Label-free and Reagent-free DNA Detection Based on Supramolecular Electrochemistry Hiroshi AOKI...
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Transcript of Label-free and Reagent-free DNA Detection Based on Supramolecular Electrochemistry Hiroshi AOKI...
Label-free and Reagent-free DNA Detection
Based on Supramolecular Electrochemistry
Hiroshi AOKI
National Institute of Advanced Industrial Science and Technology (AIST), Japan
November 29th-30th, 2010
NANOJASP2010
Barcelona, Spain
Contents
1. Introduction
2. Approach based on probe conformation
3. Approach based on signal-generating/suppressing moieties
4. Summary
1
Mechanism of Biological Effect
bodybody
cellcell
environmentenvironment
nucleolusnucleolustransciption
biological biological effecteffect chemicalschemicals
gene expression
proteinsproteinsenzymesenzymes
Mechanism of Biological Effect of Chemical Substances
Gene expression allows to evaluate biological effect at a genetic level
and to collect genetic information rapidly without animal tests.
DNA microarray is widely used in a laboratory level as a comprehensive
diagnosis tool for gene expression in medical and environmental fields.
mRNAsmRNAs
DNAmicroarray
extraction
chemical exposure
cell
2
A conventional technique of oligonucleotide detection--- Spectroscopic technique based on fluorescence labeling
Background
DNA microarray
- Time-consuming and expensive process
- Affection to quantitativity
- Needing a wash for removing unbound targets
Present problemsPresent problems
fluorescentlabel
Hybridization
probe h
Probe array
target
DNA microarray scanner(company A)
Size: 60 x 90 x 60 [cm]
Example
We have studied on these problems by electrochemical approaches, expected to contribute simplifying and size-reducing the detection system.We have studied on these problems by electrochemical approaches, expected to contribute simplifying and size-reducing the detection system.
3
solid surface
Approach based on electrostaticts
Hybridization changes the surface charge, inhibiting redox reaction of the marker, enables label-free target detection.
Approach based on probe conformation
Hybridization makes the probe structure more rigid, inhibiting redox reaction of Fc, enables label- and reagent-free target detection (self-reporting DNA detection).
Approach based on signal-generating/suppressing moieties
Hybridization makes the probe structure more rigid, restoring redox reaction of Fc, enables self-reporting and “signal-on” target detection (“OFF”→“ON”).
Approaches
ON OFF
ON OFF
ONOFF
Simple and rapid electrochemical gene detection techniques
signal-generating moiety: Fc
signal-suppressing moiety: β-CD
electroactive moiety: Fc
electroactive marker: [Fe(CN)6]4-
4
Scheme of electrochemical experiments
Electrochemical measurement- cyclic voltammetry (CV)- square wave voltammetry (SWV)
target DNAsin 0.1 M NaClO4
+ 2.5 mM phosphate buffer (Na+, pH 7.0)
in 0.1 M NaClO4
+ 2.5 mM phosphate buffer (Na+, pH 7.0)
Ag/AgCl reference electrode
Pt auxiliary electrode
Modification
Incubation Measurement
after incubation for 20 min, at 65 oC, cooling down to 25 oC
5’GCA ACC TTC CCT ATT ACT CCA C 3’3’CGT TGG AAG GGA TAA TGA GGT G 5’3’ATG ACA CCA ATA ACG ACA GA 5’
Fc-PNA:DNA_1:DNA_2:
probe Fc-PNAcomplementary to Fc-PNAmismatched to Fc-PNA
Sequences for the probe and targets
5Approach based on probe conformation
anchorpeptide nucleic acid (PNA)signal
5’ 3’probe solution
electrode
- probe: 0.1 mM Fc-PNA for 2 h
- thiol: 1 mM 11-HUT for 12 h
Fc-PNA
11-HUT
Evaluation of probe surface density based on electron transfer reaction
Fc-PNA
gold electrode
CV for the electrode in 0.5 M KOH
Potential / V vs Ag/AgCl-0.4 -0.6 -0.8 -1 -1.2 -1.4
-0.5
0
0.5
1
1.5
2
CV
Scan rate: 0.1 V s-1
S-Au: 32.9 pmol cm-2 surface density of S-Au bonds
RS–Au + e– RS– + Au0
CV for the electrode in a buffer solution
Potential / V vs Ag/AgCl0 0.2 0.4 0.6
40
0
20
40
20
CV
Scan rate: 0.01 V s-1
Fc: 33.1 pmol cm-2 surface density of Fc
Fc0 Fc+ + e-
Every probe is immobilized on the surface, keeping its Fc moiety.
6Electrochemical characterization
v, Scan Rate / V s-1
50
100
150
00.1 1 10 1000.01
diffusion-like motion
surface-confined motion
Change in probe flexibility — before hybridization
Scan rate:0.2 V s-1 – 51.2 V s-1
CVs
Ep
0 0.2 0.4 0.6
0
-1
-2
1
2
Cur
rent
· (
Sca
n R
ate)
-1 /
µA
s V
-1
Potential / V vs Ag/AgCl
Dependence of CVs on scan rate (DNA_1 = 0 M)
More irreversible CVs were measured at higher scan rates, indicating the Fc moiety needs more time for diffusion to cause electron transfer.
peak potential separation, Ep, vs scan rate, v
Scheme 1: Thermal vibration
* H. Aoki and H. Tao, Analyst 2007, 132, 784-791.
Access to the surface proceeds to Fc redox reaction
bulk
surface
7Change in probe flexibility
Plot of log(anodic peak current, ipa) vs log(scan rate, v)
log(v, Scan Rate / V s-1)-2 -1 0 1 2
-2
0
2
1
-1
slope ~1
slope ~1/2
diffusion-like motion
surface-confined motion
From slopes in the plot of log ipa vs log v,
surface-confined motion
diffusion-like motion
cvv slope ~1/2,2.21
pa vi
vv c slope ~1,1. vi pa1
The change in the Fc character revealed that the Fc moiety is located at the loose end of the probes, subject to thermal vibration.
vc
CVs
ipa
0 0.2 0.4 0.6
0
-1
-2
1
2
Cur
rent
· (
Sca
n R
ate)
-1 /
µA
s V
-1
Potential / V vs Ag/AgCl
Change in probe flexibility — before hybridizationDependence of CVs on scan rate (DNA_1 = 0 M)
* H. Aoki and H. Tao, Analyst 2007, 132, 784-791.
8Change in probe flexibility
log(v, Scan Rate / V s-1)-2 -1 0 1 2
-2
0
2
1
-1
v, Scan Rate / V s-1
50
100
150
00.1 1 10 1000.01
change in flexibility
10-4 M
0 M
0 M
10-4 M
change in flexibility
Upon hybridization, the value of scan rate at which the motion changes (vc) was shifted to be lower. This suggests the decrease in probe flexibility. Detection of target DNAs using this change in Fc character
Plot of log(anodic peak current, ipa) vs log(scan rate, v)
Change in probe flexibility — after hybridizationPeak potential separation vs scan rate (DNA_1 = 0 M)
e–
before hybridization
after hybridization
Scheme 2: Change in probe flexibility
9Approach based on probe conformation
Potential / V vs Ag/AgCl0 0.2 0.4 0.6
0
1
3
2
Potential / V vs Ag/AgCl0 0.2 0.4 0.6
-3
-1
0
-2
1
3
2
DNA concentration dependenceDependence of CVs on DNA concentration
Dependence of SWV on DNA concentration
10-4 M of DNA_1
0 M
10-4 M DNA_1
0 M
SWVsStep potential: 2 mVAmplitude: 25 mVFrequency: 50 Hz
i o – i baseline
i – i baseline
CVs
(i –
i ba
selin
e)
/ (i o
– i ba
selin
e)
Concentration / M0 10-14 10-12 10-10 10-8 10-6 10-4
1.2
1
0.8
0.6
0.4
0.2
0
DNA_2 (mismatch):10–4 M
DNA_1 (complementary)Detection limit: 1.4 x 10–11 M
Sensor response dependence on target concentration (SWV)
Detection limit: 1.4×10-11 M (S/N = 3.0)
Sequence-specific DNA detection was achieved based on the change in the probe flexibility without labeling targets nor adding external markers (“self-repoting”).
Scan rate: 1 V s-1
* H. Aoki and H. Tao, Analyst 2007, 132, 784-791.
10Hybridization with target DNAs
Potential / V vs Ag/AgCl0 0.2 0.4 0.6
0
0.5
1
Repeated use of the prepared DNA sensors
Regeneration of the sensors
rehybridization(10-4 M of DNA_1)
denaturationSWVs
Step potential: 2 mVAmplitude: 25 mVFrequency: 50 Hz
Relative change in peak currents in regeneration process
- 1…1st measurement (right after prep.)- 2, 4…1st and 2nd hybridization DNA_1, 10–4 M- 3, 5…1st and 2nd denaturation ( in 2 M urea, 65 oC )- 6…mismatched DNA_2, 10–4 M
0
0.2
0.4
0.6
0.8
1
1.2
1 2 3 4 5 6
Process Number
1st measurement
DNA_1, 10–4 M
DNA_1, 10–4 M
denaturation
denaturationDNA_2, 10–4 M
The electrodes modified with Fc-DNA monolayer can be used repeatedly.
* H. Aoki and H. Tao, Analyst 2007, 132, 784-791.
11Repeated use of the sensors
Molecular beacon (PNAS, 2003, 100, 9134 (Plaxco et al.))
Change in flexibility (JACS, 2003, 125, 1112 (Anne et al.))
Electron wire (PNAS, 2005, 102, 11606 (Inouye et al.))
Aptamer (Angew. Chem., 2005, 44, 5456 (Plaxco et al.))
Aptamer (JACS, 2006, 128, 117 (O’Sullivan et al.))
“Self-reporting” probes from other research groups
Detection limit: 10 pM
Detection limit: 5 µM
Detection limit: 100 µM
Detection limit: 6.4 nM
Detection limit: 0.5 nM
Change in flexibility (Analyst, 2007, 132, 784 (Aoki et al.))
“ON” “OFF”
DNA
Fc-PNA
Detection limit: 14 pM
Improvement of sensitivity due to high Tm and flexibility in PNA
12Other contemporary probes
Almost of all reported probes were based on a “signal-off” architecture.
probe
Hybridization
electrode“OFF” “ON”
e–
Approach based on a “signal-on” architecture
Development of label- and reagent-free (self-reporting),
and “signal-on” probes.
Development of label- and reagent-free (self-reporting),
and “signal-on” probes.
Patent application: -JP patent application 2008-168546, 2009-208400, 2009-242921, 2010-193207
Hybridization
“ON” “OFF”
Fc
e–
marker : [Fe(CN)6]4–
Hybridization
“ON” “OFF”
e–
Label-free
Reagent-free
Self-reporting
Self-report&“signal-on”
Suppressing redox activity“OFF”
Fe
Inclusion complexInclusion complex
β-cyclodextrin
ferrocene
Restoring redox activity“ON”
Fe
dissociationdissociation
e–
Hybridization
DNA
“Signal-on” architectures have advantage of higher sensitivity over “signal-off” ones.
13
Improvement of sensitivity for DNA detection The use of probes emitting signals upon hybridization, i.e., “signal-on” probes, is important.
Approach based on a “signal-on” architecture
Scheme of electrochemical experiments
3.125 pmol, 5 µLCD-DNA-Fc
in 7.5 mM NaCl+ 75 mM phosphate buffer (Na+, pH 7.0)
Measurement
Electrochemical measurement--- cyclic voltammetry (CV) scan rate: 0.01 V s-1
Interdigitated arrayelectrode (carbon)
Width: 10 µmGap: 5 µmLength: 2 mmNumber: 65
5’GCA ACC TTC CCT ATT ACT CCA C 3’3’CGT TGG AAG GGA TAA TGA GGT G 5’
CD-DNA-Fc:DNA:
CD-DNA-Fc (22 mer)complementary to CD-DNA-Fc
Sequences of the probe and target
14
e– e–
Fc
β-CD
hybridizationelectrode
Detection system using probes
without anchors
enables detection in
bulk solutions.
O
HO
OHHO
O
OHO
OH
HO
O
O
HO
HO
O
O
OH
HO OH
O
O
OHHO
OHO
OOH
HO
OH
O
O
OH
OH
HO
O
beta-CD
N
OHN
OO
SOP
O
O
OO
PO O
PO
O
O
O
O
Nu
O
n
5' 3'
OP
O
O
O
HN
O
Fe
2 3
CD-DNA-FC
0.1 0.2 0.3 0.4 0.5
0
10
20
Potential, E / V
Cur
rent
, I
/ nA
0 0.1 0.2 0.3 0.4 0.5 0.6-20
-10
0
10
20
Potential, E / V
Cur
rent
, I
/ nA
Results
Dependence of CVs on target concentration
12.5 pmol
0 mol
CVs
Fe
e-
Fe
e-
Ferrocene moiety
・ Redox potential: negative shift of Δ62 mV
・ Current change: 1.3 nA 6.9 nA (5-fold) The redox activity of Fc was restored.
-6
-4
-2
0
0 0.1 0.2 0.3 0.4 0.5Potential / V vs AgAgCl
Cur
rent
/ µ
A
Step E: 2 mVAmplitude: 25 mVFrequency: 50 Hz
SWVs
e-
Fe
OH e-
Fe
OH
[ref.] SWV of HMFc ( 0.3 mM ) + β-CD ( 15 mM )
12.5 pmol
0 mol
Article: Supramol. Chem., 22, 455 (2010)
15
The probe works based on a self-reporting “signal-on” architecture.
Summary 16
e– e–
Fc
β-CD
hybridizationelectrode
“ON” “OFF”
1. Based on conformational flexibility change in probe structure, label-free and reagent-free (“self-reporting”) DNA detection was achieved.
2. Based on signal-generating and suppressing moieties in a probe, “self-reporting” and “signal-on” DNA detection was achieved.
These approaches are expected to contribute to more simple and rapid DNA detection.These approaches are expected to contribute to more simple and rapid DNA detection.
Tsukuba, 45 min from Tokyo
Mt. Tsukuba
Tsukuba Express (TX), established in 2005
National Institute of Advanced Industrial Science and Technology (AIST)
Japan Aerospace Exploration Agency (JAXA)
National Institute for Materials Science (NIMS)
University of Tsukuba
At last … What is AIST? 17
Acknowledgements
Collaborators- Prof. Emeritus Yoshio UMEZAWA (Univ. Tokyo)- Prof. Masao SUGAWARA (Nihon Univ.)- Prof. Koji TOHDA (Univ.Toyama)- Prof. Philippe BUHLMANN (Univ. Minnesota)- Prof. Sandra RONDININI (Univ. Milan)- Prof. Marcin MAJDA (UC Berkeley)
- Dr. Hiroaki TAO (AIST)- Dr. Masaki TORIMURA (AIST)- Dr. Hiroaki SATO (AIST)- Dr. Hanna RADECKA (Polish Academy of Science)- Akiko KITAJIMA, Tsutomu FIJIKAKE
Financial Supports- MEXT: Grant-in-Aid for Young Scientists (B) (Nos. 19750068, 21750085) Grant-in-Aid for Scientific Research Innovative Areas (No. 21106523)- JST: Research Grant for Promoting Technological Seeds (No. 04-015)
Thank you for your attention!
18