Electrochemical DNA sensors

1. Electrochemical DNA sensors

2. Topics • Introduction • The Molecular Structure of DNA • Principles of biosensor function • Electrochemical readout • Conclusions and perspectives

3. 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

4. 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

5. Electrochemical DNA biosensors offers: • Simple • Accurate • rapid • inexpensive platform • Independent of sample turbidity

6. The Molecular Structure of DNA a DNA strand is a polymer: 2'-deoxyribose (a five-carbon sugar), phosphoric acid four nitrogen- containing bases

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

8. 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.

9. three-dimensional structure of the DNA Watson and Crick(1953) The molecule consists of: two polynucleotide chains twisted around one another to form a double-stranded helix

11. 3' 5' Minor Major 3' 5' Stracture of DNA Major groove Minor groove 8

12. (Normal) Stracture of DNA 9

13. Structure 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 • Z-DNA: • left-handed • zig-zaggy • ~12 bp per turn • 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

14. Binding with DNA 10

15. Peptide nucleic acid

17. 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: • recognition layer • signal transducer

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

19. Electrochemical readout: Electrochemistry-based sensors offer: Sensitivity Selectivity low cost detection of selected DNA sequences or mutated genes associated with human disease.

20. Sensitive electrochemical signaling strategies: • Direct oxidation of DNA bases, • catalyzed oxidation of DNA bases, • redox reactions of reporter molecules • enzymes recruited to the electrode surface by specific DNA probe-target interactions • charge transport reactions mediated by the π-stacked base pairs have all been demonstrate.

21. 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).

22. The purine bases of DNA can be oxidized • electrochemically, and this process can be carried out using: • carbon • Gold • indium tin oxide (ITO) • polymer-coated electrodes

23. 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

24. 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)

25. 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.

27. 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.

29. Multi-Target Analysis 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.

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

31. 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.

32. 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.

33. 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-base Mismatches have been readily detected .At a 30-μm electrode, as few as ∼108 duplexes have been detected.

35. Thus DNA-mediated charge transport provides: specificity in mutation detection, sensitivity through electrocatalysis, Facile access to an array format

36. Conclusions and perspectives Despite the enormous opportunities clearly offered by electrochemical DNA sensing, some important hurdles remain: • 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.

37. 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

38. Reference: VOLUME 21 NUMBER 10 OCTOBER 2003 NATURE BIOTECHNOLOGY