity was greatest during the initial 40

minutes, which coincides with the time frame for the capture of complementary target. The loss

of the non-complementary pre-treatment material may have allowed the complementary targets

to adsorb onto the gel.

The addition of non-complementary dC20 (10% of the total DNA sample) to the running buffers

did not reduce the amount of non-selective adsorption of target. The average fluorescence

intensity following elution of captured targets was similar with and without the addition of the

dC20 in the running buffer. This suggested the need for a more rigorous pre-treatment protocol

if low concentrations of target were to be handled using DNA-modified polyacrylamide gels.

Work by other groups using the Acrydite™ immobilization chemistry in polyacrylamide gels has

made no mention of non-selective adsorption [96, 97, 187]. Those reports have used a lower

concentration of acrylamide monomer in the affinity gel, but it is not clear whether this should be

related to the trend in adsorption that was noted.

Some loss due to non-specific adsorption could be expected because the blocking agent was of

the same chemistry as the sample. The goal of this work was not to obtain 100% efficiency in

the release of the captured targets, but rather to achieve a reproducible efficiency of capture and

release. Even with an efficiency less than 100%, a correction factor may be applied to the data

for correction to achieve quantitative results, but reproducibility must be achieved for this

correction to be applicable.

Using the pre-treatment scheme, the loss due to adsorption was observed to be constant at 8 fmol

for complementary targets regardless of factors such as probe concentration, or the initial target

concentration within the range that was examined. This suggested a constant error of 8 ± 8 fmol

using the affinity gel, which provided for confidence in reproducibility of capture and elution,

and for determination of relative loss.

3.0.4 Variation of Polymer Density

The mobility of a target through a polyacrylamide gel is dependent on the density of the gel.

Therefore, it was of interest to examine the capture and elution process of affinity gels

56

manufactured using different polymer densities. However, when the density was below 12%T of

linear polyacrylamide gel, the polyacrylamide appeared to be physically unstable; elution of the

probe-modified polyacrylamide from the capillary was observed.

Figure 3.5 show images of the Cy3 channel of the capillary for a 0.45 µM Cy3-dT20 probe in a

7.5 %T linear polyacrylamide affinity gel (a) before and (b) after the introduction of

complementary target. Elution of the probe-modified polymer chains from the column was

observed following the capture of complementary targets.

Figure 3.5: Confocal fluorescence microscopy images of capillary affinity capture gel of the Cy3 channel showing the elution of 0.45 µM Cy3-dT20 probe immobilized in a 7.5%T linear polyacrylamide gel. The image was taken of the capillary in (a), following pre-conditioning and pre-treatment and (b), following

loading 1 µM Cy5-dA20 and running for 35 minutes. Injection condition: 5 µL of a sample containing 1 µM Cy5-dA20 at 267 Vcm

-1 for 60 seconds.

Electrophoresis condition: 133 Vcm-1

with 1x TBE/0.1% PVP for 35 minutes. Images were acquired using the Chipreader.

The incorporation of oligonucleotide probes into the polyacrylamide imparts a net negative

charge on the polymer chains. When subjected to an electric field, the charged polymer chains

would exhibit an electrophoretic mobility if not anchored, and would elute from the column.

The fact that this was not observed for the 12.5 %T polyacrylamide gels is attributed to the

higher degree of entanglement for higher polymer density.

57

The polyacrylamide used to manufacture the affinity gels did not contain any N,N’-methylene-

bis-acrylamide cross-linker in the monomeric solution, meaning that the gel was not chemically

cross-linked. Therefore, the polymer chains must entangle with one another in order to form

‘pores’ within the matrix for size sieving to occur. The concentration where entanglement takes

place is referred to as the entanglement concentration c*. At concentrations ten times greater

than c* (usually greater than 10% w/w [188]) the polymers will form a strongly entangled

network and are considered to behave similarly to cross-linked counterparts [189]. The 12.5 %T

linear polyacrylamide gels used may be considered a concentrated gel.

In the work reported by Mathies and Olsen, the affinity capture probes were incorporated into

lower density polymers, 5 %T and 10 %T polyacrylamide gels, respectively. However, elution

of the polymer chains was not reported. This could be due to the shorter times used for capture

and elution in these experiments. Since smaller volumes of the affinity gel were used inside the

microfluidics channels versus in the capillary, the processing time was significantly reduced.

From the experiment shown in Figure 3.5, the elution of the polymer was not observed until after

having subjected the gel to the electric field for approximately 90 minutes (30 minutes for pre-

conditioning, 25 minutes during the pre-treatment, and 35 minutes following the loading of

complementary target). By contrast, the total capture and elution process was completed in 120

seconds and 15 minutes, respectively, by Mathies and Olsen.

3.0.5 Quantity of Probe that was Immobilized in the Polyacrylamide Matrix

The amount of probe (dT20) that was incorporated into the affinity gels was examined by

confocal fluorescence microscopy. Three different concentrations of Cy3-labelled dT20 probe,

182 nM, 454 nM, and 727 nM in 12.5 %T linear polyacrylamide gels were prepared. Once the

gels were cast, the capillaries were pre-conditioned by running the capillaries at 67 Vcm-1 for 30

minutes in 1x TBE /0.1% PVP buffer. This was followed by pre-treatment with non-labelled

non-complementary target as previously described. Subsequently, a 5 µL sample of 1 µM dA20-

Cy5 was injected and was allowed to run through the capillary.

It was expected that any probe molecules that were not incorporated into the polyacrylamide gel

matrix would be eluted from the column during the pre-conditioning step. The Cy3 fluorescence

intensity measured inside the capillary channel would correspond to the amount of probe that

was successfully incorporated. Concentrations were determined using calibration data and the

58

amount of dye-labelled probe inside the gel was calculated based on a geometric volume of the

capillary of 0.59 µL. Calculation of the percentage of probe that was incorporated into the gel

was done by considering the quantity of probe in the gel in comparison to the amount of probe in

the original monomeric solution.

As shown in Table 3.1, an average of about 40% of probe molecules was incorporated into the

gels, and this result did not appear to be affected by the initial concentration of probe in the

reaction solution (in the concentration range examined).

Table 3.1: Tabulated results for extent of probe incorporation and performance in capture for three different probe concentrations.

Affinity gel: Varying concentrations of Cy3-dT20 probe (182 nM, 454 nM and 727 nM) in a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dT20 at 267 Vcm

-1 for

60 s. Electrophoresis condition: 133 Vcm-1

with 1x TBE/0.1% PVP for 20 minutes. Elution condition: 267 Vcm

-1 with 1x TBE/0.5 M NaSCN/0.1%PVP for 15 minutes at 60 °C. Error bars are propagated error

following correlation of average fluorescence intensity to concentration using a calibration curve. Original

amount of Probe (fmol)

Amount of probe (fmol)

% probe incorporated

Amount of target captured

(fmol)

% target captured versus probe

Amount of target

remaining (fmol)

% elution based on amount of

capture

107 46±6 43±6 42±9 93 8 ± 8 81

268 107±7 40±3 86±9 80 8 ± 8 91

429 179±8 42±2 159±11 89 8 ± 8 95

The efficiency of capture of target was based on the ratio of concentration of fully

complementary target that was captured versus the concentration of probe that had been

immobilized in the gel. Binding efficiencies less than 100% would suggest some binding sites

were not available for binding. Since the polymer chains were entangled, some entanglement

points might be situated between two probe sites, thereby preventing hybridization with

complementary targets. Conversely, binding efficiencies greater than 100% would suggest non-

selective adsorption or incomplete elution of any excess, unbound targets off the column.

No obvious correlation in efficiency was observed between the amount of target captured versus

the amount of probe that was available for the three different probe concentrations that were

examined. The capture efficiency appeared to be fairly high, and was in the range of 80% to

93% for the three probe concentrations that were used. A sample of 1 µM target was used for all

three cases to examine the effect of saturation of the probe sites (amount of target available to

load into the capillary was 5000 fmol).

Approximately 80 to 95% of the captured target was released after elution. The relative quantity

of target eluted was somewhat dependent on the quantity of probe that was in the gel, with higher

59

elution efficiencies being apparent for gels that contained more probe molecules. Following

elution, a constant residual retention of 8 fmol was noted. Non-selective adsorption was not

directly related to the concentration of the immobilized probe molecules in the gel.

3.0.6 Influence of Concentration of Target on the Efficiency of Capture

The ratio of amount of target-to-probe was determined and was compared to the amount of target

in the original sample. The intention was to assess the working range of the affinity gel. All

experiments were based on an electrokinetic injection for 1 minute at 533 Vcm-1 of a 10 µL

sample into a DNA-modified gel (454 nM Cy3-dT20 probe affinity gel). The target was allowed

to migrate for 10 minutes and the gel was then imaged. This was followed by elution for 5

minutes, after which the gel was imaged again. Note that not all of the available target was

actually loaded onto the column in the process of electrokinetic injection. This is the cause for

the differences observed in data for experiments related to Table 3.1 and Table 3.2. Different

loading conditions were used to collect each data set.

Table 3.2: Tabulated results for effect of concentration of sample on loading of affinity gel. Amount of target in loading of a sample was calculated based on a 10 µL of the target concentration solution used for loading. Amount of probe and target were calculated using the geometric volume of 0.59 µL for the

capillary. Affinity gel: 0.45 µM Cy3-dT20 probe in a 12.5%T linear polyacrylamide gel. Injection condition: 10 µL sample containing Cy5-dA20 at 533 Vcm


-1 with 1x

TBE/0.1% PVP for 10 minutes. Elution condition: 267 Vcm-1

with 1xTBE/0.5 M NaSCN/0.1%PVP for 5 minutes at 60°C. Error bars are propagated error following correlation of average fluorescence intensity

to concentration using a calibration curve. Amount of Target in

sample (fmol)

Amount of Probe incorporated

(fmol)

Amount of Target

Captured (fmol)

Target / Probe

Amount of Target after

Elution (fmol)

5 71 ± 6 10 ± 9 0.1 ± 0.1 8 ± 8

10 54 ± 6 14 ± 9 0.3 ± 0.2 8 ± 8

50 71 ± 6 29 ± 9 0.4 ± 0.1 8 ± 8

100 82 ± 6 16 ± 2 0.19 ± 0.02 8 ± 8

200 84 ± 6 54 ± 3 0.64 ± 0.06 8 ± 8

300 85 ± 7 41 ± 2 0.48 ± 0.05 8 ± 8

400 74 ± 6 99 ± 4 1.3 ± 0.1 8 ± 8

500 76 ± 6 78 ± 9 1.0 ± 0.1 8 ± 8

1000 75 ± 6 90 ± 9 1.2 ± 0.2 8 ± 8

2500 60 ± 6 83 ± 9 1.4 ± 0.2 8 ± 8

60

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 500 1000 1500 2000 2500

Amount of Target (fmol)

Am

ou

nt

of

Targ

et

Cap

ture

d (

fmo

l) /

Am

ou

nt

of

Pro

be (

fmo

l)

Figure 3.6: Effect of amount of target on the efficiency of capture using a 454 nM Cy3-dT20 probe

affinity gel. The ratio of captured target versus available probe was plotted against varying concentrations of Cy5-dA20 targets. Error bars are propagated error following correlation of average

fluorescence intensity to concentration using a calibration curve.

This analysis provided an indication of the working range of the affinity gel and also indicated a

non-linearity between the amount of target that was processed and the amount captured. This

was expected since a saturation point would be reached for amounts of target higher than the

amount of probe available to capture the targets. This was observed for samples containing

amounts of target greater than 400 fmoles in the original sample. The fluorescence intensity in

the affinity gel after loading a 5 fmol sample was not significantly different from the

background. The experimental data indicates that the lower limit of target that can be reliably

captured and detected by confocal fluorescence microscopy is 50 fmol.

For amounts of target greater than 400 fmol, it was observed that the ratio of target-to-probe was

greater than one. These results suggested that the column was overloaded and insufficient time

was allowed for the removal of excess target, resulting in capture efficiencies greater than unity.

3.0.7 Capture and Elution using a Non-Complementary Target

Figure 3.7 shows that non-complementary target (Cy5-dC20) was not captured by the affinity

probe gel. The average fluorescence intensity following elution corresponded to retention of

35 ± 15 fmol of material. In Figure 3.8, the plug of non-complementary target migrated through

61

the affinity gel without any significant tailing, indicating very little interaction between the

affinity gel and the target.

Figure 3.7: Confocal microscope images of capillaries examining the use of a non-complementary target in the affinity gel from (a) prior to loading the non-complementary target, (b) after loading and running for

25 minutes and (c) after the elution step was applied. Affinity gel: 0.45 µM Cy3-dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dC20 at 267 Vcm

-1 for 60 seconds. Electrophoresis condition:

133 Vcm-1

with 1x TBE/0.1% PVP for 35 minutes. Elution condition: 267 Vcm-1

with 1xTBE/0.5 M NaSCN/0.1%PVP for 25 minutes at 60°C. Images were obtained using the Chipreader.

Figure 3.8: Confocal microscope images of capillaries showing non-complementary target (Cy5-dC20)

as it moved electrophoretically through the capillary after (a) 60 seconds, (b) 120 seconds, (c) 240 seconds and (d) 420 seconds. Only the images of the Cy5 channel are shown.

Affinity gel: 0.45 µM Cy3-dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection condition: 5 µL of a sample containing 1 µM Cy5-dC20 at 267 Vcm

-1 for 60 seconds. Electrophoresis condition:

133 Vcm-1

with 1x TBE/0.1% PVP. Images were obtained using the Chipreader.

62

3.0.8 Examination of Selectivity Using a Five Base Pair Mismatch Target

A Cy3 labelled oligonucleotide target with a five base pair mismatch in the center of the 20 nt

sequence (Cy3-dA8C5A8) was run through the affinity gel. From Figure 3.9 it can be seen that

the five base pair mismatch target traveled through the affinity gel with very little interaction

with the affinity probe. No statistically significant increase in the average fluorescence intensity

was observed in the Cy3 channel following the migration of the 5 base pair mismatch targets.

Fluorescence images indicated that there was no trapping of the target, and the absence of tailing

suggested that the five base pair mismatch target traveled through the gel with very little

interaction with the affinity probe.

Figure 3.9: Confocal microscope images of capillaries examining the loading of Cy3-dA8C5A8 (5 base pair mismatch) target through an affinity capture gel. Images were taken after (a) 5 minutes and (b) 30

minutes. Affinity gel: 1.8 µM dT20 probe immobilized in a 12.5%T polyacrylamide gel. Injection condition: 5 µL of a

sample containing 1 µM Cy3-dA8C5A8 at 267 Vcm-1


-1 with 1x TBE/0.1% PVP. Images were obtained using the Chipreader. The images in (b) was

enhanced in ImageJ using the Window/Level function for better clarity.

The five base pair mismatch was not expected to be captured at room temperature conditions.

The melt temperature of the probe and fully complementary target hybrid that was examined was

37.3 °C [190]. The melt temperature for the mismatch target was calculated to be 17.4 °C (1.8

63

µM probe, 1.0 µM target, 50 mM [Na+]) using the online DNA Thermodynamics &

Hybridization Tool (biophysics.idtdna.com). This set of experiments was done at room

temperature (23 °C) and therefore was not expected to lead to formation of stable hybrids.

3.0.9 Separations of Mixtures of Complementary and Non-complementary Targets

Figure 3.10 shows the results of the passage of a mixture of 0.5 µM dT20-Cy3 (complementary)

and 0.5 µM dC20-Cy5 (non-complementary) targets through a 12.5%T linear polyacrylamide gel

containing 1.8 µM dA20 probe.

Figure 3.10: Confocal microscope images of capillaries tracking a time course experiment for loading a mixture of dT20-Cy3 and dC20-Cy5. Images shown were taken after (a) 120 seconds, (b) 240 seconds,

(c) 540 seconds and (d) 840 seconds. Affinity gel: 1.8 µM dA20 probe immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition:

5 µL of a sample containing 0.5 µM Cy3-dT20, 0.5 µM Cy5-dC20 at 267 Vcm


-1 with 1x TBE/0.1% PVP. Images were

obtained using the Chipreader.

As the sample containing the mixture of complementary and non-complementary targets

migrated through the affinity gel, selective capture of the complementary targets by the

immobilized probes was observed. This was indicated by a retardation of the electrophoretic

mobility as well as tailing of the complementary target (Figure 3.11).

64

0

10000

20000

30000

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(b)

Figure 3.11: Profile plot taken from the inlet to outlet end of the capillary from confocal microscope

images of the Cy5 channel after a 4 minute run of the (a) fully complementary (dT20-Cy3) target with (b) non-complementary (dC20-Cy5) targets in a dA20-probe affinity gel. Images were acquired using the

Chipreader.

Selectivity in a capillary gel electrophoresis experiment is expressed as the ratio of the corrected

migration time between two species [191].

)(

)(

1

2

o

o

tt

tt

−

−=α (3)

where t2 and t1 are the migration time of the first and second eluted analyte being compared, and

t0 is the void time. The migration time of a target inside the capillary is directly related to

electrophoretic mobility. In the affinity gel experiment, the electrophoretic mobility is

determined by the concentration of free affinity ligands as well as the association constant for

complex formation [192]:

][1 LKt

tA+==

ο

ο

µ

µ (4)

where µo and to are the electrophoretic mobility and migration time of the target in the absence of

the affinity ligand, µ and t are the electrophoretic mobility and migration time of the target in the

presence of the affinity ligand, respectively. The concentration of the free ligand is [L], and KA is

the association constant.

It can be seen in Figure 3.12 that both targets exhibited similar electrophoretic mobility

migrating through an unmodified polyacrylamide gel. In Figure 3.11, the electrophoretic

65

mobility of the complementary target was retarded by the presence of the affinity probe in the

polyacrylamide gel matrix, demonstrating selectivity of the affinity probe for the complementary

target.

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Figure 3.12: Profile plot taken from the inlet to outlet end of the capillary from confocal microscope

images of the Cy5 channel. Profile plots following migration of (a) Cy3-dT20 and (b) Cy5-dC20 through an unmodified polyacrylamide gel after five minutes. The profile of the entire capillary length is not shown. The distance is shown from the injection end to the elution end. Images acquired using the Chipreader.

3.0.10 Five Base Pair Mismatch in Mixture with Fully Complementary Target

Selectivity for the fully complementary target was shown using mixtures containing 1:1 (Figure

3.13) and 1:9 mixture (Figure 3.14) of the fully complementary target (dA20 – Cy5) to five base

pair mismatch target (Cy3 – dA8C5A8).

The competitive experiments demonstrated selectivity of the affinity probe towards the

complementary target in the presence of non-complementary and also 5 base-pair mismatched

targets.

66

Figure 3.13: Confocal microscope images taken from an affinity gel column containing immobilized dT20-probe after loading a 1:1 mixture of Cy3-dA8C5A8 and Cy5-dA20. Images taken after (a) 120

seconds, (b) 240 seconds, (c) 360 seconds, (d) 660 seconds and (e) 960 seconds. Affinity gel: 1.8 µM dT20 probe immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition: 5 µL of a sample containing 0.5 µM Cy3-dA8C5A8 and 0.5 µM Cy5-dA20 at 267 Vcm

-1 for 60 seconds.


with 1x TBE/0.1% PVP. Images acquired using the Chipreader. The Cy3 channel image in (e) was enhanced using the Window/Level function in ImageJ.

67

Figure 3.14: Confocal microscope images taken from an affinity gel column containing immobilized

dT20-probe after loading a 9:1 mixture of Cy3-dA8C5A8 Cy5-dA20 target. Images were taken after (a) 240 s, (b) 540 s and (c) 840s.

Affinity gel: 0.9 µM dT20 probe immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition: 5 µL of a sample containing 1.8 µM Cy3-dA8C5A8 and 0.1 µM Cy5-dA20 at 267 Vcm

-1 for 60 seconds.


with 1x TBE/0.1% PVP. Images acquired using the Chipreader.

3.0.11 Capture of 40 nt Length Targets

Figure 3.15 demonstrated the successful capture of a 40 nt length target, Cy5-dC10T20C10 using

the affinity gel.

68

Figure 3.15: Confocal microscope images of capillaries for the Cy5 channel demonstrating the loading and capture of a 1 µM 40 nt target sequence, Cy5-dC10T20C10 through the affinity gel. Images were

taken after electrophoresis following electrokinetic injection for (a) 300 s and (b) 25 min. Affinity gel: 1.8 µM dA20 probe immobilized 12.5%T linear polyacrylamide affinity gel. Injection condition:

5 µL of a sample containing 1 µM Cy5-dC10T20C10at 267 Vcm-1


-1 with 1x TBE/0.1% PVP. Images were acquired using the Chipreader.

Following elution, the average fluorescence intensity change indicated a capture of 184 ± 12

fmol of target and adsorption of 8 ± 8 fmol with a relative error of -5%. Complete removal of the

target from the gel had again not occurred for the elution conditions reported herein, and the

marginal loss of material was similar to that observed for all the other experiments that used

complementary targets.

3.1 Capture of Targets of Greater Lengths than 40 nt – Moving Towards Handling of NAs From Real Samples

Building on the preliminary findings using 20 and 40 nt length of NAs, my interest was to

capture longer DNA targets such as would be available in real samples that were prepared by

PCR, or by use of ultrasonication to shear genomic DNA into smaller fragments [122, 193–195].

It was necessary to identify the most important factors that affect the performance of the affinity

gel towards the capture and subsequent release of longer DNA targets.

69

Various gel formulations were examined to better understand the incorporation of probes for

capture of longer DNA sequences. A systematic factorial approach was implemented to assist in

the identification of the primary factors that determined optimal loading of the targets, and

concurrent reduction of non-selective adsorption for target solutions that contained mixtures of

complementary and non-complementary sequences. These studies allowed for optimization of

affinity gels for evaluation of the practical potential for handling longer oligonucleotides to

increase target concentration prior to delivery into an analytical biosensing device.

3.1.1 DNA Targets Selected for Experiments

Typical methods for the processing of DNA to generate single stranded targets after extraction

from cells have included PCR and ultrasonication [26, 28, 23, 196, 30, 29]. Examples of lengths

of DNA targets generated by PCR from real samples that have been detected with DNA

biosensors are commonly in the range of 150 - 600 nt [197–200]. The majority of the reported

targets were generated by symmetric PCR, and single stranded DNA targets were generated by

heating at 90-95 °C for 5 to 10 minutes, followed by fast cooling and storage on ice [198–200].

An example of a report that used asymmetric PCR generated single stranded targets of 168 and

340 nt lengths [197].

Larguinho et al. examined the fragmentation of genomic and plasmid DNA using a number of

different ultrasonication devices. They were able to generate fragment distributions between 100

and 5000 base for plasmid DNA, and fragments between 100 and 800 base distribution for

genomic DNA [193].

Work previously published by our group has demonstrated the ultrasonication of genomic E. coli

DNA to generate fragments of 100-400 nt in length. Additionally, experiments done by Mann

have shown that the length of DNA target generated by ultrasonication can be adjusted by

changing various factors such as ionic strength, ultrasound power, temperature of the solution

and exposure time [122].

The lengths of DNA targets chosen in this study were lengths that could be generated using

either PCR or ultrasound. Three representative lengths of DNA targets were chosen; 150 nt,

250 nt and 400 nt. The 150 nt target was the provided by Dr. Paul Piunno and is a target from

the β-actin gene. As previously mentioned in the Material and Methods section, the β-actin gene

70

is a highly conserved housekeeping gene, and typically is used to normalize molecular

expression studies. Some studies have shown that the level of expression of β-actin can change

in the presence of a disease such as Alzheimer’s disease, as well as in cancer samples [182].

The 250 nt target was generated by combining the 150 nt β-actin fragment with the 100 nt

fragment LAMA3 target also provided by Dr. Paul Piunno. The 100 nt fragment is a target for

the LAMA3 gene. This gene codes the α3A part of the Laminin-5 heterotrimer filament protein,

which is an important structural component in basement membranes. There is evidence that

Laminin-5 is expressed in invading tumor cells and can be strongly active in promoting the

migration of some tumor cells [183].

The 400 nt target was generated by the genomic E. coli DNA that was extracted from E. coli K12

strain. The target of interest was a region in the uidA gene, which is present in all E. coli strains.

This was selected because this gene was previously used by our group as a control [201].

Supplemental information regarding the synthesis of the 250 nt and 400 nt targets, as well as gel

electrophoresis and sequencing data for confirmation of the correct DNA targets can be found in

Appendix B. Experiments examining the generation of single stranded targets by heat

denaturation are included in Appendix C.

3.2 Compositions of Affinity Capture Gels

Most of the work using capillary electrophoresis in the literature has been with linear

polyacrylamide solutions instead of crosslinked polyacrylamide gels due to the advantage of

relatively facile replacement of the gels, as well as problems that arise with bubble or void

formation when casting crosslinked polyacrylamide gels in situ inside capillary columns. During

the polymerization process that creates crosslinked polyacrylamide, shrinkage of the polymer can

often result in the formation of voids inside the capillary [189, 202, 203].

It was previously shown in Figure 3.5 that the use of lower density gels resulted in the elution of

the polyacrylamide affinity gel from the capillary. The polyacrylamide affinity gel matrix was

based on the covalent attachment of negatively charged oligonucleotide probes. This imparted a

negative charge to the polyacrylamide strands. It was observed that the gel could elute from the

column under the influence of an electric field. For the longer DNA targets of interest in this

thesis, the time required to move the DNA sample through the capillary would increase, meaning

71

a significantly longer total analysis time and greater probability of loss of gel due to the applied

electric field. The elution of polyacrylamide affinity gels limits the use of linear gels at low

concentrations. Therefore, the decision was made to switch to crosslinked polyacrylamide gels

that would offer physical stability as well as an advantage in speeding the elution of longer DNA

sequences. One technical problem was that previous experiments already had indicated bubble

and void formation in the gel was apparent for recipes where the amount of crosslinker used was

2.5% w/v (approximately 20-33 %C given a %T range used in these experiments of 7.5-12.5%).

However, further investigations revealed that this was minimized when the amount of crosslinker

was decreased to 0.5% w/v (approximately 4-7 %C for the same %T range). Electroosmotic

Flow (EOF) in the capillaries was suppressed using dynamic coating with PVP. Experiments

comparing the efficacy between a dynamic and a covalent coating for suppression of EOF can be

found in Appendix D.

3.3 Selective Capture of 150 nt Target

The sequence for the 150 nt target is shown in Appendix B. The region on the 150 nt target that

is complementary to the oligonucleotide probe lies between base number 54-72 on the DNA

strand. Initial experimental work associated with this thesis began with use the of 20 nt targets.

The targets were introduced into the affinity capture column by elution chromatography; a small

amount of the sample was introduced into the capillary column and was transported by the

mobile phase (running buffer). The target interacted with the stationary phase, and could be

captured by the affinity gel. Initially, capture experiments for the longer DNA targets made use

of this method of sample introduction. However, reproducible target capture was not achieved.

When the 150 nt target was introduced in a similar fashion as the 20 nt target, offline images

scanned of the capillary showed successful introduction of the target materia, but hybridization

of the complementary target as the injected DNA targets moved through the capillary was not

observed.

It was believed that either; 1) not enough material was injected into the capillary during the

injection step since the mobility of the longer DNA targets was less than the 20 nt

oligonucleotides, or 2) that the kinetics of reassociation was slower for the longer DNA targets

and the longer targets were not efficiently captured as the sample plug moved through the

affinity gel.

72

To allow more material to be injected into the capillary, the sample solution was continuously

loaded into the column, filling the capillary with the DNA target. A period of incubation

following the injection step was introduced to allow the DNA targets time to hybridize with the

affinity probes. Any unbound material was removed from the column by electrophoresis.

Figure 3.16 demonstrates an example of selective capture and elution of a 150 nt DNA target.

The average fluorescence intensities shown correspond to: the affinity capture gel prior to the

introduction of target; following injection of the target; removal of any unbound target by

electrophoresis of the affinity gel; and elution of the captured target by heating the affinity gel to

65 °C via a water jacket surrounding the capillary.

0

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before load wash elute

Figure 3.16: Fluorescence intensity values from the Cy5 channel as measured from confocal microscope

images of the capillary taken at various times during the capture and elution experiment. Values were obtained by taking the average of the values generated from the profile plot of the confocal image. The

capillary containing the affinity capture matrix was first imaged to establish the background fluorescence signal (‘before’). A 10 µL, 1.67 µM solution of the Cy5 labeled 150 nt complementary target was then

loaded into the affinity capture gel (7.5%T/6%C, 1.8 µM β-actin probe) electrokinetically for 20 minutes at 167 Vcm

-1 (‘load’) (The fluorescence intensity following this step saturated the detector and the actual

value is not shown). The fluorescence intensity for the (‘wash’) step was taken after the entire capillary was heated to 95 °C for 5 minutes, allowed to sit for 20 minutes at 20 °C, and following the application of

a voltage of 167 Vcm-1

for 20 minutes at 20 °C. Finally the captured targets were eluted by the application of a voltage of 167 Vcm

-1 for 15 minutes at 65 °C. Images were acquired using the

Chipreader.

Figure 3.17 shows an example of a profile plots of images of the capillary obtained by confocal

microscopy following capture of the longer DNA targets (3.17(a)) versus that for the 20 nt target

(3.17(b)).

73

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b)

Figure 3.17: Profile plots from the outlet end to inlet end of the capillary from confocal microscope

images obtained for the Cy5 channel of the capillary. a) Fluorescence intensity profile of the Cy5 channel of the affinity gel following the capture of the (0.14 µM) Cy5 labeled 150 nt target. Affinity capture gel:

7.5%T, 6 %C, 3 µM β-actin probe. b) Profile of the Cy5 channel of the affinity gel following the capture of the (1 µM) Cy5 labeled 19 nt target (SMN). Affinity capture gel: 10%T, 5%C, 0.5 µM affinity capture

probe (SMN). Images were acquired using the Chipreader.

74

The shape of the fluorescence intensity profile observed for the capture of the 150 nt DNA target

(3.17(a)) is different than that observed for the 19 nt oligonucleotide target (3.17(b)), with the

latter showing a relatively homogeneous distribution profile along the length of the channel. The

experimental conditions between these two experiments were conducted differently. The

experiment shown in Figure 3.17 (b) was performed with the Cy5-labelled 20 nt target using a

sample introduction mode similar to elution chromatography. Here, the 20 nt target was injected

for a short amount of time (1 minute) from the original sample solution and then the target was

moved through the affinity capture gel by electrophoresis and is seen to be captured more

homogeneously throughout the affinity capture gel . By contrast, the experiment using the 150

nt target was run by frontal chromatography. Here, the sample solution containing the Cy5-150

nt target was used as the feed solution and was continually injected into the capillary under the

moved to the outlet end of the capillary. The sample solution was replaced with the run buffer

and electrophoresis was conducted.

Retarded diffusion results in the shape observed of the profile plots in Figure 3.17(a). The

electrophoretic mobility of the complementary target as it moves through the affinity capture gel

is decreased due to interaction the target has with the immobilized probe. Livshits et. al. have

proposed that DNA moves through such affinity gels via a mechanism called "retarded

diffusion". As the DNA targets move through the gels, they will continuously associate and

dissociate with the immobilized capture probes. A higher probe concentration or the formation

of fully complementary hybrids would result in the material taking a longer time to move

through the gel. This model applies for a system where the DNA is moved through the gel by

diffusion and will be discussed further in Section 3.5.6 [204].

In the system examined in this thesis, the DNA is being moved through the capillary gel by

electrophoresis, but will still association and disassociation of the target with immobilized

probes. After the complementary target is loaded onto the column, those strands that form partial

duplexes with the probe will be retarded to a lesser degree versus those that form full duplexes

with the immobilized probe. Over time, targets that form partial duplexes with the probe as well

as targets that are near the outlet end of the capillary will elute out of the capillary column first,

resulting in very little fluorescence intensity observed near the outlet end of the capillary in the

fluorescence image in Figure 3.17(a). Those targets which form full duplexes with the

immobilized probe and closer to the inlet end of the capillary will have traveled the shortest

75

distance in the capillary during the wash step, and is seen as higher fluorescence intensity

observed near the inlet end of the capillary in Figure 3.17(a). What is observed in Figure 3.17(b)

is the fluorescence intensity observed is the material that formed perfect duplexes remaining in

the capillary column following the wash step. Here, the material has remained in the inlet end of

the capillary, indicating that its mobility has been reduced to the largest degree and that

population is expected to be purely composed of the complementary material. Fluorescence

images taken of the Cy3 labeled probes showed a homogeneous fluorescence, confirming that

the probes remained inside the capillary column.

Figure 3.18 shows profile plots of confocal images taken of the affinity capture gels following

the washing step for capture experiments that used a probe length that had been reduced to 10 nt.

The shorter probe sequence would result in weaker association and lower thermodynamic

stability, and would affect the ability of the capture probe to retard the movement of the DNA

targets. From Figure 3.18, the use of a 10 nt probe caused a shift in the position profile of the

captured material that was consistent with a reduction in affinity. The position of the

complementary material appears to be shifted forward along the capillary when the shorter probe

was used versus the 19 nt probe again due to the decrease in mobility expected between the

different probes.

From Equation 4, the degree of retardation of electrophoretic mobility is dependent on

association constant. Since the shorter probe is expected to have a smaller affinity binding

constant than the 19 nt probe, the overall result is a smaller effect on electrophoretic mobility of

the complementary target with the shorter probe. For the same amount of electrophoresis time

during the wash step, the population of DNA targets that form a full duplex with the immobilized

probe will have a smaller decrease in electrophoretic mobility and appears to have moved further

along the capillary.

76

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beta-actin probe (19 nt) beta-actin probe (10 nt)

Figure 3.18: Profile plots from the outlet end to inlet end of the capillary from confocal microscope

images obtained for the Cy5 channel of the capillary. Profile plots for the capture of a (0.14 µM) 150 nt target using a 20 nt length probe and a 10 nt length probe. Affinity capture gel: 7.5%T, 6 %C, 3 µM

affinity capture probe (β-actin). Images acquired using the Chipreader.

The use of the longer DNA targets in the affinity capture gels may raise issues not observed

previously with the 20 base oliogonucleotide targets. A reduction of the amount of target that

could be captured might arise from:

1) The reassociation of target with complementary sequence in the sample solution. Additionally,

the target and its complementary strand could decrease the availability of the probe through non-

specific interactions such as adsorption.

2) A longer target could possess secondary structures such as hairpins through intra-strand

interactions.

Figure 3.19 shows 3 of 15 possible hairpin structures of the 150 nt target, as calculated using the

OligoAnalyzer 3.1 software provided by Integrated DNA Technologies. The melt temperature of

these hairpin structures ranged from 32.6 to 40.1 °C.

77

Figure 3.19: Examples of hairpin structures as calculated by OligoAnalyzer software. Settings used for calculations were 25 °C, 50 mM Na

+ concentration, suboptimality 50% and maximum foldings 20. Probe

region is highlighted in the drawn box.

The target region that is complementary to the probe lies between base number 54-72 on the

DNA strand, and is highlighted in the drawn box in Figure 3.19. Therefore, it is possible that the

target adopted hairpin structures during the injection step, ultimately limiting the availability of

conformations of target that were suitable for capture by hybridization.

Figure 3.20 shows a histogram of all the partial interactions possible between the target, probe

and complementary strand as calculated using the OligoAnalyzer software (www.idtdna.com),

sorted by the number of base pairs interacting between the two sequences.

78

0

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1 to 9 10 to 19 20 to 29 30 to 39 40 to 49 50 to 59

Number of Interacting Base Pairs

Fre

quency o

f P

art

ial In

tera

ction B

etw

een N

ucle

ic A

cid

Str

ands

Target-Target Target-Complement Target-Probe Complement-Probe

Figure 3.20: A histogram of the number of partial interactions possible between the target, its complementary strand and the probe as calculated by OligoAnalyzer software. The number of

interactions was binned by the number of base pairs forming the interactions. Calculation conditions were oligonucleotide concentration = 0.25 µM, [Na

+] = 50 mM.

As can be seen in Figure 3.20, a large number of possible partial interactions exist between the

target strands and probes, ranging from 2 base pairs to 50 base pairs. This is reasonable

considering that the target and its complementary strand are relatively long, increasing the

likelihood of partial interactions. It is important to note that the interacting bases may not

necessarily be in a conserved sequence. Some interactions are between the ends of the two

targets, while others are between bases where the alignment of the two targets are shifted from

one another. Based on this, it is reasonable to assume that the target will participate in

interactions other than with the probe. Therefore, not all of the target that is injected into the

affinity capture gel may actually be available for hybridization, resulting in a reduction of the

amount of material that can be captured.

In one set of experiments the capillary column was heated to 95 °C following injection of sample

onto the column. This was done to drive hybridization to completion by first disrupting all

79

interactions between the target and other sequences present, and to denature any strands that may

have reannealed after the initial heat denaturation step. It is likely that heating the capillary to

95 °C denatured the target-probe hybrids as well, and upon cooling the targets were still able to

form partial interactions. A difference in the capture profile from that shown in Figure 3.17

would indicate that heating could free target for preferential hybridization with probes, resulting

in a more homogenous fluorescence intensity profile. However, this was not seen in the data of

Figure 3.21. Even if targets were captured along the capillary column, dissociation and

reassociation would occur during electrophoresis as described by Livshits, and targets captured

near the elution end of the capillary would still be eluted from the column during the time frame

of the washing step.

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heat no heat

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Figure 3.21: Data for comparison of the amount of 150 nt target (100 nM) captured by the affinity capture

gel after the capillary was heated to 95 °C for 5 minutes following the injection relative to the amount captured by an unheated column. Affinity capture gel: 7.5%T, 6 %C, 3 µM β-actin probe. Error bars are

propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.

3.3.1 Comparison of Capture of 150 nt DNA Targets Using Complementary and Non-complementary Probe

Figure 3.22 compares the amount of material captured based on correlation of the average

fluorescence intensity to a calibration curve. The data is collected following the ‘wash’ step, and

represents the difference between affinity gels that are complementary and non-complementary

80

to the 150 nt target. It is clear from this data that much more target was captured with the

complementary probe than the non-complementary probe, supporting the conclusion that the

capture was selective.

Figures 3.23 and 3.24 demonstrate the selective capture of the 250 nt and 400 nt length targets by

affinity capture gels containing complementary and non-complementary probes. The

complementary region on the 250 nt target is between base number 25 to 43; the complementary

region on the 400 nt target is between base number 162 to 184. The sequences for the target can

be found in Appendix B. From these sets of data, it can be observed that there is a difference

between the amount of material remaining in the capillary when a complementary and non-

complementary target bound. The amount of material remaining for uidA and SMN may

represent some interaction of the uidA with the SMN probe. The uidA probe is selective towards

the uidA gene in E. coli K12, while SMN is a probe for the spinal muscle neuron gene. The SMN

probe was meant to be a non-complementary probe to the 400 nt target. The two probes were

used for complementary and non-complementary probe due to similar GC content as well as melt

temperatures.

-20

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100

120

Beta-Actin Probe Non Beta-Actin Probe

Am

ount

of

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et

Captu

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fmol)

Figure 3.22: Difference in the amount of target retained after the ‘wash’ step from capture and elution

experiments between affinity capture gels that were complementary (3 µM β-actin) and non-complementary (3 µM non-β-Actin) to a 150 nt length DNA target (20 nM). The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to

concentration using a calibration curve.

81

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Beta-Actin Probe Non Beta-Actin Probe

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ount

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fmol)


experiments between affinity capture gels that were complementary (3 µM β-actin) and non-complementary probe (3 µM non-β-Actin) to a Cy5 labelled 250 nt DNA target (10 nM). The experimental

conditions are the same as in Figure 3.17 except the last step in the ‘wash’ step was applied for 40 minutes rather than 20 minutes. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error


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400

uidA SMN

Am

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experiments between affinity capture gels that contained complementary (3 µM uidA) and non-complementary (3 µM SMN) to a Cy5 labelled 400 nt DNA target (130 nM). The experimental conditions are the same as in Figure 3.17 except the last step in the ‘wash’ step was applied for 50 minutes. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average

fluorescence intensity to concentration using a calibration curve.

82

3.4 Performance of the Affinity Gel for the Capture DNA Targets

Figure 3.25 shows the amount of complementary 150 nt target captured by the affinity capture

gel to different concentrations of the 150 nt target in the original sample solution and Figure 3.26

shows the response to a mixture of the 150 nt and non-complementary target. The conditions

that were selected for the gel formulation and capture conditions were based on criteria for

optimization that were identified from a quarter-fractional factorial for gel formulation and

analysis of stringency conditions using a three-level factorial analysis: Affinity capture gel:

12.5%T, 1%C, 3 µM probe. Capture conditions: Incubation time: 5 min, injection voltage: 222

Vcm-1, wash voltage: 222 Vcm-1, wash temperature 40 °C, wash buffer: 1xTBE/PVP/25% v/v

formamide.

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140

0 0.1 0.2 0.3 0.4 0.5 0.6

Original Amount of Target in Sample (pmol)

Am

ount

Captu

red (

fmol)

Figure 3.25: Amount of 150 nt target captured onto affinity capture gel as a function of concentration.

Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 150 nt target in 1XTBE/PVP, 20 minute electrokinetic injection at 133 Vcm

-1. Incubation time: 5 min. Wash Step:

electrophoresis at 133 Vcm-1

for 25 minutes at 25 °C with 1x TBE/PVP buffer. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to


83

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1.31/0.71 0.48/0.71 0.32/0.71 0.16/0.71

Amount non-complementary/complementary in original target solution

(pmol)

Am

ount

of

Mate

rial C

aptu

red (

fmol)

non-complementary complementary

Figure 3.26: Amount of 150 nt target captured from samples in mixtures of complementary and non-

complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 150 nt target and non-complementary target in 1XTBE/PVP, 20 minute electrokinetic injection at 133 Vcm

-1. Incubation time:

5 min. Wash Step: electrophoresis at 133 Vcm-1

for 25 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP buffer. The data was obtained from confocal fluorescence images (Chipreader) of the

capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.

From Figure 3.25, the amount of complementary target retained by affinity capture gel did not

show a linear response, and may indicate saturation of the immobilized probe. In Figure 3.26

examines the affect of different amounts of non-complementary target in a constant amount of

complementary target. The results indicate non-selective adsorption of the non-complementary

target on the affinity capture gel as well as a saturation effect for the non-complementary target

at approximately 0.48 pmol, which might indicate a maximum level of the non-complementary

target being non-selectively adsorbed. There might also be a suppression of the amount of non-

complementary target at the highest non-complementary content used (at 1.31 pmol non-

complementary).

Originally, a Cy3-labelled 100 nt sequence (LAMA3) was selected for use as the non-

complementary target in these sets of experiments. The interaction of the 100 nt sequence that

was non-complementary to the probe was examined. Figure 3.27 shows the level of retention of

84

the non-complementary target was not influenced at different formamide concentrations in the

buffer during the wash step.

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

0 10 25

%formamide (v/v)

Flu

ore

scen

ce In

ten

sit

y (

AU

)

Figure 3.27: Comparison of amount of material retained by the 150 nt complementary target when

treating with 100 nt non-complementary target. Affinity capture gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 40 nM complementary

(150 nt Cy5-β-actin) and 45 nM non-complementary targets (100 nt Cy3-LAMA3) in 1xTBE/PVP, 20 minute electrokinetic injection at 133 Vcm

-1. Incubation time 5 min. Wash Step: electrophoresis at

133 Vcm-1

for 25 minutes at 40 °C with 1xTBE/PVP with different concentrations of formamide. Data was derived from images obtained from epifluorescence images of Cy3 channel (Alpha). Error bars represent

1 standard deviation of three trials.

Based on the results, it was observed that the fluorescence intensity resulting from the 100 nt

non-complementary target used originally was retained to a small degree and did not vary at

different stringency conditions examined. This indicates a small amount of non-selective

adsorption. Since the objective of this experiment was to examine the change in non-

complementary level with increasing formamide to increase stringency conditions, and the use of

the 100 nt target was not influenced by stringency conditions, the 100 nt non-complementary

was not used for these experiments.

Therefore a mixture of longer non-complementary PCR target containing targets of 500 nt and

greater was used to examine the affect of different stringency conditions in the factorial

experiment. Figure 3.28 shows the agarose gel electrophoresis of the targets. This target was

obtained by circumstance rather than by design, and originated from a contaminated DNA

template used for PCR. Therefore, the actual sequence was not known. The sequence was

85

assumed to be non-complementary to the probe sequence used. Results summarized below in

Table 3.5 shows a similar quantity of non-complementary target captured using a different probe

(uidA), suggesting that the non-complementary target was non-complementary to the probe

sequences used in these experiments.

Figure 3.28: 1% Agarose gel electrophoresis for non-complementary target used in efficiency

experiments. Lane 1: DNA Ladder. Lanes 2 and 3: non-complementary target used in factorial analysis. Run conditions: 100 V, 1 hour, 1x TBE buffer.

Figures 3.29 - 3.31 indicate the quantity of complementary material captured by the affinity

capture gel for the 150, 250 and 400 nt targets in a mixture of 150 nM (1.5 pmol) non-

complementary targets shown in Figure 3.28. This concentration of non-complementary target

was chosen as the maximum amount of material tested that previously had demonstrated the

highest level of retention in the capture gel. Again, a non-linear response was observed, which

may indicate saturation of the immobilized probe.

The results between Figures 3.25 and 3.29 represent data for samples containing complementary

150 nt target and samples containing 150 nt complementary target and non-complementary,

respectively. It was observed that samples containing the same quantity of complementary

86

material, a higher amount of complementary target was captured when the sample contained the

complementary target alone versus when the non-complementary target was present. This could

be due to a number of effects. Since experiments conducted containing mixtures was washed at

high stringency conditions (25%v/v formamide at 40 °C), loss of some complementary target

would be expected. Additionally, the presence of the non-complementary target can also add

additional sources of interactions between the complementary target strand and immobilized

probe, occupying these targets for hybridization. It would be expected the same trend for the

other two targets examined.

0

20

40

60

80

100

120

0 0.1 0.2 0.3 0.4 0.5


Am

ount

Captu

red (

fmol)

Figure 3.29: Amount of material captured for 150 nt target in mixture of constant concentration of non-

complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 150 nt target and

1.5 pmol of non-complementary target in 1XTBE/PVP, 20 minute electrokinetic injection at 133 Vcm-1

. Incubation time: 5 min. Wash Step: electrophoresis at 133 Vcm

-1 for 25 minutes at 40 °C with 25%v/v

formamide/1x TBE/PVP buffer. The data was obtained from epifluorescence microscope images (Alpha) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error


87

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1 1.2


Am

ount

Captu

red (

fmol)

Figure 3.30: Amount of material captured for 250 nt target in mixture of constant concentration of non-

complementary target. Affinity Capture Gel: 12.5%T/1%C, 3 µM β-actin probe. Injection step: 10 µL of 250 nt target and

1.5 pmol of non-complementary target in 1XTBE/PVP, 30 minutes electrokinetic injection at 133 Vcm-1

. Incubation time: 5 min. Wash Step: electrophoresis at 133 Vcm

-1 for 40 minutes at 40 °C with 25%v/v

formamide/1x TBE/PVP buffer. The data was obtained from epifluorescence microscope images (Alpha) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error


0

50

100

150

200

250

300

350

400

450

0 0.2 0.4 0.6 0.8 1 1.2


Am

ount

Captu

red (

fmol)

Figure 3.31: Amount of 400 nt target captured in mixture of non-complementary targets onto affinity

capture gel as a function of concentration. Affinity Capture Gel: 12.5%T/1%C, 3 µM uidA probe. Injection step: 10 µL of 400 nt target and 1.5 pmol of non-complementary target in 1XTBE/PVP, 34 minutes electrokinetic injection at 133 Vcm

-1. Incubation

time: 5 min. Wash Step: electrophoresis at 133 Vcm-1

for 50 minutes at 40 °C with 25%v/v formamide/1x TBE/PVP buffer. The data was obtained from epifluorescence microscope images (Alpha) of the

capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.

88

Tables 3.3-3.5 presents a summary of the performance of the affinity capture gel in terms of

recovery (amount recovered relative to the amount of complementary target in the original

sample solution) and purity (relative amount of complementary target versus the total amount of

DNA target (complementary and non-complementary) in the original sample solution). It is

important to note that these results are based on elution of the entire capillary without

concentration as achieved by heating of the entire capillary at once. Therefore, the volume of

material eluting through was assumed to be the volume of the capillary (350 nL). For

comparison, Section 3.6 will describe the performance of the affinity capture gel for the same

targets are concentrated by step elution.

Table 3.3: Summary results for Recovery and Purity for mixture containing 150 nt and 1.5 pmol non-

complementary targets by affinity capture gel. Recovery and purity of the original solution and by selective capture are presented. The recovery and purity were calculated based on removal of material from the affinity capture gel by elution of the entire capillary (no concentrating). Experimental conditions

shown in Figure 3.29.

150 nt Without selective

purification By selective purification (no concentrating)

Amount of

comple-mentary target in original sample solution

Recovery (%)

Purity (%)

Amount of Complemen-

tary target captured

Amount of Non-

complemen-tary material

captured

Recovery (%)

Purity (%)

Enhance-ment

400 fmol 100 23.12 5.52 ± 0.7

fmol 12.4 ± 2 fmol 1.4 ± 0.2 31 ± 6 1.5 ± 0.3

10 fmol 100 0.75 470 ± 160

amol 12.4 ± 4 fmol 4.7 ± 1.6

3.6 ± 1.5

5 ± 2

5 fmol 100 0.37 86 ± 50 amol 12.4 ± 4 fmol 1.7 ± 1.0 0.77 ±

0.4 2.0 ± 1.3

1 fmol 100 0.08 0 0 0 0 0

89

Table 3.4: Summary results for Recovery and Purity for mixture containing 250 nt and 1.5 pmol non-complementary targets by affinity capture gel. Recovery and purity of the original solution and by

selective capture are presented. The recovery and purity were calculated based on removal of material from the affinity capture gel by elution of the entire capillary (no concentrating). Experimental conditions




Amount of


Recovery (%)

Purity (%)



Amount of Non-


captured

Recovery (%)

Purity (%)

Enhance-ment

1 pmol 100 42.92 10 ± 4 fmol 13 ± 4 fmol 1.0 ± 0.5 44 ± 22

1.1 ± 0.5

500 fmol 100 27.32 6.0 ± 0.9 fmol 16 ± 3 fmol 1.1 ± 0.2 27 ± 6 1.1 ± 0.2

250 fmol 100 15.82 6 ± 2 fmol 14 ± 2 fmol 2.6 ± 0.8 31 ± 11

2.2 ± 0.7

100 fmol 100 6.99 1.8 ± 0.7 fmol 14 ± 2 fmol 1.8 ± 0.7 11 ± 5 1.8 ± 0.8

50 fmol 100 3.62 0.7 ± 0.2 fmol 14 ± 2 fmol 1.4 ± 0.5 4.8 ± 1.7

1.5 ± 0.5

10 fmol 100 0.75 0.32 ± 0.1

fmol 14 ± 2 fmol 3.2 ± 1.3

2.2 ± 0.9

3.3 ± 1.4


complementary targets by affinity capture gel. Recovery and purity of the original solution and by selective capture are presented. The recovery and purity were calculated based on removal of material from the affinity capture gel by elution of the entire capillary (no concentrating). Experimental conditions




Amount of


Recovery (%)

Purity (%)



Amount of Non-


captured

Recovery (%)

Purity (%)

Enhance-ment

1 pmol 100 42.92 22 ± 4 fmol 14 ± 2 fmol 2.2 ± 0.4 61 ± 13

1.5 ± 0.3

500 fmol 100 27.32 11 ± 3 fmol 14 ± 2 fmol 2.3 ± 0.5 44 ± 12

1.8 ± 0.5

250 fmol 100 15.82 10 ± 2 fmol 14 ± 2 fmol 4.4 ± 0.9 44 ± 10

3.1 ± 0.7

100 fmol 100 6.99 9.2 ± 0.4 fmol 14 ± 2 fmol 9.2 ± 0.4 40 ± 5 6.3 ± 0.7

50 fmol 100 3.62 2.8 ± 0.7 fmol 14 ± 2 fmol 5.7 ± 1.3 17 ± 5 5.2 ± 1.5

10 fmol 100 0.75 0.69 ± 0.20

fmol 19 ± 2 fmol 7 ± 2

3.5 ± 1.1

5.3 ± 1.6

1 fmol 100 0.08 35 ± 30 amol 12 ± 2 fmol 3.5 ± 2.9 0.28 ± 0.24

4.3 ± 3.6

90

Based on the results presented, the lowest quantity of material that could be processed and

purified with the affinity capture gel was 5 fmol, 10 fmol and 1 fmol for the 150 nt, 250 nt and

400 nt targets, respectively. The improvement in purity through delivery of the material by

affinity capture and elution ranged from a factor of 1.1 to 6.3. However, the recovery of the

method was low, and ranged from 2-10%.

3.5 Examination of the Effects of Varying Gel Formulation on Performance

The conditions used to assess the performance of the affinity capture gel were determined based

on optimization as was identified by factorial analysis. Factorial analysis allows for an efficient

systematic multiplexed approach to identify important factors that influence analytical

performance, and furthermore allows identification of interactions between different factors that

have impact on the analytical response.

Two independent factorial experiments were performed to assess and improve the recovery and

purity of the affinity capture gel. A quarter fractional 2-level 5 factor factorial experiment was

carried out to determine the effects of differences in gel formulation on the performance of the

affinity capture gel. Different gel formulations included variation of the quantities of the

following components: the total monomer concentration (%T), the amount of crosslinker (%C),

the oligonucleotide probe concentration, and the concentrations of TEMED and APS radical

initiator. Performance considerations included the amount of probe that was incorporated

(Appendix E) and the amount of target that was captured (Appendix F). The amount of material

eluted from the capillary column following the elution step (electrophoresis at 167 Vcm-1 for

15 minutes at 65 °C) was determined by measuring the fluorescence intensity remaining in the

capillary. Correlation of the measured fluorescence intensity with the calibration curve resulted

in negative concentration values. Therefore, the amount of material remaining was set as zero,

and no differences were observed for the gel formulations examined.

A three level factorial analysis was then completed to determine optimal conditions for capture

of targets and selectivity. Conditions that were examined included incubation time, wash

voltage, wash temperature and formamide content in the elution buffer (Appendix G).

91

The factorial experiments screened for the impact of a large number of factors to identify

dominant trends. The affinity capture gel was meant to be used on a series of targets of different

lengths. Therefore, the intent was to identify general trends rather than optimization of

conditions for a single target length. An exhaustive optimization and exploration of the response

surface was not performed.

The next sections will provide an overview of some of the primary factors that were identified by

the factorial experiments. Primary or significant factors are identified at the 95% confidence

interval. Detailed reports and calculations generated from the factorial experiment are included

in the appendix.

3.5.1 Affect of Gel Formulation on the Quantity of Probe that was Incorporated

The amount of oligonucleotide probe immobilized into the affinity capture gel was determined

on a relative basis by measuring the change in fluorescence intensity following polymerization of

the affinity capture gel, and also following pre-conditioning of the affinity capture gel. This

latter step was considered significant because materials such as unreacted monomer,

oligonucleotide probes and other compounds were removed by electrophoresis. The change in

fluorescence intensity measured following the preconditioning step more accurately indicates the

amount of probe immobilized into the polyacrylamide gel matrix.

A factorial analysis was carried out to examine the influence of factors changing gel formulation.

The factors examined were the monomer and crosslinker content, the amount of oligonucleotide

probe, and the concentrations of the radical initiator system (TEMED and APS). Processing of

the factorial data showed no significant influence (at 95% confidence) (Appendix E) of the

factors on the amount of probe incorporated. The average quantity of oligonucleotide probe

incorporated into the affinity capture gel was 88% ± 9% of the amount initially in the mixture.

This result suggested that the conversion efficiency of the oligonucleotide modified monomers

into the polyacrylamide gel was not affected by differences in the gel formulation for the factors

and concentration ranges examined at the 95% confidence limit. This also implies that the

polymerization efficiency was similar across the different gel formulations

92

The change in fluorescence intensity was reported rather than the absolute fluorescence intensity

due to a possible reaction between the fluorophore and the radicals as well as possible

differences in the degree of light scatter. Initially, the factorial analysis on the absolute

fluorescence intensity of the capillary following polymerization showed a negative result on the

amount of probe by the radical initiator (APS) used (Appendix E2). This result was attributed to

a possible reaction between the fluorophore and radical initiator and also was possibly due to

changes in the physical structure of the gel, leading to changes in the extent of light scatter.

Figure 3.32 shows the percentage of fluorescence intensity lost of the Cy3 labelled

oligonucleotide probe following polymerization after 20 minutes with different concentrations of

TEMED and APS as compared with the fluorescence intensity of the solution before the addition

of the radical initiator. The fluorescence intensity lost was greatest when the TEMED/APS ratio

used was at 4%/10%. At this ratio, the concentration of TEMED and APS were 0.22 M and 0.44

M. For all the other ratios tested, the concentration of TEMED was in excess of the

concentration of APS. A larger APS concentration than TEMED may result in excess APS

available to react with the oligonucleotide probe directly.

0

10

20

30

40

50

60

70

80

90

10% / 10% 10% / 4% 4% / 10% 4% / 4%

%v/v TEMED/ %w/w APS Ratio

Perc

enta

ge F

luore

scence I

nte

nsity

Rem

ain

ing (

%)

Figure 3.32: The relative fluorescence intensity following reaction between a 1 µM Cy3 labeled

oligonucleotide with different ratios of TEMED/APS radical initiator system after a period of 20 minutes. The loss was calculated relative to the in initial fluorescence intensity measured of the solution

immediately following the addition of the radical initiator. Error bars represent 1 standard deviation of three trials.

93

Additionally, UV-VIS spectra collected before and after polymerization (Figure 3.33) show

differences in light scattering of the gel. An apparent increase in absorbance above 280 nm is

due to increase in light scattering as the polymer gel is formed [205]. As can be observed, all gel

formulations exhibit some scattering, but scattering was most pronounced when the

concentration for 7.5% and 5%C polyacrylamide gels. The disappearance of the signal at 275

nm has been previously used to track the disappearance of the C=C double bond as the

acrylamide and bis-acrylamide monomers are incorporated into the growing polymer chains

during polymerization [206].

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

200 300 400 500 600 700 800

wavelength (nm)

ab

so

rba

nc

e (

AU

)

before polymerization after 20 min polymerizationa)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

200 300 400 500 600 700 800

wavelength (nm)

ab

so

rba

nc

e (

AU

)

before polymerization after 20 min polymerizationb)

94

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

200 300 400 500 600 700 800

wavelength (nm)

ab

so

rba

nc

e (

AU

)

before polymerization after 20 min polymerizationc)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

200 300 400 500 600 700 800

wavelength (nm)

ab

so

rba

nc

e (

AU

)

before polymerization after 20 min polymerizationd)

Figure 3.33: UV-VIS spectra of bulk polyacrylamide gels before and after polymerization for 20 minutes

as a function of different gel formulations. a) 12.5%T/5%C, b) 12.5%T/1%C, c) 7.5%T/5%C, d) 7.5%T/1%C

The results from Figure 3.32 and 3.33 demonstrate that absolute fluorescence intensity data from

the factorial analysis might be influenced by reaction with radicals and light scattering and may

not necessarily be representative of the amount of probe present in the gel. Therefore, the

relative difference in fluorescence intensity before and after pre-conditioning was used to

determine the amount of probe incorporation. The fluorescence intensity inside the capillary

following the polymerization of the affinity capture gels was obtained. Since nothing had been

done to the capillaries at this point, the amount of probe inside should still be equal to the

amount added in the original solution. Differences in the fluorescence intensity between

different capillaries would be the result of undesired reactions by the radical initiator as well as

differences in local environment, but not due to difference in the amount of oligonucleotide

95

probes incorporated into the capillary. Therefore, the factorial analysis was carried out to

examine if differences in gel formulation would result in a difference in the percentage of probes

incorporated into the gel. Since none of the factors showed a significant result, the average

percentage of incorporation was determined to be 88%.

It is important to note that the total quantity of probe incorporated into the gel is determined on

the concentration of oligonucleotide probe in the pre-polymer solution. The results highlighted

in the previous paragraph indicate that for the concentration range examined, the percentage of

probe incorporated into the gel was not influenced by the gel formulation. This suggests that for

an a initial probe concentration of 3 µM and 0.5 µM, the final probe concentration inside the

affinity capture gel after polymerization pre-conditioning would be 2.64 µM and 0.44 µM.

3.5.2 Effect of Radical Initiators on Oligonucleotide Sequence

The results from the previous section indicate that the radicals may react with the fluorophore

attached to the oligonucleotide target. This raises the issue of possible effects of the radical

initiator on the oligonucleotide sequence, with potential cleavage of the probe or structural

changes of the nucleotides.

Persulfate radical anion is a strong oxidizing agent and can attack organic species aside from the

acrylamide monomer [207–209]. Sulfate radical anions are generated from thermal homolysis of

the sulfur-sulfur bond. Sulfate radical anion may also be converted to hydroxyl radical in

aqueous solution, as shown in the reactions below.

⋅→ −−4

282 2SOOS

⋅+→+⋅ −− OHHSOOHSO 424

⋅++⋅→ −−−OHHSOSOOS 44

282

The rate of hydrolysis of sulfate radical anion at neutral pH is slow (k=107 M-1s-1) compared to

rate of reaction with most monomers (k=108-109 M-1s-1) and becomes significant when the

monomer concentration is very low. The persulfate radical anion initiates polymerization by

attacking the vinyl group of the monomer either through direct addition to the double bond or an

96

electron transfer to generate a radical anion in the monomer species. The formation of a radical

anion can lead to chain propagation through either a radical or anionic mechanism [210].

⋅−−−→=+⋅ −−CXYCHOSOCXYCHSO 2324

⋅++→=+⋅ +−− CXYCHSOCXYCHSO 22424

Since the sulfate radical anions initiate polymerization by attacking vinyl bonds, the sulfate

radical anion can also attack the double bonds present in all the nucleotide bases [209, 211, 212].

Again, this can proceed either through an electron transfer to the double bond or the addition of

the sulfate radical to the nucleobase. Elimination of SO42- produces a one-electron oxidized

species [212].

It has been reported that guanine is the most easily oxidized of the nucleic acid bases [213]. The

dominant reaction of the sulfate radical anion is the one-electron oxidization of guanine, with a

minor contribution from adenine [213]. The reaction is proposed to first produce an addition-

adduct, followed by a rapid loss of SO42- to give the one electron oxidized radical species [207].

The one electron oxidation produces a positive charge, and guanine is the major site for this

positive hole localization. The cationic radical of guanine is often deprotonated to become a

neutral radical species at pH 4.5 to 9.5 [207, 209, 213].

Figure 3.34 shows possible reaction product(s) between the nucleobases and SO4- radical. It is

also possible for the other components besides the nucleobase to be damaged. Figure 3.35

demonstrates the transfer of the radical from the nucleobase to the sugar moiety for ribonucleic

acid. The lone electron can cleave the nucleobase from the sugar, releasing it from the

oligonucleotide strand, or can cleave the phosphate backbone, breaking the oligonucleotide

strand [211, 214, 215]. Attack of the anion radical directly on the negatively charged phosphate

backbone is unlikely as there is electrostatic repulsion [213]. It has been shown that the SO4-

radical preferentially attacks the nucleobase rather than the deoxyribose or phosphate [211–213].

Studies done on polyribonucleotide polyA and single stranded DNA of mixed base sequence

with sulfate radical anions showed a strand breakage efficiency (yield of strand breakage for total

radical induced) of less than 5% strand breakage [213]. This was due to inefficient transfer of

the radical from the guanine base to the sugar moiety as shown in Figure 3.35 when the radical

97

of attack was the sulfate radical anion [213]. Radiation induced radicals interacting with guanine

have also demonstrated inefficient strand breakage [213].

Figure 3.34: Reaction products between the sulfate radical anion and (a) adenine, (b) guanine, (c)

cytosine and (d) thymine. Adapted from [207].

98

Figure 3.35: Scheme of the reaction between the radical and the nucleotide base that can lead to either

removal of the nucleoside base or strand cleavage. Adapted from [213].

The inclusion of TEMED in the polymerization of acrylamide serves as a catalyst for the

generation of radicals with APS [216–218]. The TEMED/APS combination acts as a redox pair,

where a one electron reduction of TEMED by persulfate generates one TEMED radical and one

sulfate radical anion [219, 220]. This enhances the polymerization rate by a factor of 3 [216].

Although there have been a lot of studies on the reaction between sulfate radical anion and DNA,

the reaction of the TEMED radical with DNA does not appear to be as well studied. Possible

reaction of the radical initiator system with the oligonucleotide probes used in our experiment

will be explored in the next section.

3.5.3 Cleavage of Oligonucleotide Probe by Radicals

The following experiment examined the possibility of cleavage of the oligonucleotide probes due

to reaction with the radical initiators. A 5 µM sample of Cy5 labeled oligonucleotide was mixed

with different ratios of TEMED and APS for five minutes, followed by the addition of 12 mM of

99

hydroquinone to the solution. Hydroquinone has been reported as a free radical scavenger [221,

222]. The reaction mixtures were then injected into a 12.5%w/v linear polyacrylamide gel

capillary and the products were tracked by capillary gel electrophoresis. Figure 3.36 shows the

electrophoretogram of a mixture of 12 nt (2 µM Cy5 - dT4A3T5), 19 nt (0.5 µM Cy5 - SMN

Target) and 20 nt (1 µM Cy5-dC20) oligonucleotides.

0

0.5

1

1.5

2

2.5

300 320 340 360 380 400 420 440

Time (s)

Flu

ore

scen

ce In

ten

sit

y (

AU

)

12nt

19 nt

20 nt

Figure 3.36: Electrophoretogram for a solution containing 2 µM 12nt, 0.5 µM 19 nt and 1.0 µM 20 nt

oligonucleotides. Cy5 labeled targets. Injection: 10 µL sample volume, 142 Vcm-1

, 4 s. Run condition: 142 Vcm

-1 in 1xTBE/PVP buffer.

Figure 3.37 shows the electrophoretograms for the solutions of 19 nt oligonucleotide targets

(Cy5-SMN) after reacting with different amounts of TEMED and APS for five minutes and

quenched with hydroquinone. Figure 3.37(a) shows the electrophoretogram of the 19 nt

oligonucleotide mixed with hydroquinone (no radical initiator).

100

0

0.2

0.4

0.6

0.8

1

1.2

400 420 440 460 480 500

Time (s)

Flu

ore

scence I

nte

nsity (

AU

)

a)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

400 420 440 460 480 500

Time (s)

Flu

ore

scence I

nte

nsity (

AU

)

b)

101

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

400 420 440 460 480 500

Time (s)

Flu

ore

scence I

nte

nsity (

AU

)

c)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

400 420 440 460 480 500

Time (s)

Flu

ore

scence I

nte

nsity (

AU

)

d)

102

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

400 420 440 460 480 500

Time (s)

Flu

ore

scence I

nte

nsity (

AU

)

e)

Figure 3.37: Electrophoretograms of the reaction products between a 19 nt Cy5 labelled target and different amounts of TEMED/APS. Injection: 10 µL sample volume, 142 Vcm

-1, 4 s. Run condition:

142 Vcm-1

in 1xTBE/PVP buffer. a) Control b) TEMED/APS in 10%/10%. c) TEMED/APS 10%/4%, d) TEMED/APS 4%/10%, e) TEMED/APS 4%/4%

A summary of the migration times of the various peaks is presented in Table 3.6. The results

show, based on migration time, a peak corresponding to the original 19 nt oligonucleotide target

as well as the appearance of a second, slower moving peak after reaction with the radical

initiator for 5 minutes. The intensity of this second peak is higher for cases where a higher

concentration of persulfate was used, suggesting that this second peak is a reaction product

between the oligonucleotide and the sulfate radical anion.

Table 3.6: Summary of the migration times of the peaks observed from the CGE experiment. The sequence of the oligonucleotide target used in these experiments: 5’ Cy5 - ACA GGG TTT CAG ACA

AAA T 3’. Error represents 1 standard from three trials. Reaction mixture Migration

Time (s)

19 nt and hydoquinone only (no radical initiator)

457 ± 11

457 ± 18 19 nt + TEMED/APS 10% v/v/10%w/v 471 ± 13

443 ± 11 19 nt + TEMED/APS 10% v/v/4%w/v 461 ± 13

437 ± 11 19 nt + TEMED/APS 4% v/v/10%w/v 455 ± 14

440 ± 14 19 nt + TEMED/APS 4% v/v/4%w/v 458 ± 17

103

It is possible that this second peak is the one electron oxidation of the guanine base present on

the oligonucleotide strand. The one electron oxidation of guanine can add a positive charge to

the oligonucleotide sequence, assuming the base is not deprotonated. The resulting

oligonucleotide product would migrate slower (smaller charge density) and observed as the

secondary peak. Since the purpose of this the examination was to examine possible cleavage

reaction products, further analysis of the second peak was not performed.

Guanine is the base most susceptible to radical attack, cleavage would be expected to have the

highest probability to at these sites. Fragments that are of at least 3 bases and 12 bases in length

would be observed in the electrophoretograms if cleavage had occurred. The absence of peaks

migrating faster than the original 19 nt peak from the electrophoretograms suggests that cleavage

did not occur, or occurred in amounts too small to be detected by fluorescence. This agrees with

experiments performed on single stranded DNA where only low levels of cleavage was observed

[209].

3.5.4 Examination of Damage to Nucleobases by Radical

Damage to the nucleobases by the radical initiator was also considered. If the fidelity of the

probe sequence was compromised by reaction with the radical initiator, then this would

negatively affect the selectivity as well as the capture efficiency of the oligonucleotide probes.

Previous experiments have shown the ability of the affinity capture gels to selectively capture

complementary oligonucleotide targets. Selective capture by the affinity gel was also observed

for 150 nt and as well as other targets complementary to the probe. Additionally, displacement

chromatography experiments demonstrated an ability to discriminate a system where a 1 bp

mismatch was present, and an increase in displacement was observed (Appendix B4).

Experiments performed using a probe that was 10 nt in length also demonstrated the successful

capture of the 150 nt target. The use of the TEMED/APS radical initiator system has also been

reported by other groups for the immobilization of oligonucleotide sequences onto gel matrices

[97, 151, 100]. Possible negative effects of the sulfate radical anion on the oligonucleotide

targets were not reported to be an issue. These results and observations already strongly suggest

that the probe sequence was not significantly damaged by the radical initiators.

Melt curves were obtained using oligonucleotide probes that had been subjected to reactions with

the radical initiator system to further examine whether there was evidence of damage of the

104

nucleobases. A shift in the melt temperature would be observed if damage to a significant

proportion of nucleobases had occurred.

The radical initiator was allowed to react with the oligonucleotide probes for 20 minutes, and the

target strand was introduced and allowed to hybridize for an additional 20 minutes prior to

determining the melt temperature. Hydroquinone was not added to stop the reaction since it

showed a large UV absorbance in the region of interest, obscuring the absorbance from the

DNA. Additionally, APS and TEMED exhibited an absorbance peak in the UV region, which

changed as a function of temperature. Therefore melt curves of solutions containing just APS

and TEMED were first run as controls and this background signal was subtracted from the

spectra obtained for the melt curve experiments. Figure 3.38 shows a representative melt curve

where the oligonucleotide probe was not reacted with the radical initiator (control). Figure 3.39

presents melt curves where the oligonucleotide probe was reacted with the different

concentrations of TEMED and APS.

1

1.1

1.2

1.3

1.4

1.5

1.6

45 50 55 60 65 70

Temperature (C)

norm

aliz

ed a

bsorb

ance (

AU

)

Figure 3.38: Representative melt curve for a sample of 0.3 µM 19 bp duplex (SMN) in 1 x TBE. Error

bars represent 1 standard deviation of three trials.

105

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

40 45 50 55 60 65 70

Temperature (C)

Norm

aliz

ed A

bsorb

ance (

AU

)

a)

1

1.2

1.4

1.6

1.8

2

2.2

2.4

45 50 55 60 65 70 75

Temperature (C)

Norm

aliz

ed A

bsorb

ance (

AU

)

b)

106

0.9

1.1

1.3

1.5

1.7

1.9

2.1

50 55 60 65 70 75

Temperature (C)

Norm

aliz

ed A

bsorb

ance (

AU

)

c)

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

1.4

45 50 55 60 65 70

Temperature (C)

Norm

aliz

ed A

bsorb

ance (

AU

)

d)

Figure 3.39: Melt curves for a sample of 0.3 µM 19 bp duplex in 1 x TBE where the probe was reacted with different amounts of TEMED and APS for 20 minutes prior to addition of the complementary target. a) TEMED/APS 10%/10%, b) TEMED/APS 10%/4%, c) TEMED/APS, 4%/10%, d) TEMED/APS 4%/4%.

Error bars represent 1 standard deviation of three trials.

The melt temperature was determined by taking the first derivative of the melt curve and taking

the temperature point at the inflection point between 50 and 70 °C. The results are presented in

Table 3.7.

107

Table 3.7: Summary of melt temperatures of the oligonucleotide duplex following reaction with the different radical initiator ratio. Error represent 1 standard deviation of three trials.

Concentration of

TEMED/APS added

Melt Temperature

(°C)

Control 58.5 ± 3

10%/10% 62.5 ± 1.7

10%/4% 59.5 ± 3.5

4%/10% 62.5 ± 2.3

4%/4% 57.5 ± 1.7

The sequence of the oligonucleotide target examined was: 5' ATT TTG TCT GAA ACC CTG T

3'. Guanine is predominantly the base attacked by sulfate radical anion, and 3 such nucleotides

were available. The expected change in melt temperature for 3 base pair mismatches would be a

reduction of around 5 to 10 °C, assuming a 1-3 °C shift per 1% mismatch [223]. Such shifts of

melt temperature were not observed. However, it does not preclude the possibility that the any

possible damage only resulted in partial loss of hydrogen bonding ability (i.e., 1 or 2 hydrogen

bonds between GC versus 3 hydrogen bonds normally).

3.5.5 Examination of Conditions that Affect Capture of Complementary Targets

This section examines various factors that affect the amount of material captured by affinity

capture gels, and that influence the discrimination between complementary and non-

complementary target. This work focused on:

1) A fractional factorial design to determine effects of different gel formulations on the amount

of target captured. The design matrix for this analysis was the same as used previously to

examine the amount of probe incorporated into the polyacrylamide gel matrix. The effects were

measured as the relative difference between the concentration of target injected and

concentration of target retained.

2) The discrimination between complementary and non-complementary target on the affinity

capture gel. This was examined at different stringency conditions (temperature, and increasing

formamide content in buffer) using a 3 level 2 factor factorial experiment. Two responses were

measured: the percent recovery and purity.

108

After a year of work it became obvious that some polyacrylamide gels failed either right after

polymerization or during the pre-conditioning step. Failure was observed in the form of voids

inside the polyacrylamide gel (gel did not fill continuously inside the capillary), which prevented

a current from being carried across the capillary. It was also noted that even if gels did not fail

immediately, discontinuities in the gel developed following 1-2 hours of use. These mechanical

failures of gels were observed to be more frequent for gels made with the higher monomer

concentrations used in the factorial experiment (about 50% of the time) than in lower monomer

concentration gels (less than 10%). The amide group of polyacrylamide chains is hydrolytically

unstable above pH 7; hydrolysis of the amine group forms charged carboxylic acid groups along

the polyacrylamide, transforming it to polyacrylate. EOF can occur along the charged surface,

causing matrix swelling, distortion and collapse of the gel matrix [224, 225]. Additionally,

volume changes during the gelation phase can also lead to shrinkage of the gel versus the

original volume of the pre-polymerization mixture, resulting in voids in the gel matrix [226,

227].

Mechanical failure was not encountered for gels made with linear polyacrylamide (no

crosslinker). Therefore, the factorial experiment to examine stringency conditions was

conducted using 12.5% T linear polyacrylamide gels. Factors identified as significant in

adjustment of stringency conditions were expected to be largely independent of gel formulation,

with the results being indicative of other gel formulations.

Table 3.8 summarizes the factors identified as significant (at 95% confidence) and whether a

positive or negative effect was observed. Increases in monomer and crosslinker content

increased the concentration of the target injected. Increasing monomer level increased the

quantity of target captured by the gel. A negative effect was observed for increasing the amount

of crosslinker and concentration of TEMED. Negative interaction effects were also observed

between the monomer and crosslinker content and monomer content and concentration of APS.

The use of higher temperature and increasing formamide content increased the stringency and

improved the purity of the sample that was recovered from the capillary affinity capture gel.

109

Table 3.8: Summary factors which were identified as significant from factorial analysis (95% confidence). The (+) and (-) after each factor denotes whether the effect was positive or negative. Response Measured:

Concentration of target Injected

(Appendix F2)

Amount of Target

Captured (Appendix F3)

Percent Recovery (Appendix

G2)

Percent Purity (Appendix G3)

Total Monomer (+)

Total Monomer (+)

None Formamide (+)

Crosslinker content (+)

Crosslinker content (-)

Temperature x Formamide (+)

Probe (+) Probe (+)

TEMED (-)

Total Monomer x APS (-)

Significant Factors:

Total Monomer x Crosslinker

content (-)

3.5.6 Affinity Capture of Complementary Targets with Probes that are Immobilized in 3D Gel Supports

The immobilization of probes into a 3D gel matrix for DNA diagnostics has been reported and

studied extensively by Mirzabekov et al., and termed as MAGIChip (Microarrays of Gel-

immobilized Compounds on a chip). In the MAGIChip technology, pads of polyacrylamide gels

containing oligonucleotide probes that have been covalently incorporated are spotted onto a solid

substrate surface with dimensions of approximately 100 µm x 100 µm x 20 µm (0.2 nL) [228].

The MAGIChip has been used for discrimination of duplexes and mismatched 126 nt targets

using immobilized oligonucleotide probes of 17-26 nt length [204, 229–233].

The suspension of oligonucleotide probes inside the 3D gel matrix allows the probes to be

spaced out from one another. This may alleviate issues of steric hindrance associated with

immobilization of oligonucleotide probes on a 2D interface. Hybridization kinetics inside 3D

gels have been observed to be similar to that of solution phase hybridization [204, 231–233].

The method for discrimination of complementary and non-complementary targets used with the

affinity capture gel in this thesis was that used in the MAGIChip system. In the MAGIChip

system, the gel pads are first incubated with solutions containing DNA targets. During this step,

DNA targets move through the gel by diffusion, with some delay due to interactions with the

immobilized probe. This increases the time required for the targets to move into and saturate the

gel pads, given by τH,:

110

sol

DHKh

Km

+=

1ττ (5)

where τD is the characteristic time for the targets to move into the gel by diffusion alone, m is the

immobilized probe concentration, K is the thermodynamic association constant and hsol is the

target solution concentration. This process responsible for slowing the time required to saturate

the gel pads is termed "retarded diffusion".

Following this step, the gel pads are washed and incubated with buffer, and the targets diffuse

out of the gel pads. The rate at which the DNA targets diffuses out of the gel is governed by the

degree of interaction between the DNA targets and immobilized probe. The loss of signal J(t) as

targets are removed from the capture gel by diffusion at time t is described by:

W

t

oeJtJτ

−

≅)( (6)

where Jo is the initial fluorescence signal following reaching hybridization at thermodynamic

equilibrium of hybridization, τw is the characteristic washing time for the target to wash off the

probe immobilized gel:

KmDW ττ = (7)

where τD is the characteristic time of the target by diffusion alone, m is the probe concentration

and K is the association constant.

From Equation (6), discrimination between complementary and mismatch/non-complementary

targets is achieved based on the difference in the association constant between perfect and

mismatch/non-complementary targets [204, 229–233]. Targets that form perfect duplexes are

delayed to a larger degree versus those which form non-perfect duplexes. The ratio between

fluorescence intensity of perfect duplexes and mismatches increases as a function of time.

Increasing stringency conditions during the wash step (increase wash temperature, denaturant)

can reduce the total time needed to discriminate between complementary and mismatched targets

[229, 234].

111

In our system, DNA targets are moved through the affinity capture gel by electrophoresis, and

the time required for targets to move through the gel is based on its electrophoretic mobility,

which can be modified by the presence of the immobilized probe given by Equation (4) (Section

3.0.9).

Figure 3.40 demonstrates such a difference in rates by tracking the fluorescence signal of

labelled complementary target in a gel containing complementary and non-complementary

probes for the gel system used in this work. Here, an average fluorescence intensity was

calculated from quantitative values generated from profile plots from confocal microscope

images taken of the capillary at different times during the washing process. This tracks the rate

at which the complementary target elutes off the capillary and how the immobilized probe affects

such rate. These results demonstrate that the probe immobilized gel system presented in our

thesis follows Equation (6), and behaves similarly to the MAGIChip 3D gel system.

y = 0.947e-0.0201x

R2 = 0.9803

y = 1.0062e-0.0409x

R2 = 0.9966

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50 60 70 80

Time (min)

No

rmali

zed

Flu

ore

scen

ce (

AU

)

complementary probe non-complementary probe

Figure 3.40: Decrease in average fluorescence intensity as labelled DNA targets wash out of the

capillary following injection of the DNA target into affinity capture gels containing complementary (�) and non-complementary (x) probe. Fluorescence intensity is normalized against the initial fluorescence

intensity. Experimental conditions: Affinity capture gel: 10%T, 5%C, 2 µM β-actin probe, 2 µM non-β-actin probe.

Injection conditions: 10 µL, 250 nM Cy5- 50 nt target, electrokinetic injection at 133 Vcm-1

for 20 minutes. Incubation Time: 5 mins. Wash conditions: 133 Vcm

-1 at 25 °C with 1xTBE/PVP. The data was obtained

from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars represent 1 standard deviation of three trials.

The Mathies group has also used similar affinity capture gel chemistry inside an integrated

microfluidic device for the purification of PCR amplicons for downstream sequencing [96]. The

112

capture gel chamber used in their microfluidic device was 1 mm x 1 mm, and samples were

delivered into the chamber via a 100 µm wide channel.

In their system, Mathies demonstrated the dynamic capture of DNA target as it was introduced

electrokinetically into the capture gel by optimizing the electric field and hybridization

temperature. The change in free DNA (S) is governed by the on/off rate of the duplex formed

between the target and immobilized probe as well as the electrophoretic velocity of the free DNA

target by electrophoresis [96]:

),()(]:[]][[),( txSx

xECSkCSktxSt

sbf∂

∂−+−=

∂

∂µ (8)

where S is the concentration of free ssDNA, C is the concentration of the immobilized probe, µ is

the mobility of the DNA, E(x) is the field strength at position x and kf and kb are the association

and dissociation rate constants, respectively.

Binding of DNA targets was maximized by changing temperature such that the association

constant was maximized relative to the dissociation constant while not exceeding the melt

temperature. Electrokinetic stacking was also used to increase the concentration of DNA targets

during injection into the affinity capture gel. The geometry of the affinity capture chamber was

such that the DNA targets experienced a field drop from 600 Vcm-1 to 60 Vcm-1 as the DNA

targets moved from the 100 µm channel into the 1 mm wide capture chamber. This resulted in

electrokinetic stacking of the DNA targets due to a drop in electrophoretic mobility [96].

However, this work only reported the use of the purification method on PCR reaction products,

where the major component was expected to be the target of interest and discrimination with

non-complementary target may not have been of substantial concern.

The next sections of this thesis expand on such earlier studies by Mathies and others. A

systematic examination of different gel formulations on the amount of target captured was

carried out. The influence of gel formulation on the capture of DNA targets has not been

reported in the literature. Differences in gel formulation may 1) change the effect of

electrokinetic stacking during the injection step, 2) change how the oligonucleotide probes are

incorporated, and therefore the accessibility of probes to target for hybridization, and 3) change

how the DNA targets migrate though the gel.

113

3.5.7 Effects of Gel Formulation on the Concentration of Target Injected

Electrokinetic stacking may increase the concentration of the DNA target as it is introduced into

the affinity capture gel. Changes in the gel formulation resulted in changes in the electrophoretic

mobility of the DNA targets through the gel, increasing the concentration of the target injected

into the gel during electrokinetic injection.

The concentration of the dye-labelled 150 nt target injected into the capillary was observed to be

dependent on the gel formulation. Based on the factorial analysis (Appendix F1), the factors

which had a significant effect (95% confidence) were the concentrations of monomer,

crosslinker and oligonucleotide probe.

Figure 3.41 shows the concentration levels for DNA target injected into the affinity capture gel

as a function of different gel formulations. The data of Figure 3.41 show that the concentration

of material injected into the capillary increased as the total amount of monomer and crosslinker

increased.

0

50

100

150

200

250

300

7.5%/1% 7.5%/5% 12.5%/1% 12.5%/5%

Gel Formulation (%T/%C)

Concentr

ation o

f T

arg

et

Inje

cte

d (

nM

)

Probe (0.5 uM) Probe (3 uM)

Figure 3.41: Concentration of Target Injected. Values corrected for differences in fluorescence intensity

as a function of different gel formulations. Affinity capture gel: Conditions as prescribed in factorial design. Injection: 10 µL of 170 nM (low probe) 500 nM (high probe) target. Electrokinetic injection at 133 Vcm

-1 for 20 minutes. The data was obtained

from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence

intensity to concentration using a calibration curve.

114

The positive effect observed for increased oligonucleotide probe loading was due to the sample

containing a larger concentration of target; the target-to-probe ratio was adjusted to be the same

at the different probe concentrations that were examined.

Figure 3.42 shows the difference in electrophoretic mobility of the DNA targets through the

different gel formulations, demonstrating a correlation between electrophoretic mobility and the

quantity of target that was injected. A larger difference in electrophoretic mobility between

DNA target in solution and gel would stack the targets, resulting in an increase in the

concentration.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

7.5%/1% 7.5%/5% 12.5%/1% 12.5%/5%


DN

A M

obili

ty (

x 1

04 c

m2/V

s)

Figure 3.42: Electrophoretic mobility of 150 bp target using different gel filled capillaries with different gel formulations. Gel formulations used were as previously prescribed, with radical initiator concentrations of

TEMED/APS of 10%/10%. Mobility calculated based on the time required to travel 2.6 cm along the capillary.

Injection conditions, 10 µL, 0.5 µM Cy5-150 bp target, 86 Vcm-1

, 15 s. Run conditions, 143 Vcm-1

, 1x TBE/PVP buffer. PMT gain 400 mV. Error bars represent 1 standard deviation of three trials.

The quantity of material introduced into a gel filled capillary by electrokinetic injection, Q, is

given based on the following equation [235]:

)1(2 απµ

−= rCL

tVQ

iiep (9)

where µep is the electrophoretic mobility of the DNA target, Vi is the injection field strength, ti is

the injection time, L is the capillary length, C is the concentration of the target solution, r is the

115

radius of the capillary and α is a correction term to account for the interstitial space, geometric

obstruction factor and volume fraction of electrolyte component of having a gel filled capillary.

Changes in electrophoretic mobility of the analyte will affect the quantity of material introduced

into the capillary. Since the mobility of the targets through the different gel formulations were

not the same, the injection time was adjusted so that enough time was allowed for the DNA

target to move through the entire length of the capillary. This reduced any differences in the

quantity of material injected between the different gel formulations.

3.5.8 Effects of Gel Formulation on the Quantity of Target Captured

Following injection of DNA targets into the gel, unbound material was removed from the

capillary column by 'washing' the capillary with buffer. The affinity capture gel was 'washed' to

remove any unbound or non-complementary material. From the factorial analysis (Appendix

F3), a positive effect was considered significant (95% confidence) for increasing monomer

content, while a negative effect was observed on increasing the amount of crosslinker, TEMED

concentration and an interaction between monomer content and crosslinker and monomer

content and APS concentration.

The response levels for the different gel formulations are presented graphically in Figure 3.43. It

is important to note that the wash time was adjusted based on the difference in electrophoretic

mobility of the targets. This avoided variations that would have occurred as a result of

insufficient time for material to wash off the gel.

The observed results can be due to two possibilities: 1) the accessibility of the oligonucleotide

probes that are incorporated into the gel is different at the different gel formulations, and 2)

changes in the gel structure might affect the conformation of the DNA targets as they migrate

through the porous gel.

116

0

50

100

150

200

250

300

350

400

450

7.5% / 1% 12.5% / 1%


Am

ount

of

Targ

et

Captu

red (

fmol)

Probe (0.5 uM) Probe (3 uM)a)

0

50

100

150

200

250

300

350

400

7.5% / 5% 12.5% / 5%


Am

ount

of

Targ

et

Captu

red (

fmol)

Probe (0.5 uM) Probe (3 uM)b)

Figure 3.43: Amount of material captured by affinity capture gel. Values were corrected for differences

in fluorescence intensity as a function of different gel formulations. Affinity capture gel: Conditions as prescribed in factorial experiment. Injection: 10 µL of 170 nM (low probe) or 500 nM (high probe) Cy5-150 nt target. Electrokinetic injection at 133 Vcm

-1 for 20 minutes.

Incubation time: 5 mins at 10 °C. Wash Step: electrophoresis at 133 Vcm-1

for 25 mins at 10 °C with 1xTBE/PVP. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries

and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.

117

3.5.9 Effects of Polymerization of Polyacrylamide

Polymerization of acrylamide in the presence of crosslinker occurs initially at many nucleation

sites where the monomer material is incorporated into growing centers. These gel centers have a

higher density of crosslinker than the remainder of the polyacrylamide gel. This is due to the

higher reactivity rate of the crosslinker as compared with acrylamide monomer. The increased

crosslinker content creates gel centers which are densely crosslinked [206, 217, 236–238].

Crosslinks are formed between growing polymer centers that are in close proximity as monomer

material is depleted. This second step forms the porous network within the polyacrylamide gel

[205, 217, 237, 239–241].

The dimension of the pore network (average pore size, volume occupied by the pores, pore

surface area) can be influenced by the initial monomer content. For example, gels with a larger

average pore size can be obtained where the initial monomer concentration or radicals is low. A

smaller number of growth centers is generated, and the centers are allowed to grow to a larger

size prior to being crosslinked to one another. This results in a pore network with a larger

average pore size. This is shown schematically in Figure 3.44(a). Conversely, gels with smaller

average pore size can be obtained when the number of growth centers is increased (i.e.

increasing monomer or radical concentration). The growth centers are smaller when crosslinked

together, resulting in a pore network with smaller average pore size. This is shown

schematically in Figure 3.44(b).

It is important to note that polyacrylamide gel consists of a distribution of pores of different

dimensions. Kremer et al. examined the mode, mean and variance of pore sizes formed for

different concentrations of cationic polyacrylamide gels [242]. The initial monomer

concentrations they examined were higher than used in our experiments (20%-30%T, 0.4-

1.2%C). General trends observed were that the mode, mean and variance in pore sizes decreased

with increasing acrylamide and crosslinker concentration. Increasing total monomer

concentration from 20 to 30%T (at 0.3%C) decreased the mode from 7.3 nm to 2.8 nm, mean

pore size 16.8 nm to 10 nm and variance from 400 nm to 169 nm. Increasing crosslinker content

from 0.4 to 1.2%C (at 15%T) showed a decrease from mode 9.9 nm to 5.4 nm, mean 12.4 nm to

6.7 nm and variance from 204 to 64 nm [242].

118

Figure 3.44: Schematic representation of polyacrylamide gel and how oligonucleotide probes are

incorporated into the gel. a) indicates how gels with large pores are formed and b) indicates how gels with small pores are formed. ssDNA with hairpin structures are presented in the center and illustrates the difference between how DNA might move through the different gel structures; a) unhindered via Ogston,

and b) stretched by reptation.

For the affinity capture gels used in this thesis, the oligonucleotide probe may not be accessible

for hybridization with target if these are incorporated inside gel centers. Conversely, if they are

incorporated onto the surface area inside the pore network, then they are available for interaction

with target. Changes in the initial monomer concentration can influence the size of the gel

centers, changing the proportion of oligonucleotide probes present on the surface of the pore

network versus inside the gel centers. This can change the total quantity of probe available for

hybridization.

Additionally, changes in the average pore size can affect how the DNA targets migrate through

the polyacrylamide gel. The mechanism by which DNA targets move through a porous gel is

dependent on the radius of gyration and the pore size. The volume occupied by a strand of DNA

is taken as a sphere with a radius given by the radius of gyration, Rg, of the DNA. The radius of

gyration is the root-mean-square of the distance of the center of mass of the polyelectrolyte and

the individual monomer segments. If Rg is smaller than the pore size of the polyacrylamide gel

(Figure 3.44a), then the DNA moves unhindered through the gel by the Ogston process, where

the gel acts as a sieve and movement is based on the probability that the DNA molecule will find

119

a pore large enough to accommodate its passage through the gel [243–246]. If Rg is larger than

the pore size (Figure 3.44b), then DNA moves by a reptation process, and must weave through

the gel by stretching and relaxing as it moves through the pores [243–246]. Migration of the

ssDNA through the gel by reptation may eliminate any secondary structures, opening up the

target region so that it available for hybridization with the immobilized probe.

In solution, ssDNA can adopt a discrete number of conformations, dependent on temperature, pH

and salt concentration [244, 247]. Most commonly, ssDNA will form intramolecular base pairs,

either with bases in close proximity (short range folding) or with bases further apart on the strand

(long range folding). Short range folding is kinetically favoured and expected to dominate under

all conditions over long range interactions. Short range base pairing also prevents these bases

from participating in long range associations. Additionally, forming long range base pair

interactions will contract the negatively charged DNA molecule, increasing charge repulsion.

Long range interactions also decrease the conformational entropy. At alkaline or low ionic

strength conditions, ssDNA can become unfolded and behave as randomly coiled, flexible

polyelectrolytes [248–250]. The ssDNA may take on more compact forms under high ionic

strength conditions due to charge screening that is provided by the salt [248].

A number of calculated hairpin structures for the 150 nt target was previously shown in Figure

3.19 (Section 3.3). The structures predict that the complementary region of the target participate

in the formation of a hairpin structure. Ogston migration of ssDNA through the polyacrylamide

gel would retain these hairpin structures, leaving the target region unavailable for hybridization.

Conversely, if the pore size of the polyacrylamide gel was smaller than the Rg of the ssDNA, the

ssDNA would extend and weave through the gel. This would linearize the target region, making

it available for hybridization with a consequently higher amount of target captured.

From the results of the factorial experiment, affinity capture gels made with higher monomer

concentrations resulted in a larger quantity of DNA target captured versus gels made with lower

monomer concentrations. The results might be due to a combination of a difference in the

availability of the oligonucleotide probe inside the affinity capture gel and differences in how the

ssDNA targets move; with Ogston migration in gels prepared from lower monomer

concentrations, and by reptation when moving through gels prepared using the higher monomer

concentrations.

120

3.5.10 Effect of Probe Availability As a Function of Gel Formulation

Figure 3.45 presents data about the amount of oligonucleotide captured by hybridization as a

function of different gel formulations.

0

200

400

600

800

1000

1200

12.5%/5% 12.5%/1% 7.5%/5% 7.5%/1%


Am

ount

of

Targ

et

Captu

re (

fmol)

Figure 3.45: Amount of target captured for a 19 nt probe/target pair at different monomer and crosslinker

levels as examined in the factorial analysis. Affinity capture gel: 0.5 µM SMN probe. TEMED and APS used were at 10% w/v and v/v, respectively.

Injection: 10 µL, 0.5 µM Cy5 complementary target. Electrokinetic injection at 133 Vcm-1

for 10 min. Incubation time: 5 mins at 10 °C. Wash step: electrophoresis at 133 Vcm

-1 for 15 min at 10 °C with

1xTBE/PVP. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following

correlation of average fluorescence intensity to concentration using a calibration curve.

The shorter 19 nt target was used to assess whether the availability of oligonucleotide probe

altered with gel formulation. The use of 19 nt target avoided issues of sterics and possible

differences in migration mechanism through the gel in contrast to the 150 nt ssDNA. The

amount of material captured is assumed to reflect the availability of probes that are accessible for

hybridization. No statistically significant differences were observed in the concentration of

target captured for the different gel formulations, suggesting that the amount of probe available

was not altered. Assuming that the quantity of target was sufficient to saturate all available

probes, and that only hybrids remained after washing, then the percentage of probe incorporated

was 40% ± 5% of the original amount of probe in the original pre-polymerization mixture.

121

3.5.11 Effect of Gel Formulation on Migration of DNA Targets

An estimate of the average pore size was attempted using the different gels that were made. The

intention was to establish some evidence about the processes of migration that occurred in the

different gel formulations. The calculation of pore size was based on the method reported by Lo

and Ugaz [251]. The pore size can be estimated by identifying the critical DNA fragment size

where the mechanism of migration through a particular gel transitions from Ogston to reptation

[189, 251, 245]. Under the Ogston regime, the porous gel acts like a sieve, and the plot of log of

mobility (µ) normalized by free solution mobility (µo) varies linearly with gel concentration c

with KR retardation coefficient [217, 244]:

cK R

o

−=

µ

µlog (10)

Figure 3.46 shows the relationship of log(µ/µo) for different gel formulations using known

sequences of various lengths from a dsDNA ladder. Here, the initial linear portion corresponds

to DNA fragments that move through the gel under a Ogston regime. The point where linearity

deviates is considered the point where DNA fragment begins to move through the gel by

reptation. The Rg of the DNA fragment calculated at this point can be used to provide an

estimate for pore size of the gel. Reported values of free solution mobility reported in the

literature ranged from 3.46x10-4 cm2sV-1 to 3.8 x10-4 cm2sV-1 [251–253]. The average of the

values, 3.63 x 10-4 cm2sV-1, was used as the free solution mobility used in the data shown in

Figure 3.46.

122

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0 50 100 150 200 250

Base Number

log(u

/uo)

12.5%/5% 12.5%/1% 7.5%/5% 7.5%/1%

Figure 3.46: log(µ/µo) versus number of bases for DNA fragments from a Low Range DNA Gel ladder

(Fast Ladder) using TOPRO3 in different gel formulations. Capillary Gel: Different gel formulations as described in factorial analysis with 1 µM TOPRO-3. Injection:

10 µL of ladder incubated with 10 µM TOPRO3 (1:1), 5 seconds, 570 Vcm-1

. Run: 93 Vcm-1

in 1xTBE/PVP/1µM TOPRO3. Acquisition settings: PMT Gain 400 mV, sampling rate 1Hz.

The radius of gyration of a polymer can be approximated using equation 11 [252]:

−

−

+

−×=

−p

L

DDD

D

g

D

eL

p

L

p

L

ppLMR 16631

3)(

32

2 (11)

where p is the persistence length (3 nm (ssDNA), 50 nm (dsDNA)) [254][253], LD is the contour

length (0.34 nm/base (dsDNA), 0.43 nm/base (ssDNA)) [251]. Table 3.9 lists the estimated pore

radius for the different gel formulations.

123

Table 3.9: Determination of pore size from the Ogston plot presented in Figure 3.46. The range of DNA fragments where log(µ/µo) deviates from linearity is assumed to be the size range where DNA transitions

from Ogston to reptation. The radius of gyration of the dsDNA fragments was calculated by Eq (11), where persistence length for dsDNA was 50 nm, and contour length of DNA was 0.34 nm/base.

Gel Formulation

(%T/%C)

Fragment range where DNA deviates from Ogston

(bp)

Rg (nm) = Pore radius

12.5%/5% 20-35 1.9-3.4

12.5%/1% 35-50 3.4-4.7

7.5%/5% 75-100 7-9.2

7.5%/1% 100-150 9.2-13.4

The gel concentrations for some of the gel formulations used in this work in a similar range as

reported by other groups. Lo and Ugaz reported a pore radius of 5.57 nm at 12%T/1.3%C and a

radius of 9.6 nm for a gel at 6%T/0.7%C gel [251]. Rousseau also reported pore sizes for gels

higher than 8%T/5%C to be under 10 nm [255]. Tombs examined pore size of gels by

electrophoresis of protein markers through different gels. Based on the diameter calculated by

molecular weight, they reported a pore diameter of 4 nm and 7 nm at 12.5%/5% and 7.5%/5%,

respectively [256].

Pore size has been directly examined by others by means of electron microscopy. Rüchel and

Blank obtained SEM images of the freeze-dried PAAm gels, and reported pore sizes based on

SEM images to be on the order of 2-15 µm for gels in 2.5%T-4%T/5% C gels [257–259]. These

values are several orders of magnitude larger than those determined by electrophoretic methods.

Results from SEM images imply that large particles such as a 130 nm diameter virus particle and

E. coli cells (0.5x2 µm) could migrate through the gels. However, it was demonstrated that these

particles could not migrate through the gel. E. coli could only move through very dilute, liquid

agarose like gels (0.03%). It is theorized that although larger pores are observed under SEM,

they are interconnected by much smaller channels as determined by the electrophoresis results

[259].

The radius of gyration of the 150 nt ssDNA was calculated to be 7.7 nm. At the lower monomer

concentration, the pore size of the gel is larger than the Rg of the ssDNA. This results in the

DNA targets moving through the gel relatively unhindered. This limits the portion of the DNA

where the target region is accessible to the probe. However, at the higher monomer

concentration gels, the pore size is less than the DNA, meaning that a larger proportion of the

124

ssDNA targets must reptate through the gel, eliminating secondary structure and increasing the

availability of the target region for hybridization.

A compromise takes place between availability of the target region for hybridization and the

quantity of DNA that can migrate through the gel. As previously mentioned, increasing

crosslinker content will decrease mode and average pore size of the gel. It is possible that this

can create pores so small that the DNA target no longer can access oligonucleotide probes in that

area of the gel. Additionally, pores might be of such dimensions that only a limited number of

DNA strands can fit inside. Decreasing the size of the pore would further reduce the number of

targets that might enter, reducing the contact of probe with target. This can lead to a decrease in

the amount of target captured for gel concentrations where the crosslinker content was increased.

Additionally, the radical initiator can also influence the final polyacrylamide gel structure [216,

217]. It was noted from the factorial analysis that a negative effect was observed for increasing

TEMED concentration as well as an interaction effect between monomer concentration and APS

was present. TEMED catalyzes the generation of radicals with APS and enhances the

polymerization rate by a factor of 3 [216–218]. Increasing the number of radicals present in the

pre-polymer solution can initiate a larger number of such growing radical centers during the

initial polymerization phase, resulting in gels with smaller average pore size, and may further

limits accessibility of the DNA target to regions of the gel.

Relative changes in probe density for the different gels can be calculated based on the

accessibility of 19 nt target and the average pore size. These relative changes are listed in Table

3.10. The calculation was done based on the number of pores that can fit in a cube of volume

with sides 1000 x 1000 x 1000 nm cube for a gel of the lowest monomer concentration

(7.5%T/1%C). The number of pores was determined assuming that they were packed side by

side in the cube. Probe density was calculated based on the amount of probe available from data

obtained for 19 nt oligonucleotide target binding from Figure 3.45.

125

Table 3.10: Probe density based on 0.5 µM probe in the original monomer solution. Values were calculated based on the number of pores that could fit in a 1000x1000x1000 nm cube at the lowest gel

formulation (7.5 %T/1 %C). The calculation assumes that the pore volume for the remaining gel formulation was the same as the lowest monomer concentration and number of pores for the remaining

gel formulations were calculated as such. The amount of probe was determined based on previous experiments examining the percentage of probe incorporated, and the accessibility of the probe from

experiments performed with 19 nt targets in Figure 3.45.

Gel Formulation

(%T/%C)

Number of Pores (x10

5)

Volume total (x10

8

nm3)

Surface Area Total (x10

8

nm2)

Probe density( x

10-21

nmoles/nm

2)

Ratio probe

density (versus

7.5%/0.1%)

7.5%/1% 0.52-1.6 5.2 1.2 - 1.7 1.81-3.24 1

7.5%/5% 1.6-3.6 5.2 1.7 - 2.2 3.24-4.48 0.69-0.76

12.5%/1% 12-32 5.2 3.3 - 4.6 6.67-8.77 0.35-0.37

12.5%/5% 32-180 5.2 4.6 - 8.3 8.77-12.8 0.21-0.25

For the other gel formulations, the number of pores present in the gel was calculated assuming

they occupied the same volume as the lowest gel concentration. For example, the volume

occupied by the pores was 5.2x108 nm3 at 7.5%T/1%C. It was assumed that the pores in the

7.5%T/5%C gel would occupy the same volume, and the number of pores was the pore volume

divided by the volume of each pore. Surface area and probe density for the other gel

formulations were determined assuming that the total pore volume was the same for all gel

formulations. Plieva reported a 7% decrease in pore volume for the same magnitude increase in

monomer concentration due to water vapour uptake [260]. The values of probe density therefore

represent the maximum probe density. Since the pore volume decreases with increasing

monomer concentration, it is expected that the actual probe density is lower than that calculated.

Based on the relative changes in probe density, gels with smaller pores result in larger total

surface areas, but the probe density decreases for increasing gel concentration, suggesting that

higher gel concentrations should lead to a decrease in amount of target captured. However, as

shown in Figure 3.43 an increase in the concentration of target captured was observed for the

higher monomer concentrations. This results suggests the importance of forcing ssDNA strands

to migrate by reptation, opening the target region for interaction with the immobilized probes

and thereby increasing the amount of material retained by the affinity capture gel.

3.5.12 Effect of Stringency Conditions on Percent Recovery and Purity during Washing Step

Following injection of target into the affinity capture gel, discrimination between complementary

and non-complementary targets occurred during the washing step. Here, targets inside the

126

affinity capture gel are moved out of the capillary by electrophoresis. Targets which interact

with the immobilized probe experience a modification of electrophoretic mobility as indicated by

Equation (4) (Section 3.0.9), and will take longer to move through the capillary compared to

targets which do not interact with the probe. Over time, the ratio of the amount of

complementary to non-complementary target remaining inside the capillary should increase. The

rate at which this increase occurs can be modified by changing the stringency conditions of this

washing step. Increasing stringency can decrease the time required to achieve a defined

signal/noise ratio [229].

The effect of increasing stringency by increasing temperature and addition of formamide into the

buffer on percent recovery and purity was examined using a three level factorial experiment.

These experiments were done using target solutions containing complementary and non-

complementary targets. The data from the factorial analysis (Appendix G3) confirmed that

increasing formamide content improved purity, and that there was an interaction effect between

formamide and temperature.

Figure 3.47 shows the percent recovery and percent purity for mixtures of targets as a function of

different temperature and formamide content. An improvement in percent purity as formamide

and temperature was increased was due to improvement of removal of non-complementary

material while complementary targets were retained.

Temperature can effect the dissociation/association rate of the DNA duplex and the stability of

the DNA duplex. The dissociation/association rates both increase with temperature [96, 229].

Glazer et al. examined association rates (ka) and dissociation rates (kd) at 22 and 45 °C for

perfect duplexes and a mismatch (20 nt). In this work the values of ka were similar for both the

complementary and the mismatched target, and increased by a factor of 3 at the higher

temperature. The kd of the mismatched target was larger than the complementary target by a

factor of 2, and both increased by a factor of 24 with increasing temperature [261]. Bishop et al.

developed a mathematical model based on the finite element method, and demonstrated that the

association constant increased by an order of magnitude from 320-340 K, while the dissociation

constant changed from 10-10 to 10-4 for the same temperature change for a 20 bp duplex [262].

The goal during the washing step was to maximize recovery by ensuring that the bound

complementary targets did not dissociate from the immobilized probe. It was desirable to keep

127

the dissociation constant low while still being able to discriminate between complementary and

non-complementary target. Therefore, the washing step was also examined at a low temperature

(10 °C). This was thought to reduce dissociation of complementary target, which might provide

a positive effect on recovery. However, from the factorial analysis, temperature alone did not

have a significant effect on recovery.

Temperature and formamide in combination can be used to disrupt the stability of the duplex

formed between target and probe, with formamide forming competing hydrogen bonds with the

DNA targets [263]. Increasing stringency in the capillary would weaken the stability of duplexes,

reducing the interaction between target and probe, allowing for materials to move along the

capillary column.

It has been reported that formamide lowers the melt temperature by 0.63 °C per percent

formamide added [264]. The amounts used in this work therefore results in a drop of 6.3 °C at

10 % v/v and 15.8 °C at 25%, resulting in an expected melt temperature for the complementary

target from 65 °C to 59 °C and 49 °C, respectively. The highest temperature condition during

the wash step was 40 °C. This was still well below the melt temperature for the highest

formamide content that was used so as to minimize the amount of complementary material that

could be lost. Formamide concentrations less than 30% v/v have been shown to not affect

association constants [265].

128

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0%v/v formamide 10%v/v formamide 25%v/v formamide

Recovery

(%

)

10 C 25 C 40 C

0

20

40

60

80

100

120

0% v/v formamide 10% v/v formamide 25%v/v formamide

Purity

(%

)

10 C 25 C 40 C

Figure 3.47: Percent recovery and percent purity of sample following washing of the affinity capture gel.

Data represents the average from the duplicates defined in the factorial design. Affinity capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and

12 nM of non-complementary target. Electrokinetic injection at 181 Vcm-1

for 20 minutes. Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm

-1 for 25 minutes at 10 °C, 25 °C, 40 °C

using 0%, 10% and 25% v/v formamide of 1xTBE/PVP. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries and values were obtained from the profile plot function. Error bars are propagated error following correlation of average fluorescence intensity to


129

Figures 3.48 and 3.49 show the profile plots from confocal microscope images of the affinity

capture gels showing the position of the non-complementary target and complementary target in

the capillary following the wash step at the different stringency conditions of 0%v/v formamide;

10 °C and 25%v/v formamide; 40 °C, respectively. It was observed that some complementary

was lost at the higher stringency, but a larger proportion of non-complementary target was lost in

the same amount of time. As stated previously, discrimination between complementary and non-

complementary targets is based on the degree of interaction between the DNA strand and

immobilized probe. Figure 3.48 shows the efficacy of increasing the stringency condition on the

non-complementary target component. Here, increasing formamide content decreases the degree

of duplex formation with the probe, which allowed the non-complementary target to be removed

from the capillary following the same wash time. Additionally, in figure 3.49, it can be observed

that the complementary target is still retained in the capillary even after increasing formamide

content. However, some complementary material was removed and the plug representing the

complementary material has moved forward through the capillary. This suggests an effect of

formamide on the complementary target, where it has disrupted some base pair formation and the

decrease in mobility is not as large.

In this experiment, the non-complementary target was longer (500 nt) than the complementary

target (150 nt). Therefore, the expected mobility was slower than the 150 nt complementary

target. The measured mobility of the non-complementary target was 50 ± 3 µm/s, and that for

the 150 nt target was 69 ± 1 µm/s through an unmodified polyacrylamide gel of the same

monomer concentration.

The change in mobility of the DNA targets through the affinity capture gel versus mobility

through unmodified gel is related to the degree of interaction between complementary and non-

complementary targets. The mobility of targets was calculated by observing the centre of the

fluorescence peak along the capillary and the time applied during the wash step. The mobility

was calculated as 9.4 µm/s for the complementary target and 12 µm/s for the non-complementary

target. From Equation (4), the Ka was calculated as 6.3x106 M-1 and 3x106 M-1 for the

complementary and non-complementary target, respectively. This was calculated for the lowest

stringency condition.

130

The conclusions from the data presented in Section 3.5 are that a larger amount of target was

captured by gels that were made with higher acrylamide monomer concentrations due to the

ssDNA target moving through the gel by reputation. The gel formulation that showed the

maximum amount of target captured was at 12.5 %T total monomer concentration and 1%

crosslinker content, which is a balance between availability of the target sequence and the

quantity of probe that is accessible in pores. The maximum purity was achieved when the

stringency condition was set at 25%v/v formamide and 40 °C temperature during the wash step.

It is these conditions that were applied in experiments examining the purification of targets of

150, 250 and 400 nt length in the presence of non-complementary target as presented previously

in Section 3.4.

131

0

1000

2000

3000

4000

5000

6000

7000

8000

0 0.5 1 1.5 2 2.5 3 3.5 4

Distance (cm)

Flu

ore

scen

ce In

ten

sit

y (

AU

)

a)

0

500

1000

1500

2000

2500

3000

3500

0 0.5 1 1.5 2 2.5 3 3.5 4

Distance (cm)

Flu

ore

scen

ce In

ten

sit

y (

AU

)

b)

Figure 3.48: Fluorescence profile plots of capillaries taken from the outlet to inlet end generated from

confocal microscope (Chipreader) images tracking the non-complementary target following affinity capture and a subsequent wash step using two different stringency conditions.

Affinity capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and 12 nM of non-complementary target. Electrokinetic injection at 181 Vcm

-1 for 20 minutes. Incubation time: 5

minutes at 10 °C. Washing step: electrophoresis at 181 Vcm-1

for 25 minutes, at (a) 10 °C with 1xTBE/PVP and (b) 40 °C with 25%v/v formamide/1x TBE/PVP.

132

0

2000

4000

6000

8000

10000

12000

14000

0 0.5 1 1.5 2 2.5 3 3.5 4

Distance (cm)

Flu

oro

scen

ce In

ten

sit

y (

AU

)

a)

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 0.5 1 1.5 2 2.5 3 3.5 4

Distance (cm)

Flu

ore

scen

ce In

ten

sit

y (

AU

)

b)

Figure 3.49: Profile plots of capillaries from the outlet to inlet end from confocal microscope (Chipreader) images of the complementary target following affinity capture the wash step in the affinity capture and a

subsequent wash step using two different stringency conditions. Affinity capture gel: 12.5% AAm, 1 µM β-actin probe. Injection: 10 µL of 136 nM 150 nt target and 12 nM of non-complementary target.

Electrokinetic injection at 181 Vcm-1

for 20 minutes. Incubation time: 5 minutes at 10 °C. Washing step: electrophoresis at 181 Vcm

-1 for 25 minutes, at (a) 10 °C with 1xTBE/PVP and (b) 40 °C with 25%v/v

formamide/1x TBE/PVP.

133

3.6 Selective Concentrating of Oligonucleotide Targets by Step Elution From Affinity Gels

An examination was completed of the potential for collecting and concentrating the captured

targets from affinity gels by step elution using heating. In previous work, captured targets were

eluted by heating the entire capillary column above the melt temperature of hybrids during

electrophoresis. The target was released from the affinity gel column in a volume at least equal

to the geometric volume of the capillary, i.e. a relatively large volume. The interest in the use of

step-elution was to stack targets into a smaller volume than that of the capillary, thereby

increasing the concentration of the targets on elution from the capillary.

In step elution, only one portion of the capillary is heated at a time during electrophoresis.

Hybridized targets in the heated region denature and the single-stranded material that is released

from the gel collectively moves in the affinity capture gel under the electric field. The heater is

then advanced along the capillary, sequentially denaturing any hybridized targets in a new region

of the capillary while previously denatured targets are carried along by electrophoresis. By

appropriately matching the rate at which the resistive heater was moved across the capillary to

the mobility of the oligonucleotide targets, the eluting targets could be stacked into a smaller

volume than that of the affinity capture capillary, increasing the concentration of the targets.

Figure 3.50(a) presents data that represents the elution of captured oligonucleotide targets from

an affinity capture gel by step elution. The electrophoretogram tracks the fluorescence intensity

of the Cy5-labelled targets through a window in the capillary column near the elution end as a

function of time. The peak that was observed is indicative of the stacked oligonucleotide targets

eluting from the capillary column. Figure 3.50(b) represents a control experiment using identical

elution conditions, for a gel that made use of non-complementary probes. The complementary

probe used in these experiments was for the SMN sequence and the non-complementary probe

was the β-actin probe.

Table 3.11 summarizes the results of the concentrating effect of step elution as a function of the

length of the eluted gel, i.e., the length of the capillary that was subjected to the step elution

process. The integrated peak area, peak height and peak width of the eluting peak was calculated

using Origin Pro 8.0. The integrated peak area represents the quantity of material eluted during

the stacking experiment. The volume of the eluted material was calculated from the peak width

134

and mobility of the oligonucleotide target. The enhancement factor was calculated as the

quantity of material eluted (peak area) contained in the volume of the eluting peak divided by the

concentration of the same material eluted in the original volume for the length of capillary

eluted. Typically, the enhancement factor is reported as the ratio of peak heights after

amplification and before amplification. However, these experiments were conducted using a low

concentration (50 nM) of probe and target. This was due to saturation of the PMT detector by

the peak of enriched material eluting off the capillary without the use of neutral density filters

and lowering sensitivity. At the same instrument setting, no signal above background noise was

observed from dye-labelled targets captured on the capillary prior to step elution. Rather than

correcting for the signal by developing an instrument correction factor, enhancement ratio was

calculated using peak area, and would minimize the introduction of additional sources of error.

135

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0 50 100 150 200 250

Time (s)

Flu

ore

scence I

nte

nsity (

AU

)

a)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0 50 100 150 200 250 300 350 400 450 500

Time (s)

Flu

ore

scence I

nte

nsity (

AU

)

b)

Figure 3.50: Electrophoretograms comparing the relative fluorescence intensities (concentrations) of short oligonucleotide target by step elution from complementary and non-complementary probes. a)

Affinity capture gel: 50 nM SMN probe, 10% LAAm. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150 Vcm

-1. b) Affinity capture gel: 50 nM β-actin probe, 10% LAAm. Target injection: 10 µL 5 µM

Cy5-SMN target for 1 min at 150 Vcm-1

. Capture step: 10 min, 1xTBE/PVP running buffer, 150 Vcm-1

. Concentrating step: coverage length: 25 mm; step size: 250 µm; step rate: 86 µms

-1; Voltage: 96 Vcm

-1;

Acquisition settings: ND 4, 8 and 16 filters, PMT gain 400 mV. Sampling rate: 10 Hz.

136

Table 3.11: Summary of results of the concentrating effect of step elution of complementary target as a function of elution length. Volume of the eluting targets was calculated based on the peak width and

mobility of the oligonucleotide, which was 86 µms-1

at 96 Vcm-1

. Error represent 1 standard deviation of three trials.

Affinity capture gel: 50 nM SMN probe, 10% LAAm affinity capture gel. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150 Vcm

-1. Capture: electrophoresis for 10 min at 150 Vcm

-1 in 1x TBE/PVP

running buffer. Step elution for concentrating effect: coverage length: 12.5, 25 and 37.5 mm; step size: 250 µm; step rate: 86 µms

-1; Voltage: 96 Vcm

-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain

400 mV. Sampling rate: 10 Hz

Step Elution Length

Volume of

Capillary Eluted

(nL)

Peak Area (AUs)

Peak Width at half max

height (s)

Peak Height (AU)

Volume (nL)

(based on peak width

and mobility)

Concentration of eluted targets in original volume (AU/nL)

x10-3

Conc. of stacked targets (AU/nL)

Enhance-ment Factor

12.5 mm

98.5 0.32 ± 0.07

6.27 ± 0.98

0.047 ± 0.01

4.2 ± 0.9 3.2 ± 0.7 0.08 ± 0.01

24 ± 4

25 mm 197 0.58 ± 0.11

4.15 ± 0.63

0.13 ± 0.03

2.8 ± 0.6 2.9 ± 0.6 0.21 ± 0.06

71 ± 12

37.5 mm

296 0.45 ± 0.26

1.24 ± 0.2

0.5 ± 0.3

0.84 ± 0.43 2.0 ± 0.9 0.81 ± 0.50

393 ± 73

Examining the data in Table 3.11, it can be noted that the peak widths increased for decreasing

elution lengths. Peak widths should increase as the eluting targets move through the capillary

due to longitudinal band broadening. However, as shown schematically in Figure 3.51a, the

experiment was set up so that the eluting targets travelled the same distance for the different

elution lengths examined; the final peak widths were expected to be similar if longitudinal

diffusion was the major contributor to band broadening. Therefore, the data indicates that

application of the heating element also affects band broadening of the oligonucleotide targets as

they travel through the capillary column.

The application of the heating element increases the temperature of the gel at one region of the

capillary (effective heating zone of approximately 0.8 mm). Increasing the temperature of the

gel increases the viscosity of the gel in that area and creates a temporary local discontinuity

where the mobility of the oligonucleotide targets was higher in that region of the capillary [266–

268]. Once past the heated region, the eluted target band would slow as viscosity of the solution

increased. The consequence was that the targets stacked along one side of the heated zone

boundary, and this effect was anticipated to reduce some of the dilution caused by longitudinal

broadening [171–173]. Further band broadening occurred as the targets moved through the

capillary to the detector after step elution, diluting the concentrated targets.

137

Figure 3.51: Schematic diagram of different permutations of the step elution sweeps where a) the resistive heating element was started at the same point at the injection end of the capillary, and the

terminal position was varied. In b), the step elution again swept through different distances, but stopped at the same position along the capillary.

In the case of the shortest elution length (12.5 mm), the eluted targets travelled the longest from

the end of step elution to the detector when compared to the longer elution lengths. This

subjected the eluting targets to the largest degree of band broadening, resulting in the largest

peak width for the shortest elution length.

It may also be possible that stacking the oligonucleotide targets into a small contained volume on

the capillary can create its own localized field similar to what occurs in isotachophoresis. In ITP,

targets with similar mobilities stack in a concentrated zone and become the charge carrier in that

region of the capillary, and a localized field is develops in the zone [172, 173]. However, in ITP,

the analyte is prepared such that no background electrolyte is present and the analytes serve as

the charge carrier in the electrophoresis experiment and plays a role in determining the local field

strength of the channel. In our experiment, the concentration of the 1x TBE buffer used is 90

mM Tris, the maximum concentration of the stacked peak is 20 µM, which means that the charge

carrier during electrophoresis is still the 1x TBE buffer.

138

Table 3.12 summarizes the results for the step elution experiments where the resistive heating

element was stopped at the same position along the capillary for all lengths that were thermally

scanned (depicted in Figure 3.51(b)). The eluted targets travelled the same distance to the

detector after step elution in all three cases that are shown in Figure 3.51(b), eliminating

differences in band broadening that occurred outside of the heated zone.

Table 3.12: Summary of results from step elution of complementary target as determined from data obtained from the experiment depicted in Figure 3.51b. Results for integrated Peak Area, Width, Height

were calculated using Origin Pro 8.0 Volume of the eluting targets was calculated based on the peak width and mobility of the oligonucleotide, which was 86 µms

-1 at 96 Vcm

-1. Error represent 1 standard

deviation of three trials. Affinity capture gel: 50 nM SMN probe, 10% LAAm affinity capture gel. Target injection: 10 µL 50 nM

Cy5-SMN target for 1 min at 150 Vcm-1

. Capture: electrophoresis for 10 min at 150 Vcm-1

in 1x TBE/PVP running buffer. Step elution for concentrating effect: coverage length: 12.5, 25 and 37.5 mm; step size: 250 µm; step rate: 86 µms

-1; Voltage: 96 Vcm

-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain

400 mV. Sampling rate: 10 Hz

Step Elution Length

Peak Area (AUs)

Peak Width at half max

height (s)

Peak Height (AU)

Volume of Peak (nL)

Concentration of eluted targets in original volume (AU/nL)

X10-3

Conc. of stacked targets (AU/nL)

Enhancement Factor

12.5 mm

0.17 ± 0.05

0.37 ± 0.09

0.40 ± 0.09

0.25 ± 0.03

1.7 ± 0.5 0.52 ± 0.12

300 ± 29

25 mm 0.34 ± 0.07

1.2 ± 0.5

0.29 ± 0.10

0.81 ± 0.45

1.7 ± 0.3 0.36 ± 0.13

224 ± 128

37.5 mm

0.52 ± 0.39

0.46 ± 0.34

0.31 ± 0.36

0.93 ± 0.41

1.7 ± 1.3 0.59 ± 0.42

419 ± 205

The trend of peak widths observed in Table 3.12 is reversed from that observed in Table 3.11.

The shortest step elution length resulted in the least broadening. By changing the starting

position of the resistive heater along the capillary, the total capillary column processing time was

shortest for the shortest elution length. The reduction of time on the column minimized

opportunity for band broadening.

The enhancement factors reported in Table 3.12 showed no obvious trend. The enhancement

factor was not statistically different at 95% confidence for the different elution lengths that were

examined. This might be due to variations in matching of the electrophoretic mobility of the

oligonucleotide and the scanning rate of the external heater; an effect that would become more

significant when using longer capillary lengths. The shortest elution length (12.5 mm) provided

the best precision and reproducibility in enhancement factor and was used in the remainder of the

experiments. The compromise would be that use of a shorter region of elution would lower the

quantity of target that would be accessible. Experiments performed examining a range of step

139

sizes (125 to 500 µm) showed no difference on the results (peak width, area, height) of the step

elution process (Appendix H).

3.6.1 Concentrating the 150 nt, 250 nt and 400 nt Targets by Step Elution

Tables 3.13 to 3.15 present the percentage Recovery and Purity that was achieved for samples

containing 150 nt, 250 nt, 400 nt targets and 500 nt non-complementary target processed by

affinity capillary gel electrophoresis and concentrating by step elution. These experiments were

done using the same conditions as outlined in Section 3.4. The percentage Recovery and Purity

were calculated based on the amount of complementary and non-complementary target in the

eluting peak on the capillary. The results were obtained offline versus in real time since two

targets (complementary and non-complementary) were measured and it was not possible to

measure Cy3 and Cy5 channels concurrently in real-time.

Table 3.13: Summary results for Recovery and Purity from affinity capture gel for mixtures containing varying amounts of 150 nt complementary and 1.5 pmol non-complementary targets. The Recovery and

Purity were calculated from quantitative concentration data for the eluting peak by use of calibration curves. Errors represent propagated error following correlation of average fluorescence intensity to

concentration using a calibration curve. Affinity capture gel: 3 µM β-actin probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic injection for 20 min at 133 Vcm

-1. Incubation time 5 min. Wash step: electrophoresis for 25 min at 133 Vcm

-1, 45 °C,

with 25% v/v formamide/1X TBE/PVP. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms

-1; Voltage: 96 Vcm

-1; Acquisition settings (Cy5): PMT gain 500 mV, translation speed:

50 µms-1

, scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV, Pinhole: 60 µm, 1 FPS Original Target

Solution By Selective Concentration

Amount of 150 nt

Complementary Target (fmol)

Recovery (%)

Purity (%)

Amount of Complementary target captured

(amol)

Amount of Non-

complementary material

captured (fmol)

Recovery (%)

Purity (%)

Enhance-ment

10 100 0.8 106 ± 16 1.4 ± 0.3 1.0 ± 0.2 6.9 ± 1.8

10 ± 2

5 100 0.4 68 ± 5 1.4 ± 0.3 1.4 ± 0.1 4.7 ± 1.1

14 ± 1

1 100 0.08 1.39 ± 7.4 1.4 ± 0.3 0.1 ± 0.7 0.1 ± 0.5

1.5 ± 8

140

Table 3.14: Summary results for Recovery and Purity for mixture containing varying amounts of 250 nt of complementary and 1.5 pmol non-complementary targets by affinity capture gel. Errors represent propagated error following correlation of average fluorescence intensity to concentration using a

calibration curve. Affinity capture gel: 3 µM β-actin probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic injection for 30 min at 133 Vcm


-1, 45 °C,

with 25% v/v formamide/1X TBE/PVP. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 66 µms

-1; Voltage: 96 Vcm

-1; Acquisition settings (Cy5): PMT gain 500 mV, translation speed:

50 µms-1



Amount of 250 nt


Recovery (%)

Purity (%)


(amol)

Amount of Non-


captured (fmol)

Recovery (%)

Purity (%)

Enhance-ment

100 100 7 690 ± 96 0.88 ±0.2 0.7±0.1 44 ± 9 7 ± 1

50 100 3.6 290 ± 38 0.88 ±0.2 0.43 ±0.8 20 ± 5 6 ± 1

10 100 0.8 100 ± 42 0.88 ±0.2 1.0 ±0.4 10.2 ±

4.8 15 ± 6


complementary targets by affinity capture gel. Errors represent propagated error following correlation of average fluorescence intensity to concentration using a calibration curve.

Affinity capture gel: 3 µM uidA probe, 12.5 %T, 1 %C. Capture conditions: electrokinetic injection for 40 min at 133 Vcm


-1, 45 °C, with

25% v/v formamide/1X TBE/PVP. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 52 µms

-1; Voltage: 96 Vcm

-1; Acquisition settings (Cy5): PMT gain 500 mV, translation speed: 50

µms-1



Amount of 400 nt


Recovery (%)

Purity (%)


(amol)

Amount of Non-


captured (fmol)

Recovery (%)

Purity (%)

Enhance-ment

50 100 3.6 373 ± 44 1.0 ± 0.2 0.75 ± 0.09

25.7 ± 4.4

8 ± 1

10 100 0.8 48 ± 16 1.0 ± 0.2 0.48 ± 0.16

4.3 ± 1.6

7 ± 2

1 100 0.08 0.26 ± 0.30 1.0 ± 0.2 0.03 ± 0.03

0.02 ± 0.03

0.4 ± 0.4

From the results presented in this section, it was demonstrated that concentrating the 19 nt

oligonucleotide targets by step elution provides an enhancement factor of 300 ± 29 at a step

elution length of 12.5 mm as the targets elute off the capillary. Additionally, band broadening is

minimized when the targets are eluting under the heating element, due to changes in mobility as

a function of gel viscosity and the development of a local field strength as the targets are stacked.

Based on the results presented in Tables 3.13-3.15, the lowest quantity of material that could be

processed and purified with the affinity capture gel was 1 fmol, 10 fmol and 1 fmol for the 150

nt, 250 nt and 400 nt targets, respectively.

141

In comparison to the results shown previously in Tables 3.3-3.5 in Section 3.4, where the

recovery and purity of the method was calculated where the targets were not concentrated, the

recovery by selective concentrating was observed to be lower, but the purity was improved by a

factor from 6 to 15 by selective concentrating versus selective purification without any

concentrating.

3.7 Delivery of Concentrated Targets into Microfluidic DNA Biosensing Platform

3.7.1 Design Aspects for Sample Transfer from the Capillary to the Microfluidic Biosensing Platform

The affinity capture gel was intended to serve to selectively purify and concentrate

oligonucleotide targets for delivery into a microfluidic-based DNA biosensor that was previously

described by our research group [269][185]. The following experiments made use of a 19 nt

oligonucleotide target, which was the target length for which the microfluidic biosensor had been

designed. Preliminary experiments using 150, 250 and 400 nt DNA sequences that were injected

directly into the microfluidic based biosensing platform did not result in any appreciable

hybridization signal even though the short target sequence was within the longer sequence. This

was thought to be due to folding of the longer DNA targets, which would likely block

hybridization of the short target sequence with probes that were immobilized on the glass

substrate surface.

The microfluidic biosensing platform was modified to include an interconnect for delivery of the

effluent from the affinity capture gel capillary. The interconnect was designed such that the

capillary was positioned orthogonally on top of a microfluidic channel containing pads of

immobilized oligonucleotide probes. This design was chosen over a design where the capillary

would be in-plane to the microfluidic device. Figure 3.52 illustrates the physical challenge for

the interfacing of a capillary in different orientations to the microfluidic channel. Orienting the

capillary in-plane to the microfluidic channel would result in a large difference between the

diameter of the capillary and the height of the microfluidic channel. The height of the

microfluidic channel was 8 µm, while the diameter of the capillary used here was 160 µm (the

smallest diameter of fused silica capillary that was commercially available with an I.D. of 100

µm). An in-plane configuration would physically impede the delivery of material from the

142

capillary column to the microfluidic channel, with challenges including the matching of

volumetric flow and dilution of a stacked elution band at leading and trailing edges. Orienting

the capillary column to be orthogonal to the microfluidic channel eliminated these issues in the

case where the diameter of the delivery channel of the capillary was situated within the width of

the microfluidic channel (185 µm).

In order to create the orthogonal capillary-microfluidic interconnect, an empty piece of fused

silica capillary was positioned using an xyz micromanipulator stage so that the inner diameter of

the capillary was touching the microfluidic channel on the microfluidic template. PDMS was

poured over this setup and cured on a hotplate.

This method of creating an interconnect is similar to other methods reported in the literature.

Other methods typically have been reported for applications using glass-based microfluidics

chips, and involve creation of a hole that will house the capillary in PDMS or in glass [270–273].

The ports are then plugged with a fitting or flange tubing followed by insertion of the capillary

[271, 274, 275]. Some methods also glued the capillary to the interconnect with epoxy-based

resins [272, 273, 276]. The interconnects described in these publications were for connections to

microfluidic devices where fluid was moved through the system by pressure flow. The use of

pressure flow necessitated additional features for the interconnect to be leak-proof, to minimize

dead volume and to withstand pressures from 140 to 2000 kPa [270–274, 277].

143

Figure 3.52: Schematic diagram illustrating two possible orientations for creating an interconnect

between the capillary column and the microfluidic channel. The interconnect can be created by orienting the capillary (a) orthogonal to the microfluidic channel and (b) in-plane with the microfluidic channel. The

area that is shaded in blue represents the filled area of the capillary and microfluidic channel.

For the microfluidic device used in the work of this thesis, analytes were moved by

electrophoresis. Issues associated with leakage caused by pressure flow were not of significant

concern. Dead volume in the system would result in distortion of the eluting plug of

oligonucleotide targets at the interface port. This was minimized by ensuring that the template

capillary and the affinity gel capture capillaries were as flat as possible [270, 271]. Figure 3.53

shows line scans along the channel of the microfluidic device following delivery of

oligonucleotide targets. The fluorescence signal observed represents the hybridization of the

Cy5-labelled 19 nt complementary targets with the immobilized probe on the glass surface

following injection followed by washing of the concentrated target from the capillary into the

microfluidic channel. The absence of a fluorescence signal from labelled oligonucleotides at the

location of the interconnect port indicates that oligonucleotide was not lost in the interconnect

144

region. The experiment was conducted using the setup described in Figure 2.5. Briefly,

complementary 19 nt targets were captured using the affinity capture gel and concentrated by

step elution. Immediately after step elution, the capillary was trimmed to 2 cm such that the

concentrated oligonucleotide was right near the outlet end of the capillary. The capillary was

then inserted into the previously prepared biosensing platform through the interconnect made

during the casting of the PDMS chip. A potential applied across the inlet end of the capillary

and the end of the microfluidic channel delivered the concentrated oligonucleotide targets from

the affinity capture capillary into the microfluidic channel. As the concentrated oligonucleotide

targets move through the microfluidic channel, they hybridize with oligonucleotide probes

imobilized on the surface of the epoxy-modified glass slide. After the experiment was

completed, the fluorescence intensity across the microfluidic channel was scanned using an

epifluorescent microscope (Alpha). The fluorescence intensity observed corresponds to

complementary targets hybridized to the immobilized probe spots.

Figure 3.53: Line scans of the microfluidic channel of the DNA biosensing platform following delivery of

fluorescently-labelled complementary target by selective concentration. Affinity capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL 5 nM A647 SMN target, electrokinetic injection for 1 min at 150 Vcm


-1 with 1x

TBE/PVP running buffer. Concentration step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms

-1; Voltage: 96 Vcm

-1; Delivery of concentrated targets into microfluidic biosensing platform: 500 V,

10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms

-1 scan rate: 50 Hz.

The interconnect was not always functional at the time of assembly of some of the microfluidic

devices, and would fail after repeated use. Failure was typically observed for devices that had

145

been subjected to approximately 6 cycles of delivery of oligonucleotides, hybridization and

regeneration of the microfluidic chip. Confirmation that the interface was at fault was confirmed

by testing for the presence of the immobilized oligonucleotide probes. This was done by

injecting complementary oligonucleotide targets through the reservoir wells, bypassing the

interconnect. Hybridization confirmed that the immobilized oligonucleotide probes were still

present and selective. Imperfect alignment of the capillary with the microfluidic channel during

the casting process and the repetitive insertion and removal of the capillary during replicates may

have resulted in physical damage of the interconnect, most probably introducing debris that

blocked flow and prevented the delivery of stacked oligonucleotides into the microfluidic

channel.

Selective concentrating of the oligonucleotide targets was done within the capillary column

before coupling to the microfluidic platform. This was done since it was found that the mobility

of the oligonucleotide targets through the capillary was more reproducible when the electric field

was applied to the capillary alone, rather than the capillary-to-microfluidic device. Concentrating

the oligonucleotide targets relied on an accurate measure of the mobility of material moving

through the affinity capture gel. Given the manual handling that was required to define the

length of the capillary column, the positioning of the electrodes, and the positioning of the

capillary in the interconnect region, it was not a surprise that there was significant variability in

the distribution of the electric field between the capillary and microfluidic chip when these were

connected.

Since the concentrating of oligonucleotide targets was done on the capillary, it was necessary to

stop the elution while the plug of target remained inside the capillary. A portion of the capillary

was trimmed prior to interfacing with the microfluidic platform to minimize the distance the

eluted targets needed to travel from the capillary to the microfluidic device, thereby minimizing

longitudinal diffusion.

The field strength used in the capillary-microfluidic system was similar to that reported

previously in successful hybridization experiments where hybrids were formed with immobilized

19 nt probe strands in microfluidic channels. The field strength suitable for hybrid formation as

reported by Erickson et al. was in the range of 110 Vcm-1 to 350 Vcm-1. Higher voltages

146

increased stringency conditions due to increased Joule heating and shearing effects, but resulted

in substantial reduction in the extent of hybrid formation [269].

The interconnect coupled a gel filled capillary and a buffer filled microfluidic channel.

Therefore a discontinuous buffer system existed resulting in different local field drops across the

capillary and microfluidic channel. The applied voltage was influenced when the field strength

across the capillary or the microfluidic channel was measured individually. Therefore, the local

field drops across the capillary and microfluidic device were estimated based on resistance.

The voltage that was required to achieve a set current was separately measured across the

capillary and the microfluidic chip. The relative resistance of the capillary in comparison to the

microfluidic channel was determined and the field drop across the two different devices was then

estimated. The resistance of the gel filled capillary was measured to be approximately half of the

resistance of the microfluidic channel. Therefore, the field strength across the capillary and

microfluidic device at a total voltage applied of 500 V was 62 Vcm-1 and 222 Vcm-1,

respectively. The voltage across the microfluidic channel was in a range that was suitable for

hybridization of fully complementary and one base pair mismatch targets [269].

3.7.2 Delivery of Oligonucleotide Targets to the Microfluidic Biosensing Platform by Direct Injection and by Selective Concentration

A comparison was completed of the response of the DNA biosensor to quantities of

complementary targets delivered by direct injection and by selective concentrating using the

capillary column. These experiments were completed to explore whether an improvement in the

response of the biosensor could be achieved by use of the capillary column.

Figure 3.54(a) shows the profile (background subtracted) of the fluorescence intensity along the

microfluidic channel following the introduction of labelled complementary target. The

fluorescence profile is consistent with hybridization with the immobilized oligonucleotide probes

along the microfluidic channel. Figure 3.54(b) demonstrates the selectivity of hybridization by

replacing one of the oligonucleotide probe spots with a non-complementary sequence. The

complementary target was labelled with AlexaFluor 647 (A647) fluorescent dye. The A647 dye

has similar excitation and emission wavelength as Cy5 and was used without introducing any

changes to the experiment.

147

Figure 3.54: Line scans of the fluorescence intensity along a microfluidic channel of the DNA biosensing platform following delivery of complementary target by selective concentration. a) both pad probe spots

are complementary to the SMN target sequence, and b) one probe pad is complementary (SMN) and the second is non-complementary (β-actin probe).

Affinity capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: a) 10 µL 1 nM A647 SMN target, b) 10 µL 1nM A647 SMN target, 1 µM Cy3 β-actin target, electrokinetic injection for 1 min at 150 Vcm


-1 in 1x TBE/PVP running buffer. Concentration

step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms-1

; Voltage: 96 Vcm-1

; Delivery of purified and concentrated targets into microfluidic biosensing platform: 500 V, 10 minutes, 1x TB/PVP/20

mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms-1

scan rate: 50 Hz.

148

Figure 3.55 presents the response of the biosensor to the direct injection of different quantities of

complementary target through the interconnect port. The data was generated from scans

obtained similar to Figure 3.54. Here, the fluorescence intensity signal from one "pad" was

taken into account, and the average intensity of the fluorescence signal as well as the integrated

fluorescence intensity were calculated.

y = 0.053x + 0.0142

R2 = 0.9997

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14 16 18 20

Quantity of Complementary Oligonucleotide Injected (femtomoles)

Avera

ge F

luore

scence I

nte

nsity (

AU

)

a)

y = 2.1087x + 1.041

R2 = 0.9994

0

5

10

15

20

25

30

35

40

45

50

0 2 4 6 8 10 12 14 16 18 20

Quantity of Complementary Oligonucleotide Injected (femtomoles)

Inte

gra

ted F

luore

scence S

ignal (A

U)

b)

Figure 3.55: Response of the microfluidic based DNA biosensing platform to quantities of

complementary target a) average fluorescence intensity signal level and b) integrated fluorescence intensity. Different concentrations of DNA were mixed in 10% LAAm gel and injected into an empty fused silica capillary using a syringe to CE adapter. Delivery of complementary oligonucleotide into microfluidic

biosensor: 500 V, 10 minutes, 1xTB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms

-1, scan rate: 50 Hz. Error bars represent 1 standard deviation of three trials.

Known concentrations of the oligonucleotide targets were mixed with a 10% linear

polyacrylamide solution that was then injected into the same lengths of capillary used in the pre-

concentration experiments (2 cm) using a syringe outfitted with a capillary-to-syringe luer

149

adapter. The complementary targets were then delivered into the biosensor device via the

interconnect port. This approach allowed for the delivery of a known quantity of oligonucleotide

targets to the microfluidic biosensor, with subsequent hybridization under the same experimental

conditions as experienced when delivery was performed by step elution. No measurable response

was observed when quantities lower than 0.98 fmole were injected into the detection system.

Table 3.16: Summary of data for the response of the microfluidic DNA biosensing platform to delivery of complementary targets by selective concentrating using the affinity capture gel. The amount of target

injected into the affinity capture gel by electrokinetic injection from the original target solution is shown in parenthesis. The equivalent quantity was determined based on correlation to the concentration-response

curve of Figure 3.55. The enhancement factor was calculated based on the ratio of the equivalent quantity and quantity of material injected. Errors represent 1 standard of three trials expect for Equivalent Quantity Determined from Calibration Curve, which is propagated error from correlation with calibration

curve. Affinity capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL A647 SMN target,

electrokinetic injection for 1 min at 150 Vcm-1

. Capture: electrophoresis for 10 min at 150 Vcm-1

in 1x TBE/PVP running buffer. Concentrating Step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms

-1; Voltage: 96 Vcm

-1; Delivery of purified and concentrated targets into microfluidic biosensing

platform: 500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms

-1 scan rate: 50 Hz.

Original Target Concentration, and (Quantity

of Target Injected)

Average Fluorescence

Intensity (AU)

Integrated Fluorescence Signal (AU)

Equivalent Quantity

Determined from Calibration

Curve (fmol)

Enhancement Factor

0.1 nM (0.30 ± 0.04 fmol)

0.070 ± 0.01 2.4±0.4 (1.0±0.2, 0.7±0.1)

(3.3±0.8,2.3±0.4)

0.5 nM (1.5 ± 0.2 fmol)

0.13 ± 0.03 5.9±1.3 (2.2±0.5, 2.2±0.5)

(1.5±0.4, 1.5±0.4)

1 nM (3.0 ± 0.3 fmol)

0.81 ± 0.17 35±7 (15±3, 16±3) (5±1,5±1)

Table 3.16 presents the response of the microfluidic biosensing platform following the delivery

of the concentrated oligonucleotide. The values for average fluorescence intensity and integrated

fluorescence were then correlated with the calibration curve in Figure 3.55 and an equivalent

quantity was determined. This value represents the quantity of material needed to achieve the

same response if it was delivered without concentrating.

The enhancement factor is the ratio between the equivalent quantity (determined from the

calibration curve) versus the amount of material that was injected electrokinetically into the

affinity capture gel from the original target solution (in parenthesis). The amount of material

injected was calculated from steady-state fluorescence measurement of solutions of target before

and following electrokinetic injection into the capillary. Based on the results, 31% ± 4% of the

complementary target was injected into the capillary by electrokinetic injection. This value was

obtained using solutions of 10 nM A647-labeled oligonucleotide targets. Note that the

150

percentage of material injected electrokinetically into the affinity capture gel did not appear to be

affected by the concentration of the target solution.

The results indicate that the limit of detection (LOD) of the microfluidic biosensor could be

lowered to 0.1 nM or 0.3 fmol of material by delivery by concentrating the target. Based on the

results in Figure 3.55, the LOD of the biosensor by direct injection of the oligonucleotide targets

was 1 fmol of material. The enhancement affect calculated was 3. This value is lower than the

enhancement factor determined in the previous section.

A discontinuous system exists between the gel filled capillary and buffer filled microfluidic

device since the resistance in the two channels is different. There is a difference in mobility of

the oligonucleotide targets moving through gel filled capillary and the microfluidic channel. The

mobility of the oligonucleotide target through the affinity capture gel was measured at 115 ± 6

µms-1 at a field strength of 93 Vcm-1. The mobility of the oligonucleotide target through the

microfluidic channel was measured at 354 ± 27 µms-1 at a field strength of 100 Vcm-1. During

the concentrating and hybridization experiments, the oligonucleotide targets experienced an

increase in velocity as they eluted from the capillary into the microfluidic channel. This likely

caused de-stacking of the eluted material, reducing the enhancement effect.

3.7.3 Response of Microfluidic Biosensing Platform to Mixtures of Targets Delivered by Direct Injection and Following Selective Concentration

Figure 3.56 presents the response of the microfluidic biosensor for complementary and non-

complementary oligonucleotide targets by direct injection and after the targets were purified and

concentrated on the affinity capture gel. Selective concentrating on the original sample

improved the limit of detection of the biosensor to 0.5 nM of complementary material (A647-

SMN) in the presence of a 2000 fold excess of non-complementary material (Cy3-β-actin).

151

0

0.05

0.1

0.15

0.2

0.25

0.5 nM/1 uM 1 nM/1 uM 2 nM/1 uM 1 nM/0.5 uM 1 nM/2 uM

[complementary] / [non-complementary] in original target solution

Avera

ge F

luore

scence I

nte

nsity (

AU

)

direct injection selective concentratinga)

0

1

2

3

4

5

6

7

8

9

0.5 nM/1 uM 1 nM/1 uM 2 nM/1 uM 1 nM/0.5 uM 1 nM/2 uM

[complementary] / [non-complementary] in original target solution

Inte

gra

ted F

luore

scence I

nte

nsity (

AU

)

direct injection selective concentratingb)

Figure 3.56: The response of the microfluidic biosensing platform for samples containing complementary

and non-complementary target comparing delivery with and without selective concentrating. Direct Injection: mixture of A647 SMN and Cy3 β-actin target in 10% LAAm gel. Selective concentrating:

Affinity capture gel: 100 nM SMN probe, 10% LAAm gel. Target injection: 10 µL of A647 SMN and Cy3 β-actin target, electrokinetic injection for 1 min at 150 Vcm

-1. Capture: electrophoresis for 10 min at

150 Vcm-1

in 1x TBE/PVP running buffer. Concentrating step: coverage length: 12.5 mm; step size: 250 µm; step rate: 86 µms

-1; Voltage: 96 Vcm

-1; Delivery of oligonucleotide targets into microfluidic biosensing

platform: 500 V, 10 minutes, 1x TB/PVP/20 mM NaCl. Acquisition settings: PMT gain 700 mV, translation speed: 50 µms

-1 scan rate: 50 Hz. Error bars represent 1 standard deviation of three trials.

152

For these experiments, the target solutions were made in 1x TBE buffer (rather than deionized

water in the previous experiment that examined the response to only complementary target). The

targets were made in buffer to reduce the amount of non-complementary target being injected

into the capillary.

The experiments were originally performed under the same conditions as those in used for the

experiments with complementary target only. The amount of non-complementary target

remaining inside the capillary was 1.0 ± 0.2 fmole (originally 10 pmole). To further reduce the

amount of non-complementary target, the experiment was then modified to include a second

washing step for 10 minutes at 150 Vcm-1. This reduced the amount of non-complementary

target inside the capillary to 0.67 ± 0.1 fmole. The largest reduction of the non-complementary

target was observed when the sample was made in 1x TBE buffer; the amount of non-

complementary target in the capillary was reduced to below 0.1 fmol (the fluorescence intensity

was below the lowest data point for the calibration curve used). This decrease in the amount of

non-complementary target retained in the capillary was thought to be due to a decrease in the

amount of material injected into the capillary.

The quantity of material injected into the capillary by electrokinetic injection can be influenced

by the conductivity of the sample solution:

2

1

2

1

2

1

sample

sample

sample

sample

sample

sample

c

c

vel

vel

Q

Q= (12)

where Q is the quantity of material injected, vel is the velocity of the analyte ions in the system

and c is the concentration of the sample. The velocity of the analyte ions in the sample is

dependent on its concentration of the analyte and the conductivity of the buffer used. This

implies that a different quantity of material can be injected into the capillary for a sample

containing the same concentration of analyte, but made in buffers of different conductivities

[278].

More material is injected into the capillary if the conductivity of the targets in the sample is less

than that of the background electrolyte in the capillary [278, 279]. A lower conductivity buffer

can also result in stacking of the targets as they enter the capillary through field amplified sample

stacking (FASS) [163, 280, 281].

153

Deionized water is often used to provide the largest concentrating effect by field amplified

stacking [172]. This was done to help improve the amount of material injected into the capillary

for the low target concentration examined. However, when the sample contained both

complementary and non-complementary targets, a large amount of the non-complementary target

was also injected into the affinity capture gel. During the concentrating process, this led to the

concentrating of both complementary and non-complementary target in the eluent. Experiments

were done to examine the influence of increasing stringency conditions by increasing formamide

content and temperature, and by extending wash times to twice of what was initially used. Even

with these measures the data still indicated significant retention of the non-complementary target

by the gel. Increasing the conductivity of the buffer reduced the total quantity of

oligonucleotides injected, which aided in reducing the amount of non-selective adsorption of the

non-complementary targets on the affinity capture gel, and this limited the amount of non-

complementary targets being concentrated during the elution step.

The amount of material injected as measured by spectrofluorimetry was 6.0% ± 0.2% for the

complementary target and 15.8 ± 0.9 % for the non-complementary target. This was calculated

as the difference in fluorescence intensity of the sample solution in the original solution before

and after injection into the affinity capture gel.

Due to the presence of immobilized probe, the electrophoretic mobility of the DNA target inside

the get was modified as suggested by Equation (4) (Section 3.0.9), and was decreased. The

difference between Electrophoretic mobility of the complementary and non-complementary

target might have influenced the total quantity of material introduced into the affinity gel. Figure

3.57 shows an experiment using Cy3 labeled dT-20 targets. The amount of target injected was

measured as a difference of before and after injection was decreased with increasing probe

concentration inside the gel.

154

400

600

800

1000

1200

0 0.5 1 1.5 2

Concentration of Probe (uM)

Am

ou

nt

of

Targ

et

Lo

ad

ed

(fm

ol)

Figure 3.57: Effect of the probe concentration on the amount of target introduced into the affinity gel by electrokinetic injection. Affinity gel: Varying concentrations of dA20 probe (1.8 µM, 0.45 µM, no probe) in

a 12.5%T linear polyacrylamide gel. Injection condition: 5 µL sample containing 0.5 µM Cy3-dT20 at 267 Vcm

-1 for 60 seconds. The amount of target in the original sample was 2.5 pmol. Error bars

represent 1 standard deviation of three trials.

The non-complementary target remaining in the capillary was also concentrated with the

complementary target during step-elution. The amount of the concentrated non-complementary

targets in the eluent was calculated to be 0.57 ± 0.06 fmol and did not appear to vary as a

function of the amount of non-complementary target tested in the original target solution. Table

3.17 summarizes the performance of the affinity capture gel in the processing of samples

containing complementary and non-complementary.

Table 3.17: Summary of the performance of the two delivery methods. Percent recovery is based on the proportion of the amount of target delivered to the microfluidic biosensing platform from of the original

starting sample. The values for delivery by direct injection were calculated based on the concentration of the targets in the original sample. The values used for the delivery selective concentrating were

calculated based on the response of the biosensing platform. The enrichment factor is the ratio of the percent complementary target with and without selective concentrating. Errors represent propagated

error resulting from calculating derived values. Direct

Injection Selective

Concentrating [complementary] /

[non-complementary] in original target

solution

Amount complementary

/ non-complementary

target

Recovery (%)

Purity (%)

Recovery (%)

Purity (%)

Enrichment Factor

0.5 nM/1 µM 5 fmol/10 pmol 100 0.05 15 ± 5 56 ± 16 1117 ± 333

1 nM/1 µM 10 fmol/10 pmol 100 0.1 18 ± 3 73 ± 11 731 ± 114

2 nM/1 µM 20 fmol/10 pmol 100 0.2 17.3 ± 0.9 86 ± 4 429 ± 22

1 nM/0.5 µM 10 fmol/5 pmol 100 0.1 13.3 ± 0.4 70 ± 2 699 ± 18

1 nM/2 µM 10 fmol/20 pmol 100 0.1 3.5 ± 0.9 46 ± 11 457 ± 114

While step elution reduced the total quantity of complementary material being delivered into the

microfluidic biosensor in comparison to direct injection, the material delivered through the

155

interconnect port in step elution contained a higher proportion of complementary material. The

performance of the affinity capture gel in terms of percent recovery and percent complementary

target delivered was not observed to be influenced by the amount of non-complementary target

present except for the case where the sample solution contained 1 nM complementary and 2 µM

non-complementary targets. Here, the percentage of complementary material delivered from the

original sample was observed to be less than the other cases. This decrease in performance was

not due to the delivery of a large amount of non-complementary to the microfluidic chip.

However, the large amount of non-complementary material could affect the affinity capture gel

in its capture of complementary targets. Further mixtures using higher concentrations of non-

complementary material were not examined.

3.7.4 Response of Microfluidic Biosensing Platform to Delivery of Concentrated Oligonucleotide Targets With and Without Purification

An examination was completed of the response of the microfluidic biosensing platform to

concentrated samples that were processed with or without purification. Other methods of

purification of nucleic acids typically involve the capture, concentration and elution of all nucleic

acid material, which may include non-complementary targets. Here, the work examined the

response of the biosensing platform for such a scenario, where all nucleic acid targets present in

a sample were concentrated and delivered into the microfluidic biosensing platform [88, 150,

282].

In these experiments, the affinity capture gel was modified such that it contained two different

selective oligonucleotide probes, one for the A647-SMN target and the other for the Cy3-β-actin

target. The oligonucleotide length of both targets was selected to be the same so that mobility

would be similar. The microfluidic biosensing platform contained pads of immobilized probes

that were for the SMN target only. Since two targets were captured by the affinity capture gel,

both targets were concentrated during elution and delivered into the biosensing platform.

Table 3.18 reports the position of the material eluting from the capillary column corresponding

to the A647 and Cy3 labelled targets. The similarity of location of the two targets suggests that

both targets were of the same mobility and that targets were not already separated upon elution.

156

Table 3.18: Position and peak width of the eluted targets from the concentrating of the two captured targets along the capillary (from the injection end). Error represent 1 standard deviation of three trials. Affinity capture gel: 10% LAAm, 100 nM SMN and 5 µM β-actin probes. Target injection: 10 µL of 1 nM

A647-SMN and 1 µM Cy3-β-actin targets, electrokinetic injection for 1 min at 150 Vcm-1

. Capture: electrophoresis for 10 min at 150 Vcm

-1 in 1x TBE/PVP running buffer. Concentration step: coverage

length: 12.5 mm; Step rate: 86 µms-1

; Voltage: 96 Vcm-1

; Acquisition settings (Alexa647): PMT gain 700 mV, translation speed: 50 µms

-1, scan rate, 50 Hz. (Cy3): Image resolution 512 x 512, Gain 110 mV,

Pinhole: 60 µm, 1 FPS

Position after concentrating

Peak Width (µm)

A647-SMN Target 17.1 ± 0.7 mm 437 ± 100

Cy3-β-actin Target 17.3 ± 0.8 mm 542 ±109

Table 3.19 presents information about the complementary material eluting from the capillary

column during the concentrating step under two systems: the capture gel contained SMN probe,

and the capture gel contained both SMN and β-actin probes. The data suggests that capture of

the complementary target was not influenced by the presence of the second probe in the affinity

capture gel. Any differences observed between selective concentrating and non-selective

concentrating in the response of the microfluidic biosensing platform would be due to the

delivery of a larger amount of non-complementary material by non-selective concentrating of all

targets.

Table 3.19: Quantitative information of the eluted A647-SMN target during stacking from gels which containing only SMN probe (selective concentrating) and containing both β-actin and SMN probe

(concentrating only). Errors represent 1 standard deviation of three trials. Affinity capture gel: Selective concentrating: 10% LAAm with 100 nM SMN probe. Concentrating only:

10% LAAm with 100 nM SMN and 5 µM β-actin probes. Target injection: 10 µL of 1 nM A647-SMN and 1 µM Cy3-β-actin targets, electrokinetic injection for 1 min at 150 Vcm

-1. Capture: electrophoresis for 10

min at 150 Vcm-1

in 1x TBE/PVP running buffer. Concentrating Step: coverage length: 12.5 mm; Step rate: 86 µms

-1; Voltage: 96 Vcm

-1; Acquisition settings (Alexa647): PMT gain 700 mV, translation speed:

50 µms-1

, scan rate, 50 Hz.

Peak width (nL) Peak height

(AU) Peak area (AU)

Selective Concentrating

3.67 ± 0.96 0.47 ± 0.03 3.5 ± 0.95

Concentrating Only

3.0 ± 0.5 0.55 ± 0.07 3.98 ± 1.3

Figure 3.58 presents a comparison of the response of the microfluidic biosensing platform to

three methods of target preparation: without any processing (direct injection), concentrating of

all oligonucleotide targets (concentrating only) and by selective purification and concentrating of

the target of interest (selective concentrating).

The results indicated that the response of the biosensing platform for a mixture of targets

delivered by concentrating all oligonucleotide targets in the sample was similar to that observed

157

for the material on direct injection from the capillary without any concentrating. This

demonstrates the advantage of using selective concentrating.

Based on previous results shown in Table 3.2 in Section 3.0.6, it was noted that the probe sites

were saturated at target concentrations of 10% of the probe concentration. The use of higher

concentrations of non-complementary target would not be expected to recovery a difference in

the results since the probes were already saturated.

Although the response of the biosensing platform to the non-selective delivery of material was

observed to be similar to that of direct injection, it is important to note that the actual amounts

delivered were different. For example, the concentration of the material being delivered to the

microfluidic device by concentrating of all oligonucleotide targets for the initial mixture of 1 nM

complementary in 1 µM non-complementary target was determined to be 17 ± 2 nM

complementary target to 59 ± 4 µM non-complementary target.

158

0

0.05

0.1

0.15

0.2

0.25

0.5 nM/1 uM 1 nM/1 uM 2 nM/1 uM

[A647-SMN] / [Cy3-Bactin] in original target solution

Avera

ge F

luore

scence I

nte

nsity (

AU

)

direct injection concentrating only selective concentratinga)

0

1

2

3

4

5

6

7

8

9

0.5 nM/1 uM 1 nM/1 uM 2 nM/1 uM

[A647-SMN] / [Cy3-Bactin] in original target solution

Inte

gra

ted F

luore

scence I

nte

nsity (

AU

)

direct injection concentrating only selective concentratingb)

Figure 3.58: Comparison of the response of the microfluidic biosensing platform (containing probe for

SMN) for a sample containing A647-SMN and Cy3-β-actin targets as prepared by selective concentrating and concentrating of the all oligonucleotide targets. Delivery of oligonucleotide targets by selective

concentrating was done as previously described. Delivery of oligonucleotide targets by non-selective pre-concentration was done using 10% LAAm affinity capture gels which contained 100 nM SMN probe and 5

µM β-actin probe. Error bars represent 1 standard deviation of three trials.

159

Chapter 4 Future Directions

4.1 Determination of Oligonucleotide Probe Incorporated into Affinity Capture Gel

One response parameter that was examined as part of fractional factorial analysis was the amount

of oligonucleotide probe incorporated into the polyacrylamide gel. The fluorescence intensity of

a fluorophore attached to the oligonucleotide probe was measured to determine the amount of

probe remaining on-column following polymerization and pre-conditioning of the affinity gels.

However, reaction between the fluorophore and radical initiator as well as scattering of

fluorescence as a function of the polymer at different gel formulations affected the fluorescence

intensity, and an absolute measure of the quantity of oligonucleotide probe could not determined

by fluorescence.

One possible alternative method to reliably quantify probe that becomes incorporated into the gel

may be the use of radiolabelled DNA [283]. Oligonucleotides can be labelled with 32P isotopes

and a quantitative radioactive signal can be measured using scintillation counter.

Additionally, examination of samples following reaction between the oligonucleotide probe and

radical initiator by capillary gel electrophoresis for possible cleavage reaction products showed

the presence of a second, slower eluting peak, indicating some reaction had taken place. It would

be interesting to analyze the compounds in these peaks using CE-MS to confirm the identity of

the eluent in the electrophoretograms.

4.2 Further Factorial Experiments on Gel Formulations

The factorial design used in this thesis was a quarter fractional factorial design. Such a design is

typically used as an initial survey experiment in order to quickly screen for significant factors out

of a large number of factors without the need to perform a large number of experiments.

However, main factor effects can be confounded by possible interaction effects due to the nature

of the fractional factorial design. The trends identified from factorial analysis were that amount

of target captured increased as monomer content increased, while it decreased with increasing

crosslinker content. This preliminary result may suggest non-linearity in the response surface,

160

and a better model may be obtained by performing a full factorial experiment that examines a

larger range of monomer and crosslinker content.

It was proposed that changes in average pore size of the gel stretches out the ssDNA, resulting in

more targets being captured. A set of experiments could be considered to confirm this

hypothesis, and one approach may be to modify a hairpin structure into a Molecular Beacon

(MB). MBs are strands of ssDNA designed that have a stem-loop structure, and these has are

commonly used as molecular switches to detect binding of complementary nucleic acid

sequences [284, 285]. The loop portion reports the presence of a strand of complementary

oligonucleotide, while the stem consists of 5-7 nt base paired intramolecularly [284, 285].

Attached at the ends of the ssDNA strand are a fluorophore and a quencher [284, 285]. When the

MB is in its stem-loop conformation, the fluorophore and quencher are brought close together

spatially [284, 285]. Fluorescence emission by the fluorophore is reduced due to non-radiative

resonant energy transfer to the quencher. Since the resonant energy transfer is distance

dependent, separation of the fluorophore from the quencher allows for an increase in

fluorescence emission to be measured [284, 286]. It may be possible to adopt a MB motif into

the ssDNA target. Addition of a fluorophore and quencher such that it flanks the hairpin

structure present in the ssDNA can be used to measure any differences in fluorescence emission

intensity caused by folding or extension as the oligonucleotide reptates through the gel.

4.3 Improvements to Capillary-Microfluidic Platform

One issue encountered with the current design of the capillary to microfluidic platform is that of

destacking of the concentrated targets. The experimental work indicated that an enhancement

factor of about 300 of the eluted targets can be achieved. However, due to differences in

mobility and longitudinal band broadening, the stacked targets are diluted as they exit the

capillary into the microfluidic device due to a larger mobility of the oligonucleotide targets in the

microfluidic channel.

It may be possible to limit the de-stacking of the eluting oligonucleotide targets by adjusting the

velocity of the oligonucleotide targets inside the capillary and microfluidic channel. This could

be done by modifying the resistance of the channels, either by changing the geometry or the

buffer system. Changing the resistance would alter the local field drops across the discontinuous

system such that current is constant throughout the system [172, 280]. For example, increasing

161

the diameter of the capillary increases current flow, lowering resistance of the capillary [267].

The use of higher conductivity buffers can also affect mobility, as field strength is inversely

proportional to conductivity [163, 287].

Here, the system would need to be modified such that microfluidic channel has a higher

conductivity than the affinity gel capillary. The local field strength would be lower inside the

microfluidic channel such that the mobility is decreased versus the capillary, reducing the

dilution effect. An additional stacking system can also be included into the microfluidic chip to

stack the diluted targets prior to delivery to the immobilized probe pads. For example,

isotachophoresis can be employed. Targets eluted off the capillary can be stacked on a side

microfluidic channel prior to being delivered into the main microfluidic channel.

For this thesis, the length of target was quite limited due to the constraints inherent in the

biosensing platform to offer efficient hybridization. It is desirable to test the response of the

biosensing platform for delivery of the longer DNA targets. Issues of sterics that limit

hybridization efficiency may be alleviated if the oligonucleotide probes used with the

microfluidic biosensing platform were complementary to the ends of the target of interest, rather

than being complementary to the middle of the strand.

4.4 Moving Towards DNA Targets in Complex Matrices

Since the intent for the selective concentrating technique was towards the processing of real

samples, there was also an interest in the utility of this technique with samples contained in a

complex matrix. Preliminary experiments were performed where a sample of ground beef was

processed using pre-treatment steps commonly applied to such sample matrices

(homogenization, filtration, centrifugation, dialysis) to generate a complex matrix buffer.

Fluorescently labelled ssDNA oligonucleotide (20 nt) targets were used to spike these samples

and were used as samples to be processed by the affinity capture gel by electrokinetic injection.

Based on the preliminary results, it was observed that only a small portion of the DNA was could

be injected into the capillary; this small portion was retained by the affinity capture gel. This

observation could be due to injection bias. As noted in Equation 12, charged ions with faster

electrophoretic mobilities will preferentially load into the capillary during electrokinetic

injection. It can be assumed that a large number of high mobility ions are present in the complex

162

matrix. At the initial moments of electrokinetic injection, any DNA targets immediately near the

inlet end of the capillary will be injected into the capillary. However, once depleted, the higher

mobility ions will be injected preferentially, limiting the injection of DNA moving into the

capillary from bulk solution. This preliminary result suggests limitations of processing samples

of high salt content is of limited utility and that a step to remove such other high mobility species

is required.

163

Chapter 5 Conclusions

This thesis presented a method for the selective purification and concentrating of DNA targets

for delivery into a channel-based microfluidic DNA biosensing platform. The selective

purification was done by capillary affinity gel electrophoresis. Oligonucleotide probes were

immobilized by covalent attachment in polyacrylamide gels located inside fused silica

capillaries. Single-stranded DNA targets were injected into the polyacrylamide gel by

electrokinetic injection. Complementary targets were retained in the affinity capture column due

to interaction with the immobilized probe while non-complementary targets were removed from

the column. Release of the captured DNA targets was performed by step elution to concentrate

the DNA targets. The use of localized heating created an elution zone, and was applied in small

steps along the length of the capillary to stack the targets during elution. The volume of the

stacked targets was much smaller than the geometric volume of the capillary and the original

sample volume. Stacking experiments performed with 19 nt oligonucleotide demonstrated that

the targets were stacked into a volume of 0.25 nL inside the capillary from an original volume of

elution of 98.5 nL. This provided for sample in a volume that could be introduced into a

microfluidic device. The enhancement factor of the stacked targets in the capillary was 300 ±

29.

A thorough evaluation of the selective capture of a 150 nt DNA target in a complicated mixture

was completed. The investigation considered the effects of gel formulation, and different

loading and elution conditions on recovery and purity. This was addressed systematically and

guided by a factorial analysis.

From factorial design experiments, differences in gel formulation were examined for the

incorporation of probe and amount of target captured. The amount of probe incorporated into the

affinity capture gel was dependent on the original concentration used in the pre-polymer solution

and was not influenced by differences in gel formulation. It was identified that a higher amount

of complementary target was retained in the affinity capture gels made with higher

concentrations of monomer. This was thought to be a result of such gels having a smaller

average pore size than the radius of gyration of the ssDNA target examined, causing the ssDNA

target to move through the gel by reptation. It was proposed that movement through the gel by

164

reptation eliminates the hairpin structures present in the ssDNA, this made the probe region on

the target available for hybridization with the gel immobilized probe, resulting in a higher

amount of capture of the complementary target by the affinity capture gel.

This selective concentrating method was applied to a series of DNA targets of different lengths,

19 nt, 150 nt, 250 nt and 400 nt. Improvement in purity was achieved, but recovery was reduced

was reduced for longer sequences. The recovery of the method ranged from 0.5 to 4% for the

PCR targets, while it was 13 to 18% for the 19 nt oligonucleotide target. The purity was

calculated to be up to 44% for the PCR target and up to 86% for the 19 nt target. This was an

improvement in purity of 15 fold and 1100 fold in comparison to the original samples for the

PCR targets and 19 nt oligonucleotide, respectively. The lowest concentration of the 150, 250

and 400 nt targets that saw an advantage by selective concentration was 1 nM of complementary

in 150 nM non-complementary target. The lowest concentration of the 19 nt oligonucleotide

target that could be processed to see advantage in selective purification and concentration was

0.5 nM complementary target in 1 µM non-complementary target.

A capillary-to-microfluidic biosensing platform was developed and an interconnect was used to

deliver target analyte from the capillary column into the microfluidic device. This platform was

used for the delivery of purified and concentrated 19 nt targets from sample solutions containing

a mixture of non-complementary targets. The capillary column selectively concentrated the

targets, which were then delivered into a microfluidic based biosensor device for hybridization.

Performance was determined as the difference in response of the biosensor following a number

of different sample introduction methods. It was shown that selective concentrating provided the

best signal by the biosensor. The capillary column provided an improvement of purity, moving

from a value for the unseparated mixture of 0.01% to a final stacked band from the column of

50%. This correlated with a recovery that decreased from 100% to 20%. Selective concentrating

was shown to allow for the detection of a 0.5 nM in 1 µM non-complementary solution, which

was a result that could not be achieved by direct injection of sample into the biosensing system.

165

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182

Appendices

A. Factorial Design Experiment

A factorial design experiment is a method to systematically examine the effects of a number of

explanatory variables (factors) on a response. Experiments are run where the level of all the

factors is varied according to a design matrix and a response is measured. The levels for each of

the factors are changed concurrently in each treatment in a factorial experiment. This approach

is different from a one-factor-at-a-time (OFAT) approach where only one factor is changed while

all others are held constant [288, 289]. A factorial analysis can usually be completed in less

experimental runs than for OFAT and allows for interaction effects between factors to be

identified. Additionally, the level of precision obtained from a factorial experiment is greater

than that of an OFAT experiment. Table A1 shows an example of a design matrix for a three

factor, 2 level factorial experiment.

Table A1: A sample design matrix for a 3 factor, 2 level factorial experiment, using factors A,B and C.

A B C AB AC BC ABC

1 + + + + + + +

2 + + - + - - -

3 + - + - + - -

4 + - - - - + +

5 - + + - + + -

6 - + - - + - +

7 - - + + - - +

8 - - - + - + -

The two level factorial design examines the change in response for each factor between two

levels, a high and a low level. Here, the high level is denoted by ‘+’, where it can refer to a

higher concentration of reagent, and ‘-‘, referring to a low level of reagent used for each factor.

The two level factorial design is the most common since this approach minimizes the number of

runs to complete a full data set collection. The two level factorial experiment assumes that the

response surface is linear. However, the two level design can be modified to account for non-

linearity [288].

In the example given, the response due to each factor is replicated four times at each level with a

total experiment run of 8 experiments. In contrast, for an OFAT experiment, the same level of

precision would require 24 experimental runs [289].

183

Additionally, a factorial design experiment can identify interaction affects between factors.

Interaction effects are those that combine to change the response, in addition to the main factor

effects. As a hypothetical example, consider that both A and B increase the response by 20 and

10 points, respectively, when examined at the high level. The expected response for the

experiment when both factors are run at high levels would be 30. A larger or smaller response

than what is expected would indicate an interaction between the two factors at the high level, and

indicate that A and B are not fully independent variables. Interaction effects are denoted in the

design matrix in Table A1 as AB, AC, BC and ABC. For example, the column AB examines the

interaction between the factors A and B. The level for each of the interaction terms is a product

of the signs of the two main factors whose interaction is being examined. For example, in

experiment number 3, the interaction factor for A and B is ‘-‘. A ‘+’ for an interaction factor

indicates that both factors, in this case A and B, are both being examined at the same level, either

both high or both low, while a ‘-‘ means that the two factors are at different levels. A three factor

interaction is the response of the 2 factor interaction term with a main factor term. For example,

for the three-factor interaction ABC, it can be either between AB x C or A x BC or AC x B

[289].

A1. Fractional Factorial Designs

The number of experiments required for a 2 level factorial design experiment is 2k, where k is the

number of factors. As the number of factors being examined increases, the total number of

required experiments is increased. Clearly a large number of experiments requires time and can

become costly [288, 289]. The large number of experiments is required to determine the

contribution of higher order interaction terms. For a 26 design, 64 experimental runs are

required. However, only six degrees of freedom out of total 63 are used to estimate main affects,

while 42 are associated with interactions of three or more factors [288]. Oftentimes higher order

interactions are found to be negligible in contributing to the response [288, 289].

For initial screening experiments, it may be more practical to complete a sub-set of the total

number of experiments to obtain information about main and two factor interactions even if

information about higher order interactions is lost. Such fractional factorial designs are obtained

by confounding main factor terms with higher order interaction terms. The estimated effect of

the confounded term becomes the sum of the main effect and the effect of the interaction term.

184

For example, the factor D can be accommodated in the interaction term ABC. Therefore, the

effects due to the main factor and the interaction terms cannot be separated. The ABC term is

called an alias of D.

By confounding one factor with a higher order interaction term, the total number of experimental

runs needed is 2(n-1) instead of 2n. Since the total number of experimental runs is reduced by half,

this is known as a half fractional design. A quarter fractional design is obtained by confounding

two main factors, reducing the total number of experimental runs by a quarter.

A2. Design Matrix for Quarter Fractional Factorial Design

A quarter fraction design allowed for the identification of major main effects without extensive

experimentation. Here, the terms for the radical initiator systems, TEMED and APS, were

confounded with two interaction terms, AxC and BxC. This design is a 25-2, resolution III

design, meaning that some of the main effects were confounded with two-factor interactions

[288].

Aside from confounding the terms for TEMED and APS, the quarter fractional design also

introduced other confounding terms in the design matrix. In the factorial design, the term for

TEMED was confounded with the interaction term for AC, meaning D = AC. The generator

relationship is more commonly expressed as I = ACD, where I is the identity term, which

represents a column of ‘+’. A column squared gives its identity squared (AxA=A2=I). Any

column multiplied by the identity remains the same. Aliased relationships between other factors

can be determined by multiplying that factor with the generator term. For example, factor A is

aliased with the interaction term CD (IxA = ACD x A, A = A2CD, A=CD) [289]. Table A2

shows the generator term used to generate the quarter fraction as well as the aliased or

confounded terms in this design.

Table A2: Alias structure for the fractional factorial experiment.

Term Aliased Term

Generator I=ACD, I=BCE, I=ABDE

A CD + BDE + ABCE

B CE + ADE + ABCD

C AD + BE + ABCDE

D AC + ABE + BCDE

E BC + ABD + ACDE

AB DE + ACE + BCD

AE BD + ABC + CDE

185

A3. Choice of Factors and Levels

The ‘+’ levels for the factors were selected to accommodate practical constraints. The onset time

for polymerization following addition of the radical initiator was observed to decrease when the

concentration of the monomer or the initiator increased. The upper level of monomer and

initiator was chosen to allow enough time to inject the polymerization solution into the capillary

before gelation occurred. Additionally, high concentrations of monomer would mean that the

mobility of the longer DNA targets would be decreased, increasing the total processing time.

The lowest monomer concentration chosen was based on results examining EOF suppression

methods of EOF presented in Appendix C. This experiment was done to assess the effect of two

different EOF suppression methods to reduce loss of the modified polyacrylamide gel during

electrophoresis. Loss of the probe in such a manner would alter the results negatively. The

lowest gel concentration tested, 7.5%T, did not show any significant loss of the gel immobilized

oligonucleotide probes during electrophoresis and was selected as the lowest monomer

concentration for the factorial analysis.

186

B. Synthesis of DNA Targets

B1. Construction of 250 bp Target

The 250 bp target was generated by combining the 150 bp target with the 100 bp target in a

ligation reaction using DNA ligase. Prior to the ligation reaction it was necessary to replace the

hydroxyl group on the 5’ end of the PCR products with a phosphate group using T4

polynucleotide kinase and ATP. The ligation reaction involved the formation of a

phosphodiester bond between the 5’-phosphate and the 3-hydroxyl termini of two adjacent

double-stranded DNA fragments, with water being a reaction by-product [290].

The commercial oligonucleotide primers were produced by solid phase synthesis, which placed a

hydroxyl group on the 5’ end of the oligonucleotide target instead of a phosphate group [291].

During PCR, these synthetic primers bound to the DNA target, marking the region for the DNA

polymerase to provide replication. Since the synthetic primers became part of the amplicons, the

5’ end must also have a hydroxyl group. Phosphorylation of the amplicons was therefore

required to replace the hydroxyl group with a phosphate group for the ligation reaction to occur.

This was done using ATP catalyzed by T4 polynucleotide kinase.

Figure B1 shows the agarose gel electrophoresis of the products following the ligation reaction.

A profile plot of the lane containing the ligation product is also included to better highlight the

bands.

187

0

5

10

15

20

25

30

35

40

45

50

100 120 140 160 180 200 220 240

pixel number

Av

era

ge

Flu

ore

sc

en

ce

In

ten

sit

y (

AU

)

Figure B1: Top: Agarose (2% w/v) gel electrophoresis of the ligation reaction products: 1) Fermentas 100 bp DNA ladder, 2) 150 bp DNA target, 3) 100 bp DNA target, 4) to 5) reaction product of the ligation

reaction. Background subtraction using a rolling ball of diameter 100 pixel was used. Run conditions: 100 V for 1 hour in 1x TBE (pH 8.0) buffer. Bottom: profile plot of Lane 5.

188

The gel showed bands from the original starting material, plus a series of product bands that are

identified in Table B1:

Table B1: A summary of the assignment of the bands observed from the gel in Figure B1.

DNA length (bp)

Possible Assignment

87 100 bp

158 150 bp

235 100 bp +150 bp

297 150 bp +150 bp

In this reaction the two pieces of DNA used in the ligation reaction were blunt ended, which

would result in a total of four permutations in terms of how two pieces of DNA could couple:

100 bp +100 bp, 100 bp + 150 bp, 150 bp + 100 bp, 150 bp + 150 bp. From Figure B1, the

bands correspond to the original starting DNA sequences; one for the 100 bp +150 bp and one

for the 150 +150 bp. A band corresponding to the ligation of the 100 bp +100 bp was expected.

It is possible that the reaction did occur, but the yield was very low and was not observed on the

gel. The bands due to the other products were also very weak. The significant signals that were

observed for the original DNA sequences suggest that the yield of the reaction was low.

The part of the gel corresponding to the band at 250 bp was excised. The DNA was extracted

using a commercially available kit (Qiagen QIAquick PCR Purification Kit) and PCR was

performed. Since both DNA targets were subjected to the T4 polynucleotide kinase reaction, the

lane that corresponds to the desired target (250 bp) would contain two different products, 150 bp

+100 bp and 100 bp +150 bp. Each product could be amplified preferentially using a different

set of primers.

Figure B2 shows the agarose gel separation of the PCR products. The possibility of having two

different targets of the same length was noted since the target of interest could be located on

different strands depending on the arrangement of the two targets. However, since only one

target could be amplified using one set of primers, the amount of the desired target would be in a

large excess over the undesired combination.

189

Figure B2: Agarose (1% w/v) gel electrophoresis of the PCR from the bands around 250 base excised from the ligation reaction product . 1) Fermentas 100 base DNA ladder, 2) PCR Control of 100+150 bp reaction, 3)+4) PCR reaction for 100+150 bp product, 5) PCR Control for 150+100 bp reaction, 6) PCR

Reaction for 150+100 bp. Run conditions: 100V for 50 minutes in 1x TBE (pH 8.0) buffer.

B2. Construction of 400 bp Target

The choice of the 400 bp target sequence based on the uidA gene was made due to previous

experience in our group with this target. Additionally, the genomic sequence of E. coli is readily

available.

Genomic DNA from E. coli was extracted from the cells using a commercially available kit

(Qiagen DNeasy Blood and Tissue Kit). The DNA was then subjected to a double restriction

enzyme digest to cut a 1000 bp fragment from the region of the genomic E. coli DNA

corresponding to the uidA gene. The 1000 bp fragment was used as the DNA template in PCR

to generate the 400 bp target. The sequence information about the uidA gene was obtained from

the NCBI Entrez Genome Project (Escherichia coli str K12 Mg1655 K12) [292].

The initial double restriction enzyme digest was done because it was not known if PCR on the

genomic DNA target would generate the 400 bp target from the 4 Mbp length DNA. The

restriction enzymes were chosen so that the DNA would be cut at equidistant length from both

ends of the target region. Figure C3 shows the gel electrophoresis of the reaction product

190

following the double restriction enzyme digest. A broad range of targets from 424 to 4236 bp

length were detected. The results suggest that the restriction enzymes recognized and cut various

regions of the genomic DNA, resulting in generation of fragments of different lengths along with

the one of interest.

Figure B3: Agarose (0.5% w/v) gel electrophoresis of the PCR from the bands that were about 250 base

that had been excised from the ligation reaction product. 1) Fermentas 1 kpb DNA ladder, 2) E. coli genomic DNA 3) E. coli genomic DNA following double restriction enzyme digest. Run conditions: 100 V

for 1 hour in 1x TBE (pH 8.0) buffer.

A band of about 1000 bp was excised and purified using the gel purification kit. The purified

DNA was then subjected to amplification by PCR using the primers designed to amplify a 400

bp length target. The primers were selected using PrimerQuest, which is web-based software

available online from Integrated DNA Technologies. The design criteria were that the target

region was in the middle of the strand of DNA, the GC content of both primers were 50%, with a

Tm of 60 °C and a length of 24 bases. The latter three criteria were the default settings in the

PrimerQuest software and were the recommended for primer design for PCR [293, 294].

B3. Confirmation of DNA Targets

Figure C4 shows the agarose gel electrophoresis of all the DNA targets used for the experiments.

191

Figure B4: Agarose gel of the various targets used for the experiments. Left: 1% w/v agarose gel.

Lane 1: Fermentas 100 bp DNA ladder, 2: 100 bp target, 3: 150 bp target, 4: 250 bp target. Run conditions: 50 V for 2 hours in 1xTBE (pH 8.0). Right: 1% w/v agarose gel. Lane 1: 400 bp DNA target,

2: 100 bp ladder. Run conditions: 100 V for 1 hour in 1xTBE (pH 8.0)

The lengths of the DNA targets as calibrated using the DNA ladder were 96 bp, 148 bp, 273 bp

and 404 bp. Additionally, the DNA targets were sequenced to confirm that the target region was

present. Table B2 provides the sequences of the targets, and the highlighted segment indicates

the location of the conserved region representing the desired hybridization sequence.

192

Table B2: Sequence of the DNA targets. Legend R = A,G; Y = C,T; M = A,C; K = G,T; S = C,G; W = A,T; H = A,C,T; B = C,G,T; V = A,C,G; D = A,G,T; N = A,C,G,T. The sequence for the 100 nt used in the

ligation product was provided by Dr. Paul Piunno. Desired Target Length

Length Obtained (nt)

Sequence (5’ to 3’)

100 nt 79 CAC ATA ACT CGC TTG CAG TTG ACT TTG ACC GGG AGG CTG AAG AAA TGG CAC CCT TTG CTG CTG TGA ACT GTA GCC CAG A

150 nt 100 ARA YKG KYR TTT YMK WRG GTA TWW RGC AGT MMC CGC CCA GCM GGT YMG GCG CAR GGT GGC ATG GGG GRG GCA MMC CCT CGT AAT GGG CAC AGT GTG GGM Y

250 nt

187 GSM TGT AGM TYM TGA RGG TAG TCA RGC AGT TCC CGC CCA GCC AGG TCT AGG CGC AGG GTG GCA TGG GGG AGG GCA TTC CCC TCG TAG ATG GGC ACA GTG TGG GTG ATC CAC ATA ACT CGC TTG CAG TTG ACT TTG ACC GGG AGG CTG AAG AAA TGG CAC CCT TTG CTG CTG TGA ACT GTA GCC CAG A

400 nt

393 CTG CGT ATG AGT GMM WST YTS ACA TCA CCA TTG GCC ACC ACC TGC CAG TCA ACA GAC GCG TGG TTA CAG GCT TGC GCG ACA TGC GTC ACC ACG GTG ATA TCG TCC ACC CAG GTG TTC GGC GTG GTG TAG AGC ATT ACG CTG CGA TGG ATC CCG GCG TAG TTA AAG AAA TCA TGG AAG TAA GAC TGC TTT TTC TTG CCG TTT TCG TCG GTA ATC ACC ATT CCC GGC GGG ATA GTC TGC CAG TTC AGT TCG TTG TTC ACA CAA ACG GTG ATA CGT ACA CTT TTC CCG GCA ATA ACA TAC GGC GTG ACA TCG GCT TCA AAT GGC GTA TAG CCG CCC TGA TGC TCC ATC ACT TCC TGA TTA TTG ACC CAC ACT TTG CCG TAA TGA TGG ACC GCA ATA

Figure B5 shows capillary gel electrophoresis sequencing traces and the related quality graphs

for the 150 nt, 250 nt and 400 nt targets. Details for the sequence of the 100 nt target were

provided by Dr. Paul Piunno. The quality graph is a plot of the quality of each base call as a

function of the base number. The error probability of each assignment can be calculated by the

following [295]:

pQ 10log10−= (B1)

where Q is the quality of the base call and p is the estimated error probability for that base call.

For example, a base call having a probability of 1/1000 of being incorrect is assigned a quality

value of 30 [296].

The length of the targets as provided from sequencing did not match with the lengths as obtained

by gel electrophoresis.

193

a)

b)

194

c)

Figure B5: CGE traces and quality graphs of the base calls for a) 150 nt target, b) 250 nt target and c)

400 nt target.

Sanger sequencing is based on a modified version of PCR. In the reaction mixture, fluorescently

labelled dideoxy nucleotide triphosphates are mixed with normal deoxy-nucleotide triphosphates

used for building the complement strand in PCR. The dideoxy nucleotide lacks a hydroxyl group

on the 3’ position of the deoxyribose sugar that is necessary for the formation of a

phosphodiester bond with the incoming nucleotide base. The polymerase reaction for a fragment

of DNA terminates when the dideoxy-nucleotide is incorporated into the complementary strand

instead of a deoxy-nucleotide. The reactions lead to a mixture of DNA strands having different

lengths, each terminated with a fluorescent label corresponding to the base at the end of the DNA

fragment when extension of that fragment completed. The mixture of the DNA fragments are

then size separated by capillary gel electrophoresis. Detection of the fragments is done by laser

induced fluorescence where detection makes use of the four wavelengths that correspond to the

four labelling dyes.

Ideally, the electrophoretogram of the material from the sequencing reaction would have all the

individual fragments fully resolved without overlapping peaks. However, it is very common to

see the first 50 bases of the sequencing reaction appear as noisy or unevenly spaced eluents.

This can be due to the anomalous migration of the very short fragments, and may be

compounded by the changes in mobility of the DNA due to the attachment of fluorescent dyes.

195

Detection of unreacted dye-terminator molecules may also convolute the electrophoretogram

[295, 297].

Near the end of the sequencing run, band broadening due to longitudinal diffusion may result in

poorly resolved peaks. In addition, the relative change in mass when adding additional base

pairs for the longer DNA fragments in the sequencing reaction is relatively small, making it

difficult to separate out fragments of different lengths for the long reaction fragments [295, 297].

The formation of hairpin structures of the DNA fragments may alter electrophoretic mobility,

allowing folded sequences to appear earlier in the separation run. These are observed as

compression artifacts, where two overlapping peaks are observed in the trace and may lead to

problems in base calling [295, 297].

Such compression artifacts as well as poorly resolved peaks in the early portion of the

electrophoretograms were observed, and this led to ambiguities in base calling and incorrect

length information. Additionally, the primers used in the sequencing reaction were not

accounted for in the sequencing data, which further decreased the apparent length obtained from

Sanger sequencing. Therefore, the agarose gel data presented in Figure B4 is a better indicator

of the length of the DNA targets. The sequencing information confirmed the sequence integrity

for the hybridization site within the longer DNA strands.

B4. Examination of Sequence for the 150 nt Target

A difference was observed between the expected hybridization sequence and the sequencing

results that were obtained for the 150 nt target.

Expected b-actin sequence (previously determined by Dr. Paul Piunno)

AGG ATG GCA TGG GGG AGG G

b-actin sequence from sequencing (from TCAG sequencing facility)

ARG GTG GCA TGG GGG RGG C

A single base pair inconsistency was noted between the expected sequence and the sequence

determination. There was concern that a base pair mismatch had the potential to influence the

performance of the affinity gels.

196

A displacement chromatography experiment was done to confirm the selectivity of the affinity

gels. In displacement chromatography, the analyte of interest is first attracted onto the

chromatography column using an immobilized probe that has affinity to the analyte of interest.

Following this, a displacer compound is introduced into the column. This displacer compound

has a higher binding affinity towards the probe versus the analyte of interest. Therefore, as the

displacer moves through the column, it begins to displace the analyte, allowing it to elute [298,

299]. I had previously examined displacement chromatography for a 40 nt length

oligonucleotide target where the central 20 base was complementary to the strand with a 20 nt

fully complementary target [177]. Displacement chromatography using the same

oligonucleotide attachment chemistry was also used by Zangmeister et al. for the displacement

of a 20 nt target using a 10 nt displacer [300].

Figures B6 and B7 demonstrate the effect of introducing a complementary and non-

complementary displacer (20 nt) into an affinity capture gel where the 150 nt length DNA target

had already been captured. In Figure B6, a small increase in fluorescence intensity can be

observed following the introduction of a displacer that is complementary to the probe sequence

used. This suggests that some of the previously captured material had been displaced, and

moved under the influence of the applied electric field.

197

4000

5000

6000

7000

8000

9000

10000

0 0.5 1 1.5 2

distance (cm)

flu

ore

scen

ce i

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nsit

y (

AU

)

0 min

3 min

9 min

Figure B6: Profile plots from the inlet to outlet end of the capillary taken from confocal fluorescence

microscope images (Chipreader) tracking elution progress following the application of a complementary displacer to the affinity column where a Cy5 labelled 150 nt length DNA target was already captured. The displacer sequence was injected electrokinetically under the following conditions: 5 µL 20 µM, 267 Vcm

-1,

15 s; following which a potential of 167 Vcm-1

was applied. Confocal images were taken following electrophoresis for 3 and 9 minutes. Probe sequence: 5’ CCC TCC CCC ATG CCA TCC T 3’ Target

sequence: 5’ AGG ATG GCA TGG GGG AGG G 3’ Displacer sequence: 5’ AGG GTG GCA TGG GGG AGG G 3’

3000

3500

4000

4500

5000

5500

6000

6500

7000

0 1 2 3 4

distance (cm)

flu

ore

scen

ce i

nte

nsit

y (

AU

)

0 min

3 min

6 min

Figure B7: Profile plots from the inlet to outlet end of the capillary taken from confocal fluorescence

microscope images (Chipreader) tracking elution progress following the application of a non-complementary displacer to the affinity column where a Cy5 labeled 150 nt length DNA target was

already captured. The displacer sequence was injected electrokinetically Confocal images were taken following electrophoresis for 3 and 9 minutes. Probe sequence: 5’ CCC TCC CCC ATG CCA TCC T 3’ Target sequence: 5’ AGG ATG GCA TGG GGG AGG G 3’ Displacer sequence: 5’ ACA GGG TTT CAG

ACA AAA T 3’

198

The 150 nt sequence would have a lower binding affinity towards the probe than the 20 nt fully

complementary sequence. The additional length of DNA target would increase steric hindrance

and electrostatic repulsion between the target and probe versus that of the shorter oligonucleotide

displacer. Additionally, since the experiment was operated under an electric field, the longer

DNA target would experience a shearing force due to the electrophoretic force under the applied

field [269]. This would put a strain on the interaction between the hybridized target and probe.

The lack of a displacement affect when a non-complementary target was used as a displacer as

shown in Figure B7 suggests that a competitive displacement effect was present in the results of

Figure B6.

If a base-pair mismatch was introduced between the captured target and probe sequence, a much

greater displacement effect would result following the introduction of a fully complementary

displacer. This is demonstrated in Figure B8, where a significantly higher increase in

fluorescence intensity was observed. Here, a base pair mismatch was deliberately introduced

between the target and probe, while the displacer was fully complementary to the probe

sequence. Based on these displacement experiments, there is high confidence that the original

sequence that was supplied by Dr. Piunno was correct.

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 1 2 3 4

distance (cm)

flu

ore

scen

ce i

nte

nsit

y (

AU

)

0 min

3 min

9 min

bkg

Figure B8: Profile plots from the inlet to outlet end of the capillary taken from confocal fluorescence microscope images (Chipreader) tracking the progress following the application of a complementary

displacer to the affinity column where a Cy5 labeled 150 nt DNA target that contained a single base pair mismatch to the probe was already captured. Confocal images were taken following electrophoresis for 3

and 9 minutes. Probe sequence: 5’ CCC TCC CCC ATG CCA CCC T 3’ Target sequence: 5’ AGG ATG GCA TGG GGG AGG G 3’ Displacer sequence: 5’ AGG GTG GCA TGG GGG AGG G 3’

199

C. Generation of Single Stranded DNA Targets

The targets were generated by symmetric PCR, and the DNA existed in double stranded form.

Therefore, prior to the injection of the DNA sample into the capillary, the targets were first

denatured. Denaturation was accomplished by heating the DNA sample at 95 °C for 5 minutes,

followed by rapid cooling of the solution by putting it on ice. Heating disrupts the hydrogen

bonding between the complementary base pairs, separating the two strands and the rapid cooling

in ice is thought to slow the separated strands from re-annealing with one another.

Complete denaturation of PCR fragments of 0.6 kbp to 3.2 kbp have been reported in 30 seconds

at 98 °C, and 5 seconds for fragments 2 kbp and longer. Extended heat denaturation of up to

10 minutes did not appear to affect the quality of the DNA. Renaturation of the DNA in ice or at

RT was not observed in buffers without MgCl2 after 6 hours [301].

The denaturation of DNA was confirmed by measuring the hyperchromicity of DNA as it went

from double to single stranded state. The molar absorptivity at 260 nm of double stranded DNA

as calculated based on the individual molar absorptivity of the nucleotides is about 70% higher

than the molar absorptivity of the measured DNA strand [302]. The main contributor to this

hypochromicity is thought to be due to the stacking of the base pairs in the DNA [303]. The

electronic transitional dipoles of the stacked bases are ordered parallel to one another when the

DNA is in its double stranded form and the absorbance shifts to higher energy bands [303, 304].

There is an increase in absorption of bands at 200 nm and lower, while there is a decrease in the

absorption at 260 nm [305]. When DNA is heated, the interactions between the bases are

disrupted, leading to a disordering of the bases and an increase in absorption at 260 nm [304,

306]. The maximum increase in hyperchromicity due to denaturation of DNA has been reported

to be between 34-40% [302, 306, 307].

Figure C1 shows the absorbance at 260 nm in a solution of 150 bp DNA targets before and after

heating at 95 °C. A 400 µL solution of unlabeled 150 bp dsDNA target was heated to 95 °C for 5

minutes and then the absorbance was measured. The solution was then transferred to a

microcentrifuge tube and immediately put on ice for a total of 20 minutes, with the absorbance

measured at 10 minutes intervals.

200

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

initial 5 min 95 C 10 min ice 20 min ice

No

rmali

zed

Ab

so

rban

ce (

AU

)

Figure C1: Normalized absorbance of a solution of 13 µg/mL 150 bp DNA in Tris-HCl (pH 7.0) before and after denaturation by heat and following rapid cooling on ice. The absorbance values are normalized to the initial absorbance readings prior to heat denaturation. Error bars represent 1 standard deviation of

three trials.

An experiment was done using food colouring dye to confirm that the change in absorbance was

not due to a change in volume from heating the solution at 95 °C.

A decrease in absorbance following cooling in ice was observed, suggesting some of the DNA

had re-associated. This may be due to the solution not being cooled rapidly enough. The

volumes of solution used in these experiments were 400 µL. The experiment was done such that

solutions were transferred between a microcentrifuge tube and a cuvette at room temperature for

measurement. The relatively large volume of solution may have negatively impacted how

quickly the solution was cooled, allowing some of the DNA targets to re-anneal. Smaller

volumes of the target DNA solutions of up to 20 µL were used during the actual capture

experiments to allow for faster heating and cooling of the solutions.

201

0

10000

20000

30000

40000

50000

60000

70000

80000

0 20 40 60 80 100 120 140 160

Time (s)

Flu

ore

scen

ce In

ten

sit

y (

AU

)

TOPRO3 + DNA TOPRO3

Figure C2: Denaturation of the 13 µg/mL 150 bp target in Tris-HCl (pH 7.5) at 95 °C as detected using

an intercalating dye, (5 µM) TO-PRO3. Excitation: 642 nm, Emission: 660 nm. The blue trace is the response of the intercalating dye alone to temperature. Error bars represent 1 standard deviation of three

trials.

Figure C2 shows the fluorescence intensity of the DNA intercalating dye TO-PRO3 when mixed

with a solution containing the 150 bp DNA target after denaturation at 95 °C. TO-PRO3 is an

intercalation dye derived from thiazole orange [308]. Fluorescence enhancement due to

increased quantum yield is observed from interactions with double stranded and single stranded

DNA, but the enhancement is about 2 times larger for dsDNA [308, 309]. The dye interacts with

DNA by base-stacking, groove binding and electrostatic interaction with the phosphate

backbone. However, it has been suggested that intercalation is preferred at low dye-to-base pair

ratios (0.20) [310, 311]. The dye-to-base pair ratio in this work was approximately 0.1.

The fluorescence intensity decrease observed in Figure C2 was due to the drop in quantum yield

of the fluorescent dye as the double stranded DNA target denatured. Figure C2 also includes data

representing the emission of the intercalating dye in bulk solution as a function of temperature.

The termination of the fluorescence intensity change after 140 seconds suggests complete

denaturation of the target at this point. The fluorescence intensity remained higher than that for

the unassociated state of the dye due to some interaction of the dye with single stranded DNA.

202

Short, single stranded DNA targets can also be produced by the method of asymmetric PCR.

Asymmetric PCR was also investigated for target production, but it was found that symmetric

PCR followed by heating/cooling produced significantly more target.

A number of reports in the literature that examined real samples have made use of symmetric

PCR and generated single stranded DNA targets in the same fashion as used herein (by heat) [24,

35, 85, 199, 312]. Therefore, symmetric PCR was used to generate the targets in the work of this

thesis.

203

D. Suppression of Electroosmotic Flow (EOF) in Capillary Affinity Capture Gels

EOF was suppressed in these experiments because this process has the ability to physically eject

the affinity gel from the capillary. There are two common methods for the suppression of EOF

inside the capillary: covalent modification of the silanol wall on the inside of the capillary, or by

dynamically coating a polymer on the wall that prevents the generation of an electrical double

layer.

The use of a covalently immobilized coating for the suppression of EOF in a capillary was first

reported by Hjerten [313]. This work reported a bifunctional linker which was covalently

bonded onto the fused silica surface while providing a vinyl group that allowed for a layer of

polyacrylamide to be covalently bound to the wall. The bifunctional linker used was 3-

methacryloxypropyltrimethoxysilane (MPS). The methoxy groups in 3-

methacryloxypropyltrimethoxysilane reacted with the silanol groups in the glass wall, while the

free acryl group was available for polymerization with polyacrylamide to form a thin layer of

polymer covalently bound to the wall [313].

Figure D1: Structure of 3-methacryloxypropyltrimethoxysilane.

An example of the dynamic coating process is based on use of polyvinylpyrrolidone (PVP)

which is continuously present in flow solutions to suppress EOF. The structure of PVP is shown

in Figure D2.

ON

CH3

CH3 nn Figure D2: Structure of polyvinylpyrrolidone.

204

The use of PVP for the suppression of EOF in capillary electrophoresis was demonstrated by

Gao and Yeung [314]. It has been proposed that the PVP associates with the wall of the

capillary through non-covalent interactions with the silanol groups on the silica surface. PVP is

considered a hydrophobic polymer, but the hydrophilic carbonyl groups can hydrogen bond with

the silanol groups [314, 315]. The hydrophobic nature of PVP suggests that water is a poor

solvent, and therefore PVP is not easily desorbed by aqueous buffer systems [315].

Both methods for EOF suppression were examined to determine the optimal method for the

operation with the affinity gel in capillaries. Figure D3 shows the time-dependence of

fluorescence signal from fluorescently labeled probes that were incorporated in affinity gels.

Electrophoresis was run for a set amount of time, and data was collected to compare MPS and

PVP modified capillaries. No significant difference between the MPS and PVP modified

capillaries in terms of change in fluorescence signal was observed over the course of the

electrophoresis experiment. If EOF was not adequately suppressed, then a decrease in

fluorescence intensity would have been observed as the fluorescent polyacrylamide affinity gel

eluted from the column. It was concluded that both methods suppressed EOF and provided

physical stability to the polyacrylamide affinity gel in the capillary. PVP was used for the

remainder of the experiments to suppress EOF since it was the simpler method for

implementation.

205

0

1000

2000

3000

4000

5000

6000

before 15 min 500V 10 min 1000V 20 min 1000V

Avera

ge F

luore

scence I

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nsity (

AU

)MPS

PVP

Figure D3: Time-dependent experiments tracking the fluorescence intensity of the Cy3 labeled

oligonucleotide probe incorporated into the affinity gel to examine the effectiveness of two different methods for EOF suppression. The fluorescence intensity following polymerization of the gel was

measured, and then was subsequently measured following electrophoresis for a set amount of time at various applied voltages. The column contained 7.5%T 6%C polyacrylamide gel with 2 µM Cy3-β-actin oligonucleotide probe. The data was obtained from confocal fluorescence images (Chipreader) of the

capillaries. Error bars represent 1 standard deviation of three trials.

The data shown in Figure D3 was collected by fluorescence imaging of the same capillary a

number of times. It was necessary to determine any undesired affect of photobleaching of the

fluorescent dyes as a function of the number of times the capillary was scanned. Data

representing an investigation of photobleaching effects is displayed in Figure D4. A marginal

decrease in the fluorescence intensity from the capillary was observed when scanning the same

capillary a number of times. The decrease was small, representing only about a 3% loss in

fluorescence intensity after scanning the capillary four times. Therefore, the affect of

photobleaching was considered inconsequential in the interpretation of experimental results,

particularly since capillaries were scanned less than four times in all subsequent studies.

206

0.9

0.92

0.94

0.96

0.98

1

1.02

1 2 3 4

Number of Scans

Norm

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vera

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luore

scence Inte

nsity

(A

U)

a)

0.9

0.91

0.92

0.93

0.94

0.95

0.96

0.97

0.98

0.99

1

1.01

1 2 3 4

Number of Scans

Norm

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vera

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nsity (

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)

b)

Figure D4: Change in fluorescence intensity of a) Cy3 and b) Cy5 fluorescent dyes as a function of the number of times it was scanned under the confocal fluorescence microscope. The Cy3 channel was scanned at 5% laser power, and the Cy5 channel was scanned at 40% laser power. The data was obtained from confocal fluorescence images (Chipreader) of the capillaries. Error bars represent 1

standard deviation of three trials.

207

E. Factorial Analysis for Probe Incorporated into Affinity Capture Gel

E1. Table of Results

Table E1 shows the relative change in the amount of probe before and after the pre-conditioning

step based on the factorial design. Following the polymerization of the affinity capture gel, the

fluorescence intensity of the capillary was measured. The capillary was then subjected to a pre-

conditioning step where electrophoresis was run at a low voltage (74 Vcm-1) for 15 min.

Table E1: The relative difference in concentration of Cy3-labeled oligonucleotide probe measured following polymerization and after pre-conditioning of the affinity capture gels.

Std Order

Original Concentration

(µM)

Concentration After

Polymerization (µM)

Concentration After Pre-

conditioning (µM)

Percent Decrease in

Fluorescence Intensity (%)

1 9 0.5 0.36 0.36 0.32 0.28 87.32 78.59

2 10 3.0 3.08 2.74 2.89 2.25 93.86 82.00

3 11 0.5 0.42 0.43 0.40 0.44 93.76 101.51

4 12 3.0 1.98 1.58 1.63 1.39 82.22 87.93

5 13 0.5 0.26 0.31 0.23 0.26 86.52 85.67

6 14 3.0 3.15 2.94 3.07 2.47 97.26 84.03

7 15 0.5 0.51 0.44 0.37 0.38 72.75 86.06

8 16 3.0 3.48 2.32 2.81 2.54 80.60 109.25

Rather than using the absolute fluorescence intensity to identify the amount of oligonucleotide

probes incorporated into the affinity capture gel, the percentage loss in fluorescence intensity

before and after pre-conditioning was used. This loss in fluorescence intensity represents the

amount of the dye-labelled oligonucleotide probe washed off the affinity capture gel after pre-

conditioning.

E2. Factorial Analysis for Percentage of Probe Incorporated

E2.1 Analysis of Results

Results of the fractional factorial experiment were analyzed by three methods to determine

which factors had a large affect on the quantity of probe that was incorporated into the gel. The

methods were: calculation of the magnitude of effect each factor by comparison with standard

error; analysis of variance (ANOVA); and by comparison to a normal probability distribution

plot. The results were calculated using Minitab.

208

E2.2 Pareto Effects Plot

Figure E1 shows the magnitude effect of each factor in a Pareto effects plot. Here, the

standardized effect that each factor has on the response is ranked from largest to smaller. Any

effect beyond the line that is shown in Figure E1 is significantly larger than experimental error

with 95% confidence (α=0.05). The line is the t-value at the 97.5th quantile (1-α/2) for a t-

distribution with the number of degrees of freedom of the error term in the ANOVA table.

Figure E1: Pareto Chart plotting standardized effects of each factor for the percentage of probe

incorporated into the affinity capture. Factors with magnitude greater than shown by the line represent effects larger than experimental variation with 95% confidence.

E2.3 Magnitude of Effects

The following shows the raw output for the magnitude of effects as calculated by Minitab from

the fractional factorial experiment for the percentage of probe incorporated into the affinity

capture gel.

209

Estimated Effects and Coefficients for %probe (coded units)

Term Effect Coef SE Coef T P

Constant 88.084 2.407 36.59 0.000

%T -0.629 -0.315 2.407 -0.13 0.899

%C 2.355 1.178 2.407 0.49 0.638

Probe 3.120 1.560 2.407 0.65 0.535

TEMED 6.912 3.456 2.407 1.44 0.189

APS -1.644 -0.822 2.407 -0.34 0.742

%T*%C -3.559 -1.780 2.407 -0.74 0.481

%T*APS 7.128 3.564 2.407 1.48 0.177

S = 9.62889 PRESS = 4592.72

R-Sq = 41.15% R-Sq(pred) = 0.00% R-Sq(adj) = 0.00%

The magnitude of change for each factor is the difference between the averages of the responses

between the plus level of the factor and the minus level of the factor:

−+ −= yyeffectMain (E1)

The influence of interaction effects are calculated similarly [289]. To determine which factors

have a significant affect, the magnitude can be compared with the estimated standard error of the

experiment, which is calculated by:

2

2

1)( S

nEffectse

k= (E2)

Where the overall variance estimate is calculated by:

∑∑= =

−−

=

k

i

n

j

iijkyy

nS

2

1 1

22 )()1(2

1 (E3)

where n is the number of replicates for each of the 2k runs in the design, k is the number of

factors, y is the response at that run/replicate and y is the mean of the response for that

experimental run. The standard error is multiplied by the appropriate t-statistic for a particular

confidence interval and degree of freedom. Any effect larger than this value cannot be attributed

to experimental variation at the selected confidence level [288, 289].

210

E2.4 ANOVA Table

The data from the fractional factorial experiment was also analyzed by ANOVA and the raw

output from Minitab is shown below.

Analysis of Variance for %probe (coded units)

Source DF Seq SS Adj SS Adj MS F P

Main Effects 5 264.65 264.655 52.93 0.57 0.722

2-Way Interactions 2 253.89 253.893 126.95 1.37 0.308

Residual Error 8 741.72 741.724 92.72

Pure Error 8 741.72 741.724 92.72

Total 15 1260.27

Unusual Observations for %probe

Obs StdOrder %probe Fit SE Fit Residual St Resid

15 15 80.598 94.926 6.809 -14.328 -2.10R

16 16 109.254 94.926 6.809 14.328 2.10R

R denotes an observation with a large standardized residual.

ANOVA is a method for comparing whether mean responses between two or more populations

are different. For a factorial design, the comparison is between the mean response that each

factor has on the response versus that of the experimental noise that is present. This is done by

first calculating the total variance of the experiment. For clarity, the equation shown is for a two

factor, two level experiment involving A and B.

n

yySS

ki j

n

k

ijkT 2

22

1

2

1 1

2 K−

= ∑∑∑

= = =

(E4)

The variance is then distributed amongst all the different factors being examined, including

interaction terms. For example, for a two factor, two level experiment with factors A and B, the

sum of squares attributed to factor A and B is calculated by the square of the difference of the

response between the high and low levels of the factor, divided by the total number of

experimental runs:

n

BABABABASS

kA 2

][ 2−+−−+++ −−+= (E5)

n

BABABABASS

kB 2

][ 2−−−++−++ −−+= (E6)

The variance attributed to the interaction term, AB, is calculated similarly by:

211

n

BABABABASS

kAB 2

][ 2+−−+−−++ −−+= (E7)

Where k is the number of factors, n is the number of replicates performed while.

Any variance not assigned to a term in the model is assigned to the error term and is assumed to

be random error associated with the experiment. An F-statistic is used to determine whether

there is a statistical difference between the variance explained by a particular factor relative to

the variance associated with experimental error:

ABBATE SSSSSSSSSS −−−= (E8)

The sum of squares is divided by the degrees of freedom to obtain the mean square. Each term

has one degree of freedom. The total number of degrees of freedom in the experiment is 2kn-1.

The number of degrees of freedom in the error term is the remainder not assigned to a term in the

model.

An F-statistic is used to compare the variation among groups with variation within groups. The

F test is then performed considering the variation between means versus the mean square of the

error (variation within a group):

E

R

MS

MSF =0 (E9)

P-values less than 0.05 would indicate that the population means are different. This would imply

for the work of this thesis that a particular parameter for gel preparation is statistically significant

to the response [288].

E2.5 Normal Probability Plot

A further method for determining which factors are significant is based on comparison to a

normal probability plot or a normal quantile plot. Effects that are negligible are normally

distributed, with the mean being a value of zero and variance σ2 [316]. Figure 3.43 shows the

normal probability plot for the amount of probe incorporated. The seven data points seen on the

graph are the orthogonal terms in the design matrix (five main factors and the two interaction

factors) as shown in Table B2.

212

Figure E2: Normal probability plot of the effect of the factors as compared to a to a line representing

normally distributed data (in blue). Those highlighted in red are considered statistically significant. The data points are the orthogonal terms in the design matrix (five main factors and the two interaction

factors) as shown in Table A2.

E2.6 Examination of Model Adequacy

It is necessary to examine the fitted model to ensure that it provides an adequate approximation

to the true system, and to verify that none of the least squares regression assumptions are

violated.

A check of the assumption of normality is done by constructing a normal probability plot of the

residuals. If the residuals plot provides data that lies approximately along a straight line, then the

normality assumption is satisfied. When this plot indicates a problem, the response variable is

often transformed as a remedial measure.

A plot of the residuals ei versus the predicted response yi should appear randomly scattered on

the display, suggesting that the variance of the original observations is constant for all values of

y. If the variance of the response depends on the mean level of yi, then this plot will often

exhibit a funnel-shaped pattern and can suggest a need for transformation of the response

variable y.

213

A histogram plot of the residuals checks the normality of the variance. It should appear evenly

distributed around mean zero and indicates if random error is normally distributed.

A plot of residuals in time, or run order, versus each of the individual regressors is also done.

Non-random patterns on these plots would indicate model inadequacy.

Figure E3 shows all four plots. The diagnostic plots did not indicate any deviations from the

least squares model.

Figure E3: Statistical diagnostic plots of data distributions from factorial experiment that investigated

percentage of probe that was incorporated in gels.

E3. Amount of Probe Incorporated

The following is a summary of data generated by Minitab for analysis of the fractional factorial

experiment for the response labelled as ‘Amount of Probe Incorporated’. The data were taken

from the ‘Concentration after Pre-conditioning’ column in Table E1, transformed using a log10

function. The diagnostic plot of the original, unmodified data showed a funnel shape in the

residual versus predicted plot, which prompted the transformation of the data.

214

Figure E4: Pareto Chart plotting standardized effects of each factor on the amount of probe incorporated.

Factors with values greater than the line represent effects larger than anticipated by experimental variation at 95% confidence.

Magnitude of Effects

Estimated Effects and Coefficients for log probe amt (coded units)


Constant -0.06160 0.01212 -5.08 0.001

%T 0.03772 0.01886 0.01212 1.56 0.158

%C 0.02246 0.01123 0.01212 0.93 0.381

Probe 0.84750 0.42375 0.01212 34.96 0.000

TEMED 0.10395 0.05197 0.01212 4.29 0.003

APS -0.14382 -0.07191 0.01212 -5.93 0.000

%T*%C 0.06442 0.03221 0.01212 2.66 0.029

%T*APS 0.04369 0.02184 0.01212 1.80 0.109

S = 0.0484902 PRESS = 0.136319


ANOVA Table

Analysis of Variance for log probe amt (coded units)


Main Effects 5 3.00667 3.00667 0.601334 255.75 0.000


Residual Error 8 0.01881 0.01881 0.002351

Pure Error 8 0.01881 0.01881 0.002351

Total 15 3.04971

215

Figure E5: Normal probability plot of the effect of the factors as compared to a line representing normally distributed data (in blue). Those highlighted in red are considered statistically significant. The data points are the orthogonal terms in the design matrix (five main factors and the two interaction factors) as shown

in Table A2.

Figure E6: Statistical diagnostic plots of data distributions from factorial experiment that investigated the

amount of probe that was incorporated into gels.

216

F. Amount of Target Captured

F1. Data Reflecting the Quantity of Target Captured

Table F1 summarizes data about the capture of complementary target by the affinity capture gel

at various points during experiments. The responses reported were: the concentration of target

injected, concentration captured by the gel and concentration remaining inside the capillary

following elution. The fluorescence intensity measured following elution was below the lowest

value of the calibration curve and was set as zero.

Table F1: Summary of results from fractional factorial experiment on the following responses: Concentration of target in affinity capture gel following electrokinetic injection (Inject); Concentration of

targets captured following the wash step (Load); Concentration of targets remaining in the capillary following elution step (After Elute).

Standard Order Inject (nM) Load (nM) After Elute (nM)

1 9 66 74 18 19 0 0

2 10 189 208 38 42 0 0

3 11 104 95 8.5 9.5 0 0

4 12 227 216 31 32 0 0

5 13 131 119 53 65 0 0

6 14 212 235 73 92 0 0

7 15 116 148 35 45 0 0

8 16 273 254 55 48 0 0

F2. Concentration of Target Injected


experiment for the response labelled as ‘Concentration of Target Injected’. The data were taken

from the Inject column in Table F1. The diagnostic plots shown in Figure F3 did not indicate

any deviations from the least squares model.

217

Figure F1: Pareto Chart plotting standardized effects of each factor for the concentration of

complementary target injected. Factors with values greater than the line represent effects larger than anticipated by experimental variation at 95% confidence.


Estimated Effects and Coefficients for Injected (coded units)


Constant 6000.00 85.35 70.30 0.000

%T 1012.50 506.25 85.35 5.93 0.000

%C 650.00 325.00 85.35 3.81 0.005

Probe 3175.00 1587.50 85.35 18.60 0.000

TEMED -137.50 -68.75 85.35 -0.81 0.444

APS 175.00 87.50 85.35 1.03 0.335

%T*%C -37.50 -18.75 85.35 -0.22 0.832

%T*APS 262.50 131.25 85.35 1.54 0.163

S = 341.413 PRESS = 5935000


ANOVA Table

Analysis of Variance for Injected (coded units)


Main Effects 5 46311250 46311250 9262250 79.46 0.000

2-Way Interactions 2 281250 281250 140625 1.21 0.348

Residual Error 8 932500 932500 116563

Pure Error 8 932500 932500 116563

Total 15 47525000

218

Figure F2: Normal probability plot of the effect of the factors as compared to a line representing normally distributed data (in blue). Those highlighted in red are considered statistically significant. The data points are the orthogonal terms in the design matrix (five main factors and the two interaction factors) as shown

in Table A2.

Figure F3: Statistical diagnostic plots of data distributions from factorial experiment that investigated the

concentration of target injected into gels.

219

F3. Amount of Target Captured


experiment for the response labelled as ‘Concentration of Target Captured’. The data were taken

from the Load column in Table F1, transformed using a log10 function. The diagnostic plot of

the original, unmodified data showed a funnel shape in the residual versus predicted plot, which

prompted the transformation of the data.

Figure F4: Pareto Chart plotting standardized effects of each factor on the log10 of amount of

complementary target captured. Factors with values greater than the line represent effects larger than anticipated by experimental variation at 95% confidence.

220


Estimated Effects and Coefficients for log load (coded units)


Constant 1.53899 0.01223 125.79 0.000

%T 0.41781 0.20890 0.01223 17.07 0.000

%C -0.19735 -0.09868 0.01223 -8.07 0.000

Probe 0.28408 0.14204 0.01223 11.61 0.000

TEMED -0.15552 -0.07776 0.01223 -6.36 0.000

APS 0.04435 0.02217 0.01223 1.81 0.108

%T*%C 0.01100 0.00550 0.01223 0.45 0.665

%T*APS -0.06075 -0.03037 0.01223 -2.48 0.038

S = 0.0489399 PRESS = 0.194722


ANOVA Summary

Analysis of Variance for log load (coded units)


Main Effects 5 1.28146 1.28146 0.256291 107.01 0.000


Residual Error 8 0.01916 0.01916 0.002395

Pure Error 8 0.01916 0.01916 0.002395

Total 15 1.31586

Figure F5: Normal probability plot of the effect of the factors as compared to a line representing normally distributed data (in blue). Those highlighted in red are considered statistically significant. The data points are the orthogonal terms in the design matrix (five main factors and the two interaction factors) as shown

in Table A2.

221

Figure F6: Statistical diagnostic plots of data distributions from factorial experiment that investigated the

amount of target captured by gels.

222

G. Evaluation of Hybridization and Stringency Conditions

G1. Effectiveness of Gels for Purification of Target

Table G1 summarizes the results for the recovery and purity of the sample following

examination of a number of hybridization and stringency conditions using a three level factorial

analysis experiment.

Table G1: Summary of results from a 3-level factorial analysis for factors that affect hybridization and stringency. The response %Recovery (complementary target alone) was performed using solutions

containing Cy5-150 nt target only. The factors examined were A (hybridization time) and B (wash voltage). %Recovery and % Purity (complementary and non-complementary targets) were done using

solutions containing Cy5-150 nt and Cy3-non-complementary targets. The factors examined were A (wash temperature) and B (formamide content).

Standard Order

A B % Recovery (complementary

target alone)

% Recovery (complementary

and non-complementary

targets)

% Purity (complementar

y and non-complementary

targets)

1 10 1 1 1.46 1.83 1.44 1.35 66 64

2 11 1 2 2.48 1.28 1.59 0.94 75 97

3 12 1 3 1.77 1.45 1.14 1.57 85 82

4 13 2 1 1.92 2.99 1.51 1.68 62 58

5 14 2 2 1.77 1.72 1.53 1.14 89 91

6 15 2 3 1.66 1.55 1.03 1.53 86 81

7 16 3 1 1.28 1.25 1.58 1.81 61 60

8 17 3 2 1.67 1.22 1.72 1.56 68 68

9 18 3 3 1.74 2.08 1.35 0.63 88 94

G2. Hybridization Time and Wash Voltage for Samples Containing Only Complementary Targets

The following is a summary of data generated by Minitab for analysis of the three level factorial

experiments for the response identified as percent recovery on varying hybridization time and

wash voltage for samples containing only complementary target. The data were taken from the

%Recovery (complementary target only) column in Table G1. Since the experiment was based

on a three level factorial design, only the ANOVA table and model diagnostic plot was generated

by Minitab. The diagnostic plots shown in Figure G1 did not indicate any deviations from the

least squares model.

223

ANOVA Table:

Analysis of Variance for recovery, using Adjusted SS for Tests


Blocks 1 0.0027 0.0027 0.0027 0.01 0.910

Time 2 0.4707 0.4707 0.2354 1.19 0.352

Wash V 2 0.0328 0.0328 0.0164 0.08 0.921

A*B 4 1.3273 1.3273 0.3318 1.68 0.246

Error 8 1.5762 1.5762 0.1970

Total 17 3.4098

S = 0.443877 R-Sq = 53.77% R-Sq(adj) = 1.77%

Figure G1: Statistical diagnostic plots of data distributions from three level factorial experiments that investigated the %Recovery of target captured by gels.

G3. Stringency Conditions for Samples Containing Complementary and Non-complementary Targets

The following is a summary of data generated by Minitab for analysis of the three level factorial

experiments for the responses identified as percent recovery and purity on varying wash

temperature and formamide content for samples containing complementary and non-

complementary targets. The data were taken from the %Recovery and %Purity (complementary

and non-complementary targets) columns in Table G1. The diagnostic plots shown in Figure G2

and G3 did not indicate any deviations from the least squares model.

224

ANOVA Table for Percent Recovery:

Analysis of Variance for recovery mix, using Adjusted SS for Tests


Blocks 1 0.0018 0.0018 0.0018 0.02 0.898

A 2 0.0327 0.0327 0.0164 0.16 0.855

B 2 0.3777 0.3777 0.1889 1.84 0.220

A*B 4 0.3683 0.3683 0.0921 0.90 0.508

Error 8 0.8199 0.8199 0.1025

Total 17 1.6004

S = 0.320137 R-Sq = 48.77% R-Sq(adj) = 0.00%

Figure G2: Statistical diagnostic plots of data distributions from three level factorial experiments that investigated the percent recovery of target captured by gels.

225

ANOVA Table for Percent Purity:

Analysis of Variance for purity, using Adjusted SS for Tests


Blocks 1 20.06 20.06 20.06 0.60 0.462

A 2 93.78 93.78 46.89 1.39 0.303

B 2 1972.11 1972.11 986.06 29.28 0.000

A*B 4 560.89 560.89 140.22 4.16 0.041

Error 8 269.44 269.44 33.68

Total 17 2916.28

S = 5.80350 R-Sq = 90.76% R-Sq(adj) = 80.37%

Unusual Observations for purity

Obs purity Fit SE Fit Residual St Resid

3 75.0000 84.9444 4.3257 -9.9444 -2.57 R

12 97.0000 87.0556 4.3257 9.9444 2.57 R

R denotes an observation with a large standardized residual.

Figure G3: Statistical diagnostic plots of data distributions from three level factorial experiments that investigated the percent purity of target captured by gels.

226

H. Effect of Step Size in the Step Elution Process

Step elution was accomplished by moving the resistive heating element across the capillary in

discrete steps. The physical size of the heating element was approximately 0.8 mm. One

consideration was whether a length scale of steps at the size scale of the heating element would

avoid discontinuities in the heating zone. Therefore, the effect of changing the step size was

examined.

Since the step rate must be matched to the electrophoretic mobility of the DNA targets, the

heating time was also adjusted when the step size was altered. For example, the larger steps

were held for a longer time than the smaller steps before the heating element was advanced. Step

sizes were selected based on the minimum time needed for elution to take place, and the

maximum amount of time before gel breakdown. For the 19 nt targets it was observed that about

1 second was required for elution to be completed. Additionally, continuous heating of the gel at

one spot of the capillary caused the loss of current across the gel capillary in some cases. This

was observed to coincide with formation of small voids or bubbles inside the capillary where the

heater element was positioned, and occurred about 50% of the time when the gel was heated for

more than about 6 seconds. The range of step size tested was 125 to 500 µm, with heating times

of 1.1 to 4 seconds.

Figure H1 shows the peak area, height and width of eluted oligonucleotide targets obtained by

step elution from the same length of capillary for a variety of different step sizes. These results

were obtained using a step elution length of 25 mm versus the 12.5 mm stated as optimal

previously. These experiments were performed concurrently with the previous set of

experiments prior an optimal elution length was determined. The results indicate no statistically

significant differences in peak height or area for steps of the size scale of the heating element,

indicating that elution would be continuous and unfluctuating.

227

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

peak area peak height

Flu

ore

sce

nce

In

ten

sity (

AU

)

125 um 250 um 500 uma)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

peak width

tim

e (

s)

125 um 250 um 500 umb)

Figure H1: Comparison of peak area, height and width as a function of step size used during step elution

across a column length of 25 mm. Data for a) integrated Peak Area, Height and b) Peak Width were calculated using Origin Pro 8.0.

Affinity capture gel: 50 nM SMN probe, 10% LAAm affinity capture gel. Target injection: 10 µL 50 nM Cy5-SMN target for 1 min at 150 Vcm


-1 in 1x TBE/PVP

running buffer. Step elution: step size: 125 µm, 250 µm and 500 µm; Step rate: 86 µms-1

; Voltage: 96 Vcm

-1; Acquisition settings: ND 4, 8 and 16 filters, PMT gain 400 mV. Error bars represent 1 standard

deviation of three trials.

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