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