Electrochemical DNA sensors

Topics

• Introduction

• The Molecular Structure of DNA

• Principles of biosensor function

• Electrochemical readout

• Conclusions and perspectives

Introduction

Recent important technological advances enable us :

Monitor biorecognition

Study interaction events solid devices and in solution.

Development in Nanofabrication technologies

facilitated biosensing solid substrates

Recently, an impressive number of inventive

designs for DNA-based electrochemical sensing

have appeared .

These types of sensors combine:

nucleic acid layers with electrochemical transducers

Electrochemical DNA biosensors offers:

1. Simple

2. Accurate

3. rapid

4. inexpensive platform

5. Independent of sample turbidity

The Molecular Structure of DNA

a DNA strand is a polymer:

2'-deoxyribose (a five-carbon sugar),

phosphoric acid

four nitrogen- containing bases

nucleoside= Nitrogen base + Ribose nucleotide = Nitrogen base + Ribose +phosphate

polynucleotide chain

the phosphate attached to the 5' carbon of one

sugar is linked to the hydroxyl group attached to the

3' carbon of the next sugar in line.

Watson and Crick(1953)

The molecule consists of:

two polynucleotide chains twisted around one

another to form a double-stranded helix

three-dimensional structure of the DNA

Major

Minor

3'

5'

3'

5'

Major groove

Minor groove

Stracture of DNA

8

(Normal)

Stracture of DNA

9

Structure of DNAStructure of DNA

B-DNA: - right-handed

- most common form

- 0.34 nm rise

-10.5 bp per turn

- 3.4 nm pitch

- adopted in aqueous

A-DNA: - right-handed

- broader than B

- 0.26 nm rise

- ~10 bp per turn

- 2.6 nm pitch

-adopted in non-aqueous

- has “hole” down the center

Z-DNA: - left-handed

- zig-zaggy

- ~12 bp per turn

Binding with DNA

10

Peptide nucleic acid

Principles of biosensor function

The essential role of the sensor:

A suitable platform that facilitates formation of the

probe-target complex

The minimal elements of any biosensor:

1. recognition layer

2. signal transducer

How this recognition event is reported depends ultimately on the method of signal transduction, whether it be optical ,mechanical or electrochemical.

Electrochemical readout:

Electrochemistry-based sensors offer:

Sensitivity

Selectivity

low cost

detection of selected DNA sequences or mutated

genes associated with human disease.

Sensitive electrochemical signaling strategies:

1. Direct oxidation of DNA bases,

2. catalyzed oxidation of DNA bases,

3. redox reactions of reporter molecules

4. enzymes recruited to the electrode surface by

specific DNA probe-target interactions

5. charge transport reactions mediated by the π-

stacked base pairs have all been demonstrate.

Direct electrochemistry of DNA as a detection platform

Electrochemical activities of nitrogen base was discovered by Palecek group more than 50 years.

adsorption stripping voltammetry (ASV) is one of

the most sensitive methods to detect of DNA

(~40fmol) .

The purine bases of DNA can be oxidized

electrochemically, and this process can be

carried out using:

1. carbon

2. Gold

3. indium tin oxide (ITO)

4. polymer-coated electrodes

A Big Problem !!!

Significant high background current in relatively high potential required to oxidize guanine.

How to overcome this problem???

Numerical methods to improve the signal-to-

noise ratio have been developed, but more

recent designs employ physical separation

techniques to remove the sources of

background interference

An inventive strategy for capturing target

sequences:

DNA immobilized onto magnetic beads

Target hybridization

Separation of beads are magnetically

Electrochemical Oxidation free guanine and adenine

using ASV as few as 40 fmoles (2 × 1010 molecules)

Indirect electrochemistry of DNA as a detection platform:

Thorp and coworkers developed electrocatalytic oxidation of guanine using Ru(II) and Os (II) to mediate the

the methodology does provide high sensitivity

without complex instrumentation through redox-

mediated DNA oxidation.

DNA-specific redox indicator detection platforms

target DNA sequences are labeled with redox-active reporter molecules.

Appearance of the characteristic electrochemical

response of the redox reporter therefore signals the

hybridization event.

Probe modified magnetic beads are hybridized with target DNA, separated magnetically from the pool of analytes and hybridized again with the nanoparticle-labeled reporter strands.

Multi-Target Analysis

Biocatalyzed production of insoluble products has been used by Willner and colleagues to sense DNA hybridization electrochemically at probe-modified electrodes.Target

DNA-mediated charge transport electrochemistry

In these analyses, rather than serving as a

reactant, the DNA is the mediator. These assays can

provide high sensitivity and simplicity.

Intercalative probe molecules:

the DNA base pair stack mediates charge transport

to the intercalator bound at the top of the film. If the

base pair stack is intact, current can flow.

To increase the inherent sensitivity of the assay:

a coulometric readout strategy based on the

electrocatalytic reduction of ferricyanide by

methylene blue.

Using this assay ,all of the possible single-baseMismatches have been readily detected .At a 30-μm electrode, as few as ∼108 duplexes have been detected.

Thus DNA-mediated charge transport provides:

specificity in mutation detection,

sensitivity through electrocatalysis,

Facile access to an array format

Conclusions and perspectives

Despite the enormous opportunities clearly offered by electrochemical DNA sensing, some important hurdles remain:

1. Array sizes on the order of 10 have thus far been demonstrated, but more typically arrays of 50–100 sequences will be needed for clinical application, Electronic switches in the form of an on-chip electronic multiplexer may provide a possible solution for this problem.

2.the biological complexity of a genomic DNA sample

Converting genomic information to clinical

advantage can be successfully accomplished with

DNA-based sensors. Their low cost, small size and

inherent sensitivity will certainly provide important

new tools for the diagnosis of disease

Reference:

VOLUME 21 NUMBER 10 OCTOBER 2003

NATURE BIOTECHNOLOGY