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 .

Portable DNA Biosensors

DNA Detection

Labelling and fragmentation DNA amplification

Sample input

DNA extraction

SIMPLICITY

Cell identification and capture

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

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

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/

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/

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).

(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).

-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

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

Changes in Spatial Relationships

Δh = 1 nm Δh = 4.5 nm

Complementary 20mer

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).

Single-Base Mismatch Complementary

Single-base C-A

Mismatch Single-base

G-A Mismatch Probe ssDNA

A

T C G

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).

Why can we detect mismatches?

Mismatch about here

E.L.S. Wong, J.J. Gooding, Anal. Chem. 75 3845-3852 (2003).

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).

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).

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

Voltammetric Experiments in intercalator Solution

e- e-

E.L.S. Wong, J.J. Gooding, Anal. Chem. 78 2138-2144 (2006).

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).

-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).

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).

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

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).

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).

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

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

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

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

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).

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

•  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.

DNA Surface Coverage

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).

DNA Hybridisation Biosensor

F. Caruso et al Anal. Chem. 69 2043-2049 (1997)

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).

Why the different kinetic

Target ssDNA

Redox Indicator

e-

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

Experimental Procedure

Hybridization Buffer

Self Assembly onto Gold Electrode

Exposure to MCH

e- e- e- X X

In situ Experiment

In situ Experiment

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).

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’

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).

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

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

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.