electrochemical dna biosensors exploiting changes in the...
TRANSCRIPT
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Electrochemical DNA Biosensors Exploiting Changes in the Charge
Transfer Properties of DNA
e-
DNA-Modified Electrodes Cell-Surface interactions Immunosensors
NO2
CO
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
BNHCO
H2CFe
H2C
GC
Prof. J. Justin Gooding School of Chemistry The University of New South Wales
Wiring Enzymes Nanoparticle Biosensors .
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Portable DNA Biosensors
DNA Detection
Labelling and fragmentation DNA amplification
Sample input
DNA extraction
SIMPLICITY
Cell identification and capture
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Probe DNA
Hybridization
Target DNA
DNA Biosensors
E.L.S. Wong, E. Chow, J.J. Gooding, Langmuir 21 6957-6965 (2005).
Reference frequency
mass Area of quartz
€
Δf = −2.3×106 fo2 Δm
A
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Hybridization
Probe DNA Target DNA
DNA Biosensors
Label
A. Erdem et al Electroanalysis 11 586 (1999), W. Yang et al Electroanalysis 14 1299 (2002)
-400 -300 -200 -100 0 100
Potential /mV
50 µA
a
b
c
a: bare electrode b: ss-DNA c: ds-DNA
S
N
(H3C)2N N(CH3)2
+
methylene blue
Interested in detecting DNA hybridization using differences in properties of single and double strands DNA
1) by change in persistence length
2) change in charge transfer properties of DNA
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DNA Biosensors Based on Long-Range Charge Transfer
1 S.O. Kelley et al Nucleic Acids Res. 24 4830 (1999) 2 E.M. Boon et al Nature Biotech. 18 1096 (2000). http://www.geneohm.com/
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DNA Biosensors Based on Long-Range Charge Transfer
• Barton and colleagues can differentiate between ssDNA, a complete duplex and one with a mismatched. • Needed complete surface of duplexes to achieve reliability.
S.O. Kelley et al Nucleic Acids Res. 24 4830 (1999) E.M. Boon et al Nature Biotech. 18 1096 (2000). http://www.geneohm.com/
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Further Interfacial Considerations
HS-(CH2)6-OH
Target ssDNA e- X e-
Redox Indicator
O
O
SO3-
-O3S
Redox Intercalator 2,6-disulfonic acid anthraquinone
Levicky et al J. Am. Chem. Soc. 119 8916 (1997) E. Wong, J.J. Gooding, Anal. Chem. 75 3845-3852 (2003).
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(i) Rinsing (ii) Voltammetric Experiments in Intercalator Free Solution
(i) Rinsing (ii) Voltammetric Experiments in Intercalator Free Solution
Probe ss-DNA
Target ss-DNA
Redox active intercalator
6-Mercapto-1-hexanol
E. Wong, J.J. Gooding, Anal. Chem. 75 3845-3852 (2003).
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-0.4
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
-700 -500 -300 -100
Exposure of DNA Recognition Interface to Target ssDNA
0.05
0.15
0.25
0.35
0.45
-650 -550 -450 -350 -250Potential / mV
Cu
rren
t /
µA
Cu
rren
t /
µA
Potential / mV
E
t
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Exposure of DNA Recognition Interface to Target ssDNA
Potential / mV
Cu
rren
t /
µA
Cu
rren
t /
µA
Potential / mV
-0.4
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
-700 -500 -300 -1000.05
0.15
0.25
0.35
0.45
-650 -550 -450 -350 -250
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Changes in Spatial Relationships
Δh = 1 nm Δh = 4.5 nm
Complementary 20mer
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0
2
4
6
8
10
12
14
16
Exposure of DNA Recognition Layer to different target ssDNA
Cur
rent
/ µ
A cm
-2
Potential / mV
Non
-Com
plem
enta
ry
Bef
ore
Hyb
ridis
atio
n
Com
plem
enta
ry Cur
rent
Den
sity
/ µ
A cm
-2
0.05
0.15
0.25
0.35
0.45
-650 -550 -450 -350 -250
E.L.S. Wong, J.J. Gooding, Anal. Chem. 75 3845-3852 (2003).
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Single-Base Mismatch Complementary
Single-base C-A
Mismatch Single-base
G-A Mismatch Probe ssDNA
A
T C G
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Exposure of DNA Recognition Layer to different target ssDNA
Cur
rent
/ µ
A cm
-2
Potential / mV
0
2
4
6
8
10
12
14
16
Non
-Com
plem
enta
ry
Bef
ore
Hyb
ridis
atio
n
Com
plem
enta
ry
G-A
Mis
mat
ch
C-A
Mis
mat
ch C
urre
nt D
ensi
ty / µ
A cm
-2
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
-650 -550 -450 -350 -250
E.L.S. Wong, J.J. Gooding, Anal. Chem. 75 3845-3852 (2003).
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Why can we detect mismatches?
Mismatch about here
E.L.S. Wong, J.J. Gooding, Anal. Chem. 75 3845-3852 (2003).
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Further evaluation
5′-GGGGCAGTGCCTCACAACCT-p-(CH2)6-SH-3′
5′-SH-(CH2)6-p-AGGGATGCCTGGACACAAGA-3′
0
2
4
6
8
10
12
14
16
Cocktail of Target DNA
Cur
rent
Den
sity
/ µ
A c
m-2
4 µ
M C
ompl
emen
tary
4 µ
M C
ompl
emen
tary
&
4 µ
M S
alm
on T
este
s
4 µ
M C
ompl
emen
tary
&
4 µ
M S
alm
on S
perm
4 µ
M C
ompl
emen
tary
&
4 µ
M C
alf T
hym
us
4 µ
M C
ompl
emen
tary
&
4 µ
M N
on-C
ompl
emen
tary
E.L.S. Wong, F.J. Mearns, J.J. Gooding, Sensors Actuators B 111-112 515-521 (2005).
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Pros and Cons 1) Excellent selectivity
• Can detect single base pair mismatches without stringency washings
• Can detect complementary sequence from a DNA cocktail without loss in sensitivity
2) Current assay time: • Hybridisation ~ 120 min; • Detection (intercalation) ~180 min; • Total time 5 hours TOO LONG!!
3) Detection limit: • With best interface LOD = 100 nM
4) Too complicated • Three steps in the measurement
E.L.S. Wong, P. Erokhin, J.J. Gooding, Electrochem. Comm. 6 648-654 (2004) E.L.S. Wong, F.J. Mearns, J.J. Gooding, Sensors Actuators B 111-112 515-521 (2005).
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In situ experiment
-0.4-0.3-0.2-0.10.00.10.20.3
-700 -500 -300 -100
Potential / mV C
urre
nt / µ
A
-20-15-10-5051015
-800 -600 -400
Potential / mV
Cur
rent
/ µ
A
- 550 mV - 474 mV
AQDS in Solution Intercalated AQDS
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Voltammetric Experiments in intercalator Solution
e- e-
E.L.S. Wong, J.J. Gooding, Anal. Chem. 78 2138-2144 (2006).
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In Situ Assay
0
0.2
0.4
0.6
0.8
1
1.2
-700 -600 -500 -400 -300Potential / mV
Cur
rent
/ µ
A
-474 mV Increases with time
DNA Modified electrode 25 µM AQMS 4 µM complementary target ssDNA.
O
O
SO
OO -
1-
0
10
20
30
40
50
60
70
80
90
-700 -500 -300 -100 100
Potential / mVC
urre
nt /
nA
60 minutes
75 minutes
105 minutes
90 minutes
120 minutes
E.L.S. Wong, J.J. Gooding, Anal. Chem. 78 2138-2144 (2006).
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-10
0
10
20
30
40
50
60
70
80
-700 -500 -300 -100Potential / mV
Cur
rent
/ nA
Complementary Non-ComplementaryC-A Mismatch G-A Mismatch
Target DNA
Sequential Current Ratio
In Situ Current
Ratio
Comp : G-A 2.33 : 1 4.2 : 1
Comp : C-A 6.00 : 1 15 : 1
0
0.5
1
1.5
2
2.5
3
3.5
1 2 3 4
Cu
rren
t D
en
sit
y / µ
A c
m-2
Non
-Com
plem
enta
ry
Com
plem
enta
ry
G-A
Mis
mat
ch
C-A
Mis
mat
ch
Type of Target DNA
E.L.S. Wong, J.J. Gooding, Anal. Chem. 78 2138-2144 (2006).
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Performance of the in situ assay 1) Current assay time one hour
• Hybridisation and detection (intercalation) ~60 min
2) Detection limit: • With best interface LOD = 0.5 nM (1 pmol)
3) Simplicity, requires only on handling step, addition of the sample plus intercalator
4) In situ assay can also monitor hybridization in real time and be used to probe any event that affects DNA base pairing
E.L.S. Wong, J.J. Gooding, Anal. Chem. 78 2138-2144 (2006).
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Where we are going 1) Detecting pathogens (cholera) 2) Improving the system performance • Reduce detection limit
Smaller electrodes Electrodes with lower background capacitance Better surface chemistry
• More stable surface chemistry • Make more compatible with microfabrication
Silicon
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OO
OH
8 3
OO
O
8 3
OO
8 3
OH
S
OO
8 3
OH
S
HS 3'TCCAACACTCCGTGACGGGG5'6
5'AGGTTGTGAGGCACTGCCCC3'-TAMRA
OHHNR
OH2N-R
(a) (b)
Single step fabrication of DNA chips
Linford et al. J. Am. Chem. Soc. 1995, 117 3145.
SiNH4F
h*ν or heatSilicon
SiO2
SiSi
SiSi
Si
SiSi Si
Si
SiSi
Si
SiSi
Si
SiSi Si
Si
H H H H
R R
R
T. Böcking, K.A. Kilian, K. Gaus, J.J. Gooding, Langmuir, 22, 3494-3496 (2006).
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Contact angle measurements
Surface modification contact angle
74º
<10º
OO
8 3Si
O
X-ray photoelectron spectroscopy
OO
8 3
OH
S TCCAACACTCCGTGACGGGG6
Si
T. Böcking, K.A. Kilian, K. Gaus, J.J. Gooding, Langmuir, 22, 3494-3496 (2006).
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Fluorescence intensity as a function of target concentration of DNA1 for *n=4, **n=3 independently prepared samples.
0.25 cm
complementary DNA1
0.25 cm
non-complementary DNA2
T. Böcking, K.A. Kilian, K. Gaus, J.J. Gooding, Langmuir, 22, 3494-3496 (2006).
SAM modified surface
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Click Chemistry on Silicon
S. Ciampi, T. Böcking, K.A. Kilian, M James, J.B. Harper, J.J. Gooding, Langmuir 23 9320-9329 (2007).
Huisgen 1,3-dipolar cycloaddition of terminal alkynes to azides
S. Ciampi, P.K. Eggers, G. Le Saux, M. James, J.B. Harper, J.J. Gooding, Langmuir 25 2530-2539 (2009).
Oxygen free
Oxygen present
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Where we are going 1) Detecting pathogens (cholera) 2) Improving the system performance • Reduce detection limit
Smaller electrodes Electrodes with lower background capacitance Better surface chemistry
• More stable surface chemistry • Make more compatible with microfabrication
3) Using the system to understand DNA surface chemistry and disruption of DNA base pairing
Silicon
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Using the surface structure to influence the performance
Diluent < DNA Linker Diluent = DNA Linker Diluent > DNA Linker
4.94 Å
67.3 Å 70.4 Å
10.27 Å 16.96 Å
E.L.S. Wong, E. Chow, J.J. Gooding, Langmuir 21 6957-6965 (2005).
88 95 90 75 60 75
Hybridization efficiency
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Calibration Curve
0
20
40
60
80
100
-7 -6.5 -6 -5.5 -5 -4.5
MCE/3C LinkerMCH/3C LinkerMCU/3C LinkerMCE/6C LinkerMCH/6C LinkerMCU/6C Linker
Log([Target DNA])
Nor
mal
ised
Res
pons
e Ka(MCE/6C Linker) = 8.32 x 106
Ka(MCE/3C Linker) = 7.08 x 106
Ka(MCH/6C Linker) = 3.98 x 106 Ka(MCH/3C Linker) = 3.16 x 106 Ka(MCU/6C Linker) = 1.41 x 106 Ka(MCU/3C Linker) = 1.38 x 106
Ka
E.L.S. Wong, E. Chow, J.J. Gooding, Langmuir 21 6957-6965 (2005).
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Hybridization conversion x = ST/SP as a function of probe coverage SP (probes per cm2) and ionic strength CB (moles of phosphate per liter)
Gong P., Levicky R. PNAS 2008;105:5301-5306
©2008 by National Academy of Sciences
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• The saturated surface Ru(NH2)3+ is converted to DNA probe surface density with the relationship,
nFΓ0
Cha
rge
/ µC
Sqrt (Time) / s½ Steel et. al. Analytical Chemistry, 1998, 70, 4670.
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DNA Surface Coverage
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Hybridization Kinetics
-25
-20
-15
-10
-5
00 500 1000 1500 2000
time / s
Freq
uenc
y / H
z
MCE/3C Linker MCH/3C Linker MCU/3C Linker
0
0.2
0.4
0.6
0.8
1
1.2
0 500 1000 1500 2000time / s
Nor
mal
ized
Fre
quen
cyMCE/3C Linker MCH/3C Linker MCU/3C Linker
E.L.S. Wong, E. Chow, J.J. Gooding, Langmuir 21 6957-6965 (2005).
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DNA Hybridisation Biosensor
F. Caruso et al Anal. Chem. 69 2043-2049 (1997)
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0
20
40
60
80
100
0 50 100 150
Time / min
Cu
rren
t / n
A
0
20
40
60
80
100
120
0 20 40 60 80 100
Time / min
Cu
rren
t / n
A
-0.10
0.00
0.10
0.20
0.30
0.40
-700 -500 -300 -100
Potential / mV
Cu
rren
t / µ
A
time
0
10
20
30
40
0 100 200 300
Time / min
Cu
rre
nt
/ n
A
0.0
0.1
0.2
0.3
0.4
0.5
-700 -500 -300 -100
Potential / mV
Cu
rre
nt
/ µ
A
0.00
0.10
0.20
0.30
0.40
0.50
-700 -500 -300 -100
Potential / mV
Cu
rre
nt
/ µ
A
time time
E.L.S. Wong, E. Chow, J.J. Gooding, Langmuir 21 6957-6965 (2005).
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Why the different kinetic
Target ssDNA
Redox Indicator
e-
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Detecting Small Compounds Binding to DNA
cisplatin cisplatin
DNA backbone
1 2
3
4 5 6
7 8
9
HN
N N
N
O
H2N
H
R
NN
N
HN
H
R
O
NH2
PtH3N
H3N
3'
5'α
β
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Experimental Procedure
Hybridization Buffer
Self Assembly onto Gold Electrode
Exposure to MCH
e- e- e- X X
In situ Experiment
In situ Experiment
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Results
0
100
200
300
400
-700 -500 -300 -100
Potential / mV
Cu
rre
nt
/ n
A
0 minute
10 minutes
20 minutes
30 minutes
40 minutes
50 minutes
-10
10
30
50
70
-700 -600 -500 -400 -300 -200 -100 0
Potential / mv
Cu
rren
t / n
A
Background Subtraction
1 mM cisplatin
E.L.S. Wong, J.J. Gooding, J. Am. Chem. Soc. 129 8950-8951 (2007).
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Better Defined Sequences DNA Biosensor DNA Sequence
p53 5’-GGGGCAGTGCCTCACAACCT-p-(CH2)6-SH-3’
3’-CCCCGTCACGGAGTGTTGGA-5’
P4/8 5’-GGAAAAAAAAAAAAAAAAAA-p-(CH2)6-SH-3’
3’-CCTTTTTTTTTTTTTTTTTT-5’
P5/9 5’-AAAAAAAAAAGGAAAAAAAA -p-(CH2)6-SH-3’
3’-TTTTTTTTTTCCTTTTTTTT-5’
P6/1 0 5’-AAAAAAAAAAAAAAAAAAGG -p-(CH2)6-SH-3’
3’-TTTTTTTTTTTTTTTTTTCC-5’
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Influence of the GG Position
0
20
40
60
80
100
0 200 400 600 800 1000
i aft
er/i
Befo
re / %
0
0.5
1
1.5
2
2.5
3
3.5
Concentration / nM
i / µ
A cm
-2
X
X
red pink green
E.L.S. Wong, J.J. Gooding, J. Am. Chem. Soc. 129 8950-8951 (2007).
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Summary: DNA Modified Electrodes Using long range charge transfer can produce an DNA hybridisation biosensor with: • excellent selectivity • good sensitivity • which is exceedingly simple to use and • can monitor hybridisation to give complete duplexes in real time • can monitor the interaction of anticancer drugs with DNA
The Future: • Investigate small molecule binding with DNA • Investigate DNA damage • Apply to detecting pathogens • Make the system more robust and more compatible with microfabricated devices
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Acknowledgements
CURRENT
PAST
We would like to acknowledge for funding: Australian Research Council, UNSW, Metagen, CASS Foundation, DSTO, AgaMatrix, Flinders Technology, GWRDC, CSIRO
Dr.Elicia Wong
Muthu Chockalingam
Bin Guan Sook Mei Khor
Pauline Michaels Jarred Shein
Will Rouesnel
Steven Yannoulatos
Albert Ng
Dr.Paul Eggers Dr.Guozhen Liu
Dr. Alison Chou Dr.Paul Eggers
Dr.Yit-Lung Khung
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n F Γ0(probe) C
harg
e / µ
C
Sqrt (Time) / s½
• A similar strategy was used to monitor hybridization.
n F Γ0(target)
Steel et. al. Analytical Chemistry, 1998, 70, 4670.