Biosensors and Their Applications (BIEN 515)

• Introductions – Name – Background

• Review of syllabus and class objectives – Tailor this class to your individual needs!

What is a Biosensor? • Definitions

– A device used to measure biologically relevant information • Oxygen electrodes, neural interfaces,

– A device using a biological component as part of the transduction mechanism

• Antibodies • Enzymes • DNA, RNA • Whole cells • Whole organs/systems

What is a Biosensor? • Configuration

– Can be developed from any basic sensor by adding a biological component

– Usually incorporates a biomembrane • Transduction

– Electrical – Optical – Mechancial

• Mass • Acoustic

– Thermal – Chemical – Magnetic

Strain Gages, Acceler-ometers, and Gyroscopes• Strain Gages

– Function and parameters

– Types

– Fabrication

• Accelerometers– Mechanics and theory

– Strain gage type

– Capacitive

– Force balanced

– Others

– Switch arrays

– Multi-axis

• Gyroscopes– Mechanics and theory

– Vibrating beams

– Others

Strain Gages

• Gage factor is defined asrelative resistance changeover strain

• Types include:– Metal foil

– Thin-film metal

– Bar semiconductor

– Diffused semiconductor

• Implantable strain gages

• Penetrating micro-straingage probe

Accelerometers• F=ma is basic concept

• Force measured by deflection or strain

• Can be related to spring constant, F=kx

• Generally displacement of proof mass ismeasured relative to frame

• Dynamic system as described previously

• Strain gage type most basic– Strain in beam measured as proof mass deflects

beam

– Lots of configurations

• Capacitive accelerometers mostcommercialized– Torsion bar with assymetric plates

• Force-balanced capacitive used in autos– Comb of capacitors measures differential

capacitance

– Highly sensitive, typical displacement only 10nm

– Force feedback to maintain central location ofproof mass

– Force required to maintain equilibriumgeneratessignal

Accelerometers (cont)• Piezoelectric accelerometers

– Generally show no DC response• Special circuitry to create DC response

– Typically use ZnO

• Tunneling accelerometers– Highly sensitive

– More difficult to fabricate

– Requires closed loop control

– Long term drift

• Latching accelerometers– Lock in place if acceleration exceeded

• Switch arrays– Array of switches sensitive to increasing levels

of acceleration

– Simple to build

– Optimizes range of accelerometer in use

• Multi-axis acclerometers– Only one example to date

– Cross-axis sensitivity problem

– Precise alignment and low cost are advantages

• All require extensive circuitry

Gyroscopes• Measure rotation

• Couple energy from one vibrationalaxis to another due to Coriolis effect

• Two micromachined modes: Open loopvibration and Force-to-rebalance mode

• Vibrating prismatic beams– Beam driven in one direction,

deflection measured in orthogonaldirection

• Tuning forks– Large inertial mass, increased

sensitivity

– Metallic ring structure

• Dual accelerometer

• Vibrating shells– Two-axis

– Vibration in z direction

– Output in both x and y

Calorimetric Biosensors

b Principles of Operationb Types of Devicesb Specific Examplesb Applicationsb References

Calorimetry

b Used to measure heat changes involvedwith thermo-chemical processes

b In general, most biochemical reactionsrelease heat or involve somethermodynamic process

b Heat generation is proportional tosubstrate conversion

Calorimetric Biosensors

b Rely on biological molecules fortransduction or detection

b Enzymes (or even cells) immobilized on asurface

b Enzymes catalyze a reaction whichgenerates heat

b Heat is proportional to amount of substratepresent

Calorimetric Biosensor Types

b Originally highly sophisticated equipmentwhich was very expensive

b Three general types of sensors classifiedby how heat transfer taks place betweenthe reaction vessel and surroundings• Heat conduction calorimetry

• Isoperibol calorimetry

• Isothermal calorimetry

Heat Conduction Calorimetry

b Rapid heat transfer between chamber andisothermal heat sink surrounding

b Temperature changes are measured asvoltage output of a thermoelectric sensorbetween chamber and heat sink

b Heat sink kept isothermal to keep heattransfer coefficients constant

b Thus temperature of reaction chamber isproportional to heat generation

Heat Conduction Designs

b Miniaturized devices are potentiallyimplantable

b Close contact reduces need for accuratecontrol of ambient temperature

Isoperibol Calorimetry

b Most common methodb Also known as isothermal jacket

calorimetryb Nearly adiabatic (no heat transfer)b Measure temperature change in reacting

solutionb Immediate cell environment remains at

constant temperature

Isothermal Calorimetry

b Temperature of the reaction vessel is keptconstant by heating or cooling

b An adiabatic process, i.e. no heat transferb Heating or cooling proportional to reaction

components

Applications

b Online industrial monitoring and controlb Measurement of biomass in a sampleb Can perform immunoassays using enzyme-

labeled substrates- both direct andsandwich types

b Blood glucose measurements as well asmetabolites (lactate and urea)

b Heavy metal and pesticide detection

Blood and Protein Separations Using aMicromachined Electrical Field- Flow

Fractionation System

Bruce K. Galea, Karin D. Caldwellb, A. Bruno Frazierc

a Institute for Micromanufacturing, Department ofBiomedical Engineering, Louisiana Tech University

bCenter for Surface Biotechnology, Department ofChemistry, Uppsala University, Sweden

cSchool of Electrical and Computer Engineering,Georgia Institute of Technology

HSEMB 2000

Overview

• Introduction to EFFF

• Motivation for Miniaturization

• System Fabrication and Assembly

• Basic Separations

• Biological Separations

• Conclusion

• Acknowledgments

What is EFFF?• Electric field forces charged particles towards wall

• Distance from wall determined by “ζ-potential”

• Relies on laminar flow profile to perform separationfunction

• Distance from wall determines velocity throughchannel for particles of the same size

+ + + + + + + Electrode + + + + + + +

– – – – – – – – Electrode – – – – – – – ––

Top

Bottom

Sample InputSample Outputx

y

20 µµm

Theory- Force Balance

• Electrical anddispersive forcesbalance todetermine thicknessof the particle field

• Average thicknessof particle fielddetermines velocitythrough channel

ChannelWall

VelocityProfile

CompressedParticle

Field

ElectricField

Diffusion

DiffuseParticle

Field

Previous EFFF Work

• First EFFF systems used to separate proteins in1972

• First EFFF systems with non-membrane wallsbuilt in 1993 and used to separate colloids

• EFFF has not be the subject of intense scrutinywith only a few groups working on it, and mostfor only a short time

• Progress has been slow until recent years

Separation Abilities of EFFF

• Cells and cellular components

• Viruses and bacteria

• Macromolecules (protein, DNA)

• Colloids– Clays

– Polymers

– Liposomes, micelles, and vesicles

• Emulsions

• Surface modified particles

Applications of EFFF

• Virus, bacteria, and organelle separations

• Characterization of emulsions, liposomes, colloidsand other vehicles for drug administration

• Diagnostic tests and molecular separations

• Protein adsorption/ surface characteristic studies

• Environmental monitoring

• Polymer, starch, and aggregation characterization

• Research on ζ-potentials

Theory- Resolution

Resolution of 44 nm and 62 nm Particles vs Width and Applied Voltage

0102030405060708090100

800 1000 1200 1400 1600 1800

Voltage (mV)

Wid

th (

uM

)

Rs=10

Rs=5

Rs=2

Rs=4Rs=3

Rs =Resolutiond = diameter of particleD = Diffusivityw = Channel thicknessL = Channel lengthVeff = Effective voltage<v> = Average fluid velocityµ = Electrophoretic mobility

sv

effVL

Dw

d

d

sR1

6

33

8∝

><

∆

=µ

Motivation for Miniaturization

• Shorter analysis times

• Higher resolution separations

• Parallel separation channels

• Batch or continuous fabrication withreduced production costs

• Reduced power consumption

• All advantages mentioned previously

Motivation for ApplyingMicromachining to EFFF

• By reducing channel height the power of EFFFsystems can be increased

• Macro-machining techniques are the limitingfactor in reducing channel heights in EFFFsystems

• Possibility of integrated electronics and detection• Precise micromachined features reduce

instrumental plate heights

Fabrication 1• Anisotropic etching of

input and output portsin 20% KOH at 65 Cwith Si3N4 mask

• Deposit and patternTi/Au electrodes onfront of wafer

• Thick photosensitivepolyimide or SU-8used to define flowchannels

Silicon

Silicon

AuTiDetect

orContact

Polyimide

Silicon

Fabrication 2

• Remove Si3N4

membrane

• Deposit and patternTi/Au electrode onglass substrate

• Bond glass substrateto polyimide usingbiocompatible UVcurable adhesive

Silicon

Glass

TiAu

Silicon

Glass

Fabrication 3

• Completed channellooking from the top

• Cross section throughchannel showingelectrodes, polyimideand substrates

Completed Cross Section

Polyimide

Input Port

Detector Electrode Channel Electrode

Output Port

Channel

Results- Section Fabrication

Silicon substrate with input/output ports, gold electrodes and patternedSU-8

Glass substratewith titanium,gold, andplatinumelectrode

Results-System Assembly

Above- Complete device withinput/output port connections

Right- Complete systems withsample and buffer input, µ-EFFFsystem, and detectors

Basic Separation Results• Compare off-chip

detectors with on-chip

• Peaks clearly defined

• Resolution doubles

• Time for separationreduced significantly 0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15Time (min)

Det

ecto

r R

espo

nse

Void Peak 44 nm

130 nm204 nm

1.55E-07

1.59E-07

1.63E-07

1.67E-07

1.71E-07

1.75E-07

0 120 240 360 480 600 720

130 nm 261 nm

Void Peak

9.81E-04

9.82E-04

9.83E-04

9.84E-04

9.85E-04

9.86E-04

9.87E-04

9.88E-04

9.89E-04

9.90E-04

0.00 50.00 100.00 150.00 200.00Time (sec)

Det

ecto

r C

urre

nt (

A) Void

204 nm

130 nm44 nm

Separation by Surface Charge

• Particles canbe separatedby surfacecharge as wellas size

• Particles withlower surfacecharge densityelute earlier,even foridenticallysized particles

-2.0E-06

0.0E+00

2.0E-06

4.0E-06

6.0E-06

8.0E-06

1.0E-05

1.2E-05

0 200 400 600

Time (sec)

Det

ecto

r R

espo

nse

LowHigh

HighLow

Particles with Attached Proteins

• Particles withattachedproteins arealso retaineddifferentially

• Bare particleselute ahead ofparticles withprotein on thesurface thatincreases bothsize and surfacecharge

4.55E-05

4.60E-05

4.65E-05

4.70E-05

4.75E-05

4.80E-05

4.85E-05

4.90E-05

0 200 400 600 800 1000 1200

Time (sec)

Cur

rent

(A

)

Protein APlain

Bare

Protein Attached

Blood Separations• EFFF system can be used

to separate some bloodcomponents

• Cells are too large forsystem

• Proteins, organelles, andmid-sized particles retained

4.55E-05

4.60E-05

4.65E-05

4.70E-05

4.75E-05

0 500 1000 1500 2000 2500 3000

Time (sec)

Cur

rent

(A

)

WholeHomogenized

4.55E-05

5.05E-05

5.55E-05

6.05E-05

6.55E-05

7.05E-05

7.55E-05

8.05E-05

8.55E-05

9.05E-05

9.55E-05

0 500 1000 1500 2000 2500 3000

Time (sec)

Cur

rent

(A

)

Whole

Homogenized

Hemoglobin

HemoglobinComplexes

Platelets

Cells

Conclusion• Theory predicts benefits for miniaturization of

EFFF systems

• Design, fabrication and characterization of aminiaturized EFFF system was performed

• Experiment confirmed basic theory

• Basic separations demonstrated

• Separation of particles with attached proteins

• Separation of some blood components

Acknowledgments

• National Science Foundation GraduateResearch Fellowship

• Whitaker Foundation Biobased Internship

• University of Utah Technology InnovationGrant

DNA

b Contains genetic material for all livingorganisms

b Double stranded helixb Made up of 4 different nucleotides- A,T,C,Gb Each nucleotide a 5 carbon sugarb Sequences of nucleotides define proteinsb Each sequence is a “gene”

Problem

b Typical molecular analysis problemsrequire statistically significant quantitiesand must pass detection limits on the orderof millions and billions of molecules

PCR

b Technique used to produce a large numberof copies from a target DNA sequence

b Repetitive 3 step process• Denaturation (~95ºC)• Annealing (~55ºC)• Chain Extension (~ 72ºC)

Basic PCR Reagents

b Template DNAb Complementary Primers (~20 nucleotides)b Thermostable Polymerase Enzyme (TAQ)b Single nucleotides (A,C,G,T)b Buffers (pH and ionic concentrations)

PCR Applications

b Creates 2n copies (typically 30 cycles)

b Genetic analysisb Viral diagnosisb Start with just one sample molecule

Why Apply Micromachining?

b Small reagent costsb Fast cycling time

• Low thermal mass• High surface to volume ratio

b System integration• Electrophoresis• Point of care system

b Low cost

Design Considerations

b Biocompatibilityb Chamber volumeb Control systemb Bulk or surface micromachiningb Bonding method (if necessary)b Move the fluid or cycle in positionb External equipment

Mechanical Pressure Sensors

• Designs– Absolute

– Gauge

– Sealed Gauge

– Differential

Piezoresistive Pressure Sensors

• Piezoresistivity is a material property where bulkresistivity is influenced by mechanical stress appliedto material

• Common piezoresistors: Si, poly Si, SiO2, ZnO

• Typical design: 4 piezoresistors in a Wheatstonebridge on a diaphragm

• Pressure sensitivity (mV/V-bar): S = (∆R/∆P)(1/R)

Capacitive Pressure Sensors

• Capacitive sensors convert charge into change incapacitance

• Advantages:– more sensitive than piezoresistive

– less temperature dependent

• Disadvantages:– gap fabrication

– diaphragm mechanical properties

Capacitive Pressure Sensors (cont)

• Basic concept: C = ε S/d

• Sensitivity: ∆C/∆d = -ε S/d2

• Small Gaps:– larger capacitance

– easier capacitance detection

– plates may stick together

• Large Gaps:– small capacitance

– may require wafer bonding

Microphones

• Convert acoustic energy into electrical energy

• High sensitivity pressure sensors

• Types:

– Capacitive• variable gap capacitor; most common

• require DC bias

• sensitivity: 0.2 to 25 mV/Pa

• response: 10 Hz to 15 kHz

Microphones (cont)

– Piezoresistive• diaphragm with 4 pezoresistors in a Wheatsone

bridge

• sensitivity: ~25 µV/Pa

• response: 100 Hz to 5 kHz

– Piezoelectric• use piezoelectric material mechanically coupled to

diaphragm

• sensitivity: 50 to 250 µV/Pa

• response: 10 Hz to 10 kHz

DNA • Contains code for operation of all living organisms • Made up of 4 (5) bases in long chains • Each nucleotide a 5 carbon sugar • Double helix of complementary strands • Sequences of nucleotides define “genes” • Genes define proteins

RNA • Converts DNA codes into proteins • Similar to RNA with additional oxygen • Matches up with DNA strands (complementary) • Three types

– Messenger (mRNA) • Carries genetic code out of nucleus

– Ribosomal (rRNA) • Interacts with mRNA to generate protein

– Transfer • Transports amino acids to ribosome • Translates mRNA to generate protein • Series of 3 RNA bases correspond to amino acid

Proteins • Main functional units of all cells • Provide structure and function • Catalytic proteins are called enzymes • Structure of protein is critical to function

The Cell • Basic building block of

biology • Made up of smaller

organelles – Nucleus

• Site of DNA storage • RNA synthesis takes place here

– Endoplasmic Reticulum • Smooth

– Processing of phospholipids and fats

• Rough – Processing of proteins – Studded with ribosomes –

– Golgi Body • Process and sort secretory and

membrane proteins – Lysosomes

• Degrade particles and dysfunctional units

– Mitochondria • Site of ATP production (power

generation) • ATP powers most functions in

cell – Plasma membrane

• Lipid bilayer • Gatekeeper • Communication

– Cytosol • Contains structural proteins

1) Immobilization a) Adsorption

i) SAM’s b) Entrapment c) Cross-linking d) Covalent bonding

2) Transduction efficiency a) Highly dependent on immobilization method b) Loading factor

i) Usually an optimal density c) Thin layers

i) Mass transport effects d) Specificity of attachment

i) Random attachment leads to “blocked” areas ii) Orientation important

e) Confined attachment i) Allows array fabrication

3) Surface modification techniques a) Self assembled monolayers (SAM’s) b) Light directed synthesis c) Photolithography d) Micromachining e) Ink jet deposition f) Microscopic patterning g) Printing

4) Selectivity and Recognition a) Structural complements

i) Hydrogen ii) Ionic iii) Van der Waals

b) Phase partitioning c) Size or charge exclusion

5) Arrays a) Multianalyte detection and analysis b) Amplification c) Spatial resolution d) Averaging

6) Dimensional considerations a) Where is measurement being made? b) Nano-, micro-, and ultra electrodes c) Scaling considerations and limits

i) Macro analysis may not apply at microscale ii) Concentrations

d) Which dimension is important e) Controllability and repeatability

7) Calibration and Figures of Merit a) Quantifiable Performance Measures b) Calibration uses known concentrations or samples

i) Model formation c) Zero-order

i) One data point ii) Comparison to theory or empirical data

d) First order i) Array of zero order ii) Multiple measurements in time

e) Second order, etc

f) Increasing order allows better multicomponent analysis g) In situ calibration h) Figures of Merit

i) Sensitivity (1) Slope

ii) Limit of determination (1) Based on standard deviations and sensitivity

iii) Linear range iv) Selectivity

(1) Signal overlap (2) Separation schemes (3) Interferents (4) Methods to enhance

(a) Transduction (b) Molecular recognition

v) Response time (1) May depend on “normal” (2) Depends on both instrument and signal processing (3) Measured by 95% or time constant (4) How is sample introduced? (5) Equilibration (6) Refreshing time?

vi) Accuracy vii) Precision

(1) S/N viii) Confidence level

(1) Errors ix) Robustness x) Ease of use xi) Economics xii) Availability

Application Overview

• Each application has its own challenges – Applications similar to analytical chemistry

• Design and characterization (performance) heavily dependent on application – Example of oxygen – Environmental variations

• Concentrations • Media • Temperature • Pressure • Interferents • Matrix effects

– Response time

Environmental Applications • Toxic vapors

– Hydrocarbons • Pollution in both air and water

– Heavy metals – Pesticides – Herbicides

• Explosives • Lab chemicals • Food preparation • Electronic nose

– Multianalyte • Control situations

Bio-Process Control • Pharmaceuticals • Food processing • Recombinant DNA • Microbial and cell cultures • Control (typically online)

– pH – Temperature – Alcohol and sugar levels

– Dissolved gases – Nutrients – Waste removal – Product collection

• In situ measurements limited by – Sterility – Calibration – Lifetime

• Contamination a big issue

Clinical Applications • Medical diagnostics and monitoring • Trend toward decentralized and immediate results • Generally high priority • 10% GNP goes to healthcare • Difficulties

– Measurements outside of controlled environment – Miniaturization – Stability – Biocompatibility – Glucose sensors good example

Ion Selective Membranes • Allow movement of one ion while restricting another • Allow ion selective sensing • Membranes derived from polymers • Complex chemistry to derive selectivity

Acoustic Wave Devices • Mass sensitive technique • Quartz Crystal Microbalance (QCM) • Usually use antibodies • Other factors affect response

Biocatalytic Sensors • Harness enzymatic reactions • Require immobilized enzymes • Important considerations

– Enzyme load – Membrane thickness – Alternative enzymes

• Two catalytic reactions – One substrate, one product – Ping Pong (Two phase)

Current and Future • Many biosensors still not commercialized • Mostly single analyte devices • Some arrays in production • Unlikely to develop perfect biosensor • Future directions

– Improved immobilization methods – Fine tuning of molecular selectivity

• Gene and protein engineering • Replacement of biocomponents which are expensive, unstable, and difficult to derive

– New materials • Designed membranes to improve characteristics • Improve connections for molecular recognition agents

– Specific binding sites on membrane • Understanding and improving interfaces between analyte and transducer • Transducer improvement (carbon composites, etc)

– Multianalyte sensors – High density arrays (especially for genomics) – Miniaturization – Implantation – Integration of components – Small volumes

• Cell interiors • Between nerves

– High spatial resolution – Non aqueous media – Non invasive measurements – Improved material characterization and equipment – Data interpretation (neural networks) – Microseparation systems

1) Enzymes a) Catalyze reactions b) Kinetics

i) Study of reaction rates c) Groups

i) Oxidoreductases (1) Transfer electrons: H-

ii) Transferases (1) Transfer functional groups

iii) Hydrolases (1) Transfer functional group to water

iv) Lyases (1) Transfer groups to or from double bonds

v) Isomerases (1) Transfer groups within molecules

vi) Ligases (1) Transfer by joining groups (2) ATP cleavage

d) Optional conversion routes i) What is final product ii) Where is group transferred to

2) Terminology a) Cofactor or coenzyme

i) Bond with enzyme to allow function ii) When bound called prosthetic group

b) Holoenzyme i) Enzyme with bound coenzyme

c) Apoenzyme i) Enzyme without cofactor or prosthetic group

d) Activity i) Measure of purity and ability of enzyme ii) Given as units of activity per milligram iii) Unit (U) defined as:

(1) Amount to convert 1 µmole of substrate/minute (2) Given a specific pH and temperature

3) Rational design a) Range of enzymes

i) Some enzymes catalyze same reaction but with additional advantages

b) End products c) Reagents d) Consumables e) What is being measured

i) Product ii) Consumption

f) Interferents i) Can they be removed?

g) Multiple sequential reactions 4) Kinetics

a) Factors related to reaction rate b) Plots of v vs. [S]

i) Michaelis-Menten plots ii) Assembled using multiple experiments iii) Identical temp, pH, and enzyme concentration iv) Assumptions (Requirements)

(1) Soluble enzymes (2) Optimal pH for enzyme (3) Initial substrate still in high concentration (4) Increase in [E] increases plateau height

5) Reactions a) E+SàESàP+E

i) k1 is reaction rate 1 ii) k2 is reaction rate 2

iii) [ ]ESkdt

Pdv 2

][==

iv) totEkV ][2max = v) Km is [S] when ½ Vmax occurs

vi) After derivation [ ]][

][][][ 220 SK

SEkESk

dt

PdV

m

tot

+===

b) Lineweaver Burke Plot

i) Derived from above equation ii) Constants can be read right off of plot

iii) ][

111

maxmax SV

K

VVm+=

c) Eadie-Hofstree i) Specific to amperometric biosensors

ii) ][max S

VKVV m−=

d) More complex reactions also occur and modeling has been done for them

6) Immobilization Effects a) Enzymatic behavior altered b) No general trends c) Causes

i) Random orientation ii) Shielding of active site iii) Denaturation iv) Environmental effects v) Microenvironment effects

(1) Local pH (a) Possibly caused by polymer for entrapment

(2) Accessibility/tortuosity (a) Cross linking of polymer

(3) Ionic strength (4) Polarity of membrane/medium (5) Product accumulation

7) Enzyme Inhibition a) Determine concentration of inhibitor b) Competitive and noncompetitive inhibition c) Some loss of selectivity

8) Sequenced Reactions a) Improve detection

i) Easier to detect b) Amplification

i) Heat generation

c) Elimination of interferents 9) Bioaffinity sensors

a) Immunoassays b) Kinetics of “adsorption” or binding c) On and off rates d) Determine “Association” constants

i) Kassoc ii) Kdissoc iii) Typical values 104-1011 M-1

e) Similar kinetics to enzymes f) F is fraction of available sites bound g) Equilibrium based

i) Take minutes to hours

Design Considerations • Use type:

– Disposable, single use, no reagents, no training – Portable, hand held, multiple use with a disposable component,

minimal training – Batch testing or sample injection measurements (Large labs) – In situ devices

• Sterilizable • Compatible with process

– Leaching • Minimal calibration • No additiional reagents

– Research devices

Transduction Modes • Electrochemical

– Potentiometric • ADV: Easily miniaturized, easy translation • DA: Reference required, limited linear range, pH sensitive

– Amperometric • ADV: Variety of analytes, easily mini, dynamic range, selectivity, • DA: Reference required, multiple membranes can be required

– Conductimetric • ADV: Simple, easy to fab, no ref, low frequency source • DA: Non-selective

• Optical – Advantages

• No reference required • Multiple modes: intensity, phase, frequency, polarization • Real time using evanescent waves • Multianalyte arrays simple • Wide range of EM spectra

– Disadvantages • Ambient light and scattering • Limited dynamic range • Miniatuization affects magnitude of signal • Limited selection of chromophores and fluorophores

• Thermal – Advantages

• Works with all reactions • Works with all solutions • Great for offline measurments • Multiple analytes easily

– Disadvanatages • Nonselective • Thermal enzyme probes inefficient • Flow system required

• Mass – Advantages

• Good detection limit • Good for vapors • Easily arrayed

– Disadvantages • Nonselective • Wave propagation problems

Analytes • Wide range of analytes • Wide range of enzymes • Generally, systems reduced to some “standard” form • Produce easily measured products

– H2O2 – O2 – pH – NADH – Labeled antibodies or enzymes – Ions

Chemical Transduction

• Ion selective sensing – Involves transport through selective membrane

• Vary parameters and materials to get different ions – pH electrodes (some optical) – FET

• Dissolved gases – Often coupled to ion sensing through reaction – CO2 + H2O⇔H+ + HCO3

-

– Gas permeable membrane – pH change monitored using pH electrode

• Glass membrane – Selectivity improved by going to other product – NO important in cell signaling

Vapors

• Artificial nose • Important in:

– Exhalation – Anesthetics – Pollutants – Nerve gases, etc

• Requires sorption • SAWs have been used • Neural networks for determination • Enzyme inhibition

Biocatalysis

• See book Pg. 99 for typical reactions • Optical, electrical, and thermal sensing available • Multienzyme systems

– Amplification – Recycling – Sensitivity – Elimination of interferents and products – Step beyond interferents – Suitability of sensor

Bioligand Binding

• Antibodies • DNA/RNA • Lectin and carbohydrates • May have problems with dissociation and regeneration

– Increase steps for washing, etc – May be damaging

Other Considerations

• Trace concentrations – Ultralow concentrations

• Less than 10-9 M

– Fluorescence – Recycling amperometry – ELISA

• Enzyme linked immunoassays

• Temporal Resolution – Dependent on needs – Typically 1 minute is OK

• Spatial Resolution – Confined spaces – “Near” locations – Often require micropositioning and microscopic observation

– Interactions between “sites” • Cross talk • Diffusion

Mass Transport

• Analyte must reach sensor – Internal and external components – Time required

• Equilibrium • Flow rate/reaction rate

• Diffusion – Concentration gradients

• Convection – Stirring

• Migration – External field

• Mass transport changes in vivo • Partitioning and permeability • Modeling and limits

Optical Spectroscopy for Biosensing - 12/15/99 A. Definitions

1. spectrum: an array of the components of an emission or wave, separated and arranged in the order of some varying characteristic (wavelength, frequency, mass)

2. c = λν, E=� ν, �=Planck's constant=6.626×10 -34 J· s 3. absorption, absorption coefficient 4. scatter, scattering coefficient, anisotropy

B. Absorption-based spectroscopy 1. Infrared spectroscopy

a. Theory 1) All atoms in constant relative vibration 2) Frequency of incident energy = frequency of bond vibration � absorption 3) IR active: vibrations resulting in net change in dipole moment

4) Ranges a) Far IR: 50-1,000µm, 200-10cm-1

Difference, coupling b) Mid IR: 2.5-50µm, 4,000-200cm-1

Fundamental c) Near IR: 0.78-2.5µm, 13,000-4,000cm-1

Combinations, overtones (integral multiples), coupling *Combination of all factors � Unique IR spectrum for each compound

b. Practice 1) Dispersive methods 2) FT methods - Felgett (speed), Jacquinot (throughput), Connes (internal reference) advantages 3) Transmission vs. reflectance c. Quantitation (Beer's law) I=I0e

-εlc 1) Transmittance (T): Ratio of transmitted to incident intensity (I/I0) (0-100%) 2) Absorbance (A): log10(1/T)= log10(I0/I) 3) Attenuated total reflection - evanescent wave � thick or highly absorbing samples d. Applications

1) Bioreactor monitoring 2) Food quality inspection

3) Oximetry 4) Bilirubin measurement

MAJOR ADVANTAGES: simple to perform, high SNR (FT-IR), insensitive to scatter (at longer wavelengths), multi-component analysis*

MAJOR DISADVANTAGES: limited pathlength*, *high water absorption, *temperature dependence, *overlapping bands, *weak absorption at short wavelengths

Photoacoustic spectroscopy a. Theory 1) Energy absorbed converts to heat within sample 2) thermal expansion produces pressure waves b. Practice 1) Pulsed laser source a) Tuned to specific wavelength b) Scanned over range � spectrum 2) Focus modulated FTIR beam 2) Sensitive pressure sensor (e.g. piezoelectric) a) single detector vs. array b) time-based signal c. Quantitation d. Applications Strongly-absorbing samples

MAJOR ADVANTAGES: highly sensitive MAJOR DISADVANTAGES: more complex signal processing, prone to saturation at high absorption

2. Emission spectroscopy a. Theory

1) Blackbody radiation 2) Wien's displacement law

b. Practice Heated sample Emission observed by spectrometer Emission bands occur at same frequencies as absorption bands c. Quantitation Similar to absorbance d. Applications

1) Temperature measurement 2) Thermal imaging

MAJOR ADVANTAGES: good for thick samples MAJOR DISADVANTAGES: need to heat

3. Fluorescence Spectroscopy a. Theory 1) Photon absorption � Excitation of molecule to higher energy level 2) Subsequent re-emission of photon, wavelength shifted b. Practice 1) Only small number of molecules exhibit fluorescence 2) Use known "fluorophores" to "tag" target molecules c. Quantitation 1) F ~ concentration, Φ, intensity 2) Quenching - loss of intensity

3) May use energy transfer to advantage (Friday) 4) Photobleaching 5) Fluorescence Lifetime (Friday)

d. Applications 1) Analyte sensing 2) Imaging

MAJOR ADVANTAGES: highly specific, extremely sensitive, simple instrumentation, relatively cheap MAJOR DISADVANTAGES: requires use of exogenous probes, limited choice of chemistry*, *short wavelengths

C. Scatter-based spectroscopy 1. Scattering (Rayleigh) Spectroscopy

a. Theory 1) Rayleigh Scattering occurs with the interaction of light with an atom, or anything producing a refractive index mismatched boundary 2) Scattering strength depends upon size, shape, concentration, and relative refractive index of scatterers (theory by Mie)

b. Practice c. Quantitation d. Applications

MAJOR ADVANTAGES: cheap and easy MAJOR DISADVANTAGES: nonspecific for chemistry

2. Raman Spectroscopy a. Theory

1) Raman Scattering occurs with the interaction of light with a molecular bond. 2) Raman Scattering causes the wavelength of the light to shift

(higher = "Stokes", lower = "anti-Stokes"). 3) The intensity of the Raman Scattered light is much less than the intensity of the Rayleigh scattered light 4) The amount of the wavelength shift and intensity depends on the size, shape, and strength of the molecule. 5) Similar to infrared absorption bands, each Raman shift is a distinct "fingerprint" of the molecule.

b. Practice 1) In order to see the less powerful Raman shifted light, the Rayleigh light needs to be blocked.

2) A spectrograph is used to spread out the Raman shifted light (can do FT also) 3) A photo detector or CCD camera detects the Raman shifted light from the spectrograph.

c. Quantitation Raman Intensity ~ concentration, laser power d. Applications 1) O2, CO2, anesthesia, and other gases expelled from a surgical patient. 2) Emissions of gases into the air by power, oil, steel, and other companies. 3) ANY application where gases, liquids, or solids need to be identified and measured.

MAJOR ADVANTAGES: no sample prep, can get depth-resolved information, PPM detection MAJOR DISADVANTAGES: need laser&expensive filters, weak signals, high fluorescence background, scatter dependence

D. Calibration 1. Univariate a. Peak height b. Area under the curve c. Ratios 2. Multivariate Calibration

a. Classical b. Factor-based methods

3. Wavelength selection

Optical Glucose Monitors - 12/17/99 General Advantages of Optical Biosensors Potentially Noninvasive No Electrical Connections High Bandwidth - Information Density Raman spectroscopy Need low-scattering site - Eye Polarimetry Relies upon rotation of plane of polarization by "chiral" molecules Polarized laser source, polarizers aligned at 90-degrees

àintensity increases as sample rotates polarization Need low-scattering/low-birefringence site - Eye? Other species are chiral, not just glucose Near-infrared spectroscopy Many companies working on this... “Biocontrol” Many papers published No convincing evidence of specific measurements in vivo More useful in process control…monitoring cell culture growthà can measure multiple species

simultaneously Fluorescence spectroscopy

Highly-specific (Probe Required) Invasive - Probe chemistry at tip of optical fiber Minimally Invasive - Implanted glucose-sensitive particles, interrogated transdermally Glucose assay Excited with 488nm

FITC-dextran (520nm emission) and TRITC-ConA (580nm emission) Dextran competes with glucose for ConA binding sites à dextran displaced by glucose Energy transfer from FITC to TRITC à relative shift in peak intensity 520nm peak increases with glucose concentration Currently, poor reversibility with this assay (others are available) Optics of the system relatively good

*Efficiency may be increased by using two-photon excitation

Optical Biosensors • Optical means “electromagnetic” • Types of measurements

– Absorbance – Reflectance – Fluorescence (lifetime also) – Fluorescence quenching – Refractive index and refraction – Light scattering – Polarization

• Changes measured – Intensity – Frequency – Phase shift – Polarization

• May require source and sensor • Spectral components important

Types and Components • Fiber optics • Guided waves • Evanescent waves • Surface plasmons • Light emitting diodes • Total Internal Reflection Fluorescence (TIRF) • Interferometers and gratings • Charge coupled devices (CCD) • Spectroscopy

Developments • Fiber optics for remote sensing • Usage from evanescent waves generated during total internal

reflection • Miniaturization • Near field optics • What is the simplest and easiest optical sensor?

Methods • Capillary optical sensors

– Biomaterial immobilized in capillary

– Fiber optics collect light output from capillary • Reflectance interference spectroscopy

– Reflected light from thin film interferes with light reflected from other surface

– Small changes in thickness can be measured • Adsorbed or bound analyte

• Near IR – Chemometric spectroscopy – Heavily analysis dependent

Fiber Optic Devices • Wide range of optical fibers

– Application depends on light and spectral components – Match to source and detector

• Arrays of fibers can give multicomponent info and spatial resolution • Cladding (IR smaller than fiber) used to reduce signal loss down fiber • Guided waves

– Refractive index changes determineactivity – Large angles: refraction – Critical angle: Total internal reflectance – Small angle: reflection – “Cone of acceptance” or Numerical Aperture (NA) – Modes

Evanescent Wave • Total internal reflection generates interference • Creates a standing wave • Wave extends beyond the boundary of the wave guide

– Evanescent wave • Exponentially decays from surface

– Must be close – Less than 100-200 nm – Ideal for chemically bound layers

• Cladding may be stripped and replaced with biomembrane, etc

Fiber Optic Design • See page 274 of text • Optical fiber carries light to biocomponent

– Absorbance or fluorescence is measured • Light brought in by one fiber, collected by another • Evanescent wave along fiber • Planar wave guide • Fiber bundles

• Generally very versatile

Planar Waveguides • Similar to fiber optic devices • Can add coupling prisms

Near Field Sensing • Eliminates need for high energy beams • Improved resolution • Collection through small appertures • Small magnitudes • Micropositioning

Surface Plasmon Resonance • Works with thin films • Most useful for Biospecific Interaction Analysis (BIA)

– Kinetics of binding • Requires polarized light • Only on metal surface

– Sea of electrons – Waves on “sea” absorb light at specific angles

• Quantum mechanical detection • Only occurs with light at an angle

– Angle depends on film thickness and refractive index – Very sensitive

• Experimentally observed as sharp minimum in intensity measurement

SPR Applications • Gold and silver films most often used

– About 5 nm in thickness • Proteins or antibodies bound to surface • Light injected from opposite surface

– Must be polarized- controlled to thousandths of a degree • Light undergoes total internal reflection • Evanescent wave couples to opposite layer • Reflected light monitored

– Shifts indicate change in film thickness

Optical Sol-Gel Sensors • Transparent gels for entrapment of biochemicals • Allow direct spectrophotometric measurements • Reactants diffuse into gel • Absorbance or luminescence measurements possible • Response time diffusion dependent • Entrapment has little effect on biomolecules • Limited to low molecular weight analytes

Luminescence • Requires light generating molecules • High energy molecules or systems • Chemiluminescence most fundamental • Bioluminescence is enzymatically catalyzed chemiluminescence • Energetics

– Blue (400 nm) 70 kcal/mol – Red (700 nm) 40 kcal/mol

– ATP 7 kcal/mol – ROOR- 100 kcal/mol

Chemiluminescence • Most basic is luminol • Generates a peroxide in presence of oxygen • Works in any assay involving oxygen, peroxide or peroxidase • Can be linked with fluorescence to make dual measurements • All chemiluminescent systems are also fluorescent • Need highly exothermic reaction • Quantum mechanical pathway required • Usually ring structures • All use oxygen

Bioluminescence • Relatively slow (3-20 s) • Components

– Oxidant – Luciferase or photoprotein – Chemiluminescent substrate

• Luciferin • Redox and electron transport

– Cofactors • ATP or NADH

– Fluors – Binding proteins for Protection

• Two main types – Firefly

• Creates peroxide • Quantum yield nearly one

– Bacteria • Flavins as substrate • Quantum yields 10-30 %

1) Photodetectors a) Silicon based generally b) Response independent of wavelength

(above a certain point) c) Many different arrangements d) Based on electronic bandgap in

semiconductors e) Avalanche Photodiodes

i) Junction based system ii) High voltages magnify signal iii) Random process generates noise

f)

2) Charge Coupled Devices (CCD)

a) Metal – insulator – semiconductor devices (MIS)

b) Incident light generates charge on gate i) Induced voltage causes charge separation

c) CCD cameras are arrays of MIS devices i) Can be 10 microns square

d) Charge collected and moved sequentially to edge of device

e) Dyes can be applied to transistor to detect colors

f)

3) Photomultiplier Tubes (PMT)

a) Photoemissive devices b) Generally run at low pressures c) Any incoming energy over work

function of cathode generates cascade d) Noble gases in chamber can be ionize

i) Signal to noise may drop due to shot noise e) Mulitple cathodes can be included to

create cascade (PMT) i) No gas ionization for these systems

f) Extremely high gain (107) g) Very low noise

i) Noise is amplified

ii)

1) Amperometric Sensors a) Measures current flow in system

i) Spontaneously generated (1) Microbe activity

ii) Directed by an applied voltage (1) More typical

iii) Can be linked systems (1) Enzymes attached to antibodies

iv) Setups (1) Attached/ immobilized biocomponent (2) Three types shown below

(a) Also, carbon fiber electrodes

b)

2) Methods

a) Basic materials i) Au, Pt, C, Carbon composites, and conducting salts

(1) Material limits range of applied voltages ii) Solvent can also limit range of applied voltage

b) Operation i) Potential step

(1) Applied potential critical to experiment (a) Overpotential may be required

(i) Varies with material (ii) Highly empirical

(b) Scanning methods may be helpful to find optimum

(2) Current measured for ms to seconds (a) Double layer charging and electron transfer

(3) Chronoamperometry (a) Current with time

(4) Chronocoulometry (a) Current is integrated to get charge transfer

(5) Amperometry (a) Sample current at specific time

ii) Mass transport governs (1) Diffusion through membrane (2) Alleviated by allowing electrode to go to

steady state before introducing analyte

iii) iv) Can measure variety of species (see sensing

options) (1) Note limitations of each sensing option

(2)

(3)

(4) Cyclic voltammetry

(5)

1) Electroanalytical Biosensors a) Involve charge and electron transfer

i) Potential ii) Current iii) Conductance iv) Impedance

b) Most common for practical devices c) Wide variety of methods (see chart page

208) d) Amperometry

i) Current measurement e) Voltammetry

i) Current as a function of variable voltage f) Chronoamperometry

i) Current as a function of time at constant voltage

2) Electrochemistry

a) Electrochemical cell b) Galvanic

i) Spontaneous reaction generates current or voltage

c) Electrolytic i) External energy source ii) Current flows related to

oxidation/reduction levels d) Cyclic voltammetry

i) Scan between voltages e) Nernst potential and equation

i) O+neàR ii) [ ]

[ ]R

O

nF

RTEE ln+= o

iii) Standard hydrogen electrode is basis iv) Practical use, silver/silver chloride or

calomel (Hg) (1) Typically difficult in practice,

especially in vivo (2) Short duration

v) Microsystems avoid some of the problem by using low currents

3) Potentiometric sensors

a) Ideal for ions and dissolved gases that produce ions

b) No current flow (critical assumption) c) Systems, while simple in appearance,

often quite complex d) Polymer membrane

i) Limit access ii) Selective to specific ions

e) Solid state i) Must convert ions to electrons ii) Problems with stability and

reproducibility iii) Metal wires with applied films

iv)

4) Field Effect Transistors

a) Current from source to drain related to gate voltage

b) Application of membranes to gate allows selective measurements

c) Problems i) Membrane adhesion ii) pH sensitivity iii) Drift iv) Coatings can help eliminate all v) Nonlinear

1) Amperometric Sensors (cont) a) Oxygen species

i) Several potentials at which charge transfer occurs ii) Some byproducts are highly toxic iii) Addition of appropriate enzymes can limit toxicity

b) NADH i) Involve dehydrogenases ii) Potentially hundreds of related reactions iii) Requires soluble coenzyme

(1) External reagent (2) Expensive (3) Difficult

iv) Mediators improve process (1) Enhance electron transfer (2) Lower required voltage (3) Coating on electrode

c) Detection limits i) Very low detection limits

(1) Especially with amplification ii) Immunosensors with attached enzymes

2) Other Electronic Biosensors a) Conductivity and Impedance

i) Inherently non-selective ii) Require modified surfaces for selectivity iii) No reference electrodes required iv) Easy and inexpensive to fabricate

b) Most basic configuration is Wheatstone bridge c) Both time and frequency domain measurements possible d) Involve both capacitive and resistive elements

i) Magnitude and phase measurements e) Added enzyme sensors paired with reference for differential measurement

i) Several relevant enzymes have been used f) One example included pH sensitive hydrogel on surface of electrodes

i) Change in pH changed resistance through membrane g) Impedance Spectroscopy

i) Impedance sweeps ii) Similar applications to optical spectroscopy

(1) Not as “varied” 3) Bioelectric Interfaces

a) Measurement of electrical activity in the body b) Generally very low signal levels c) Non-selective d) Neural Recording Arrays

i) Attempt to access multiple neurons simultaneously ii) Attempts at cortical prostheses iii) Used for both recording (sensor) and stimulation

e) Regeneration neural electrodes f) Cell interfaces g) Cultured cell systems

Microfluidic Sensors Example University of Washington H Sensor

Methods for Fabrication KOH etching of silicon Other etching methods (High Aspect Ratio) LIGA Molding in PDMS Interesting Ideas Bubbles for added power CD systems Systems in Ceramics, Polymers, Silicon Surface tension valves Problems Valves Pumps Seals Mixing Diffusion

1) Antibodies a) Produce by body in response to antigens b) Have specific binding domains

i) Epitope or determinant c) Y shaped in general

i) Symmetric d) Heavy and light chains e) Binding site at tip of Y

i) Made up of 20-30 amino acid sequence f) Base of Y involved in activation of

complement and other immune components g) Polyclonal

i) Antibodies from a variety of immune cells that bind to an antigen

ii) Usually have different epitope h) Monoclonal antibodies

i) All produced by same cell ii) Generated by cloning iii) These are typically used in biosensors

2) Immunoassay

a) Based on Ag-Ab binding b) Homogenous

i) No washing steps ii) Free and bound antibody does not need

to be separated iii) Usually binding causes change that

allows bound and unbound antibody to be distinguished

c) Heterogenous i) Require washing and separation ii) Displacement

(1) All sites are filled with labeled antigen

(2) Unlabeled antigen displaces labeled antigen

iii) Competitive (1) Labeled and unlabeled antigen added

simultaneously (2) Amount of labeled antigen bound

gives info iv) Sandwich

(1) Capture antibody is immobilized (2) Analyte is added and bound to

antibody (3) A labeled antibody is added

generating signal (4) Requires antigen with two

determinants d) ELISA

i) Enzyme linked immunoassay

1) Random Homogenous Immunosensor Types a) CEDIA

i) Clone enzyme donor immunoassay ii) Engineered antibodies and enzymes iii) Competitive assay iv) Existence of analyte allows active

enzymes to form v) Convert chromogenic substrate to dye vi) Linear

(1) Most competitive assays are non-linear

vii) Signal proportional to analyte present b) ARIS

i) Apoenzyme reactivation immunosystem (1) Competitive assay (2) Allows enzyme activation in the

presence of antigen c) SLFIA

i) Optical system ii) Enzyme release fluorogenic substance in

presence of analyte iii) If not bound to antigen, antibody blocks

formation of fluor

d) EMIT i) Matched enzymes and antibodies ii) Antigen activates enzyme

e) ICS i) Ion Channel Switch

(1) Measure of changing conductance across a membrane

(2) Competitive assay (a) Conductance increases if analyte

present (3) Sandwich assay

(a) Conductance decreases if analyte present

f)

Thermal Sensors a) Based on heat generated b) H=Cp∆T c) ∆T not always large

i) Requires amplification ii) Multiple enzymes produce multiple

reactions iii) Good thermistors and thermopiles can

detect 10-5 K d) Specific heat can be modified using

different solvent i) Enzyme function in other solvents not well

known e) Thermistor

i) Resistor whos value changes with temperature

f) Thermocouple i) Based on Seebeck effect ii) Dissimilar metals generate voltage

difference at interface g) Thermopile

i) Serially linked thermocouples

Acoustic Devices (a) TSM- Shear Thickness Mode (b) SAW- Surface Acoustic Wave (c) STW- Surface Transverse Wave (d) FPW- Flexural Plate Wave (e) SH-APM- Shear Horizontal Acoutic

Plate Mode (f) Challenges

(i) External changes affect resonant frequency

1) DNA Hybridization Arrays a) Manufacturing

i) Mechanical printing (1) Soft lithography

ii) Ink jet iii) Photolithography

b) Applications i) Gene expression

(1) Usually looking for RNA expression (2) Differences between cells (3) Differences in time

ii) Polymorphisms (1) Change in base pairs (2) Often looking for single base pair

change iii) Comparative genomic hybridization

(1) Compare entire genomes (2) Cannot detect small deletions

c) Problems i) Sampling

(1) Homogenous cell samples (a) Types, cell cycle (b) RNA quantity for single cells

(i) Requires amplification d) Detection

i) Usually fluorescence ii) Can use multiple tags to distinguish

samples 2) PCR

a) DNA Amplification technique b) DNA collection

i) Cell lysis 3) LCR

a) Amplification technique and sensor in one (if desired)

b) Similar to PCR c) Uses DNA ligase rather than polymerase d) Binds two strands together at the end

after binding to complementary strand e) Sensing accomplished by using strands

with attached fluor i) If binding not possible, fluor is lost in

washing step

1) Chromatography a) Separation Systems b) Usually incorporate

i) Carrier (mobile phase) ii) Stationary phase iii) Sample

c) Gas Chromatography d) Liquid Chromatography

i) Ion-Exchange ii) Normal Phase iii) Reverse Phase iv) Gel Permeation v) Affinity vi) Hydrophobic

e) Size exclusion chromatography f) Electrophoresis g) Field-Flow Fractionation

i) Multiple types