electrochemical dna sensors. topics introduction the molecular structure of dna principles of...
TRANSCRIPT
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
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
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
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