Electrochemical DNA Biosensors Lecture04

INTRODUCTION Sequence-specific DNA detection: – Screening of genetic and infectious diseases – For ensuring our food safety – For criminal investigations – Field testing of microbial and viral pathogens – ….

ELECTROCHEMICAL DNA HYBRIDIZATION BIOSENSORS The major steps involved in electrochemical DNA hybridization biosensors: – The formation of the DNA recognition layer – The actual hybridization event – The transformation of the hybridization event into an electrical signal

Interfacial Immobilization Control of the surface chemistry and coverage is essential: – For assuring high reactivity, – orientation/accessibility, – Stability of the surface-bound probe, – Aavoiding nonspecific binding/adsorption events.

It was demonstrated recently that the density of immobilized ssDNA can influence the thermodynamics of hybridization and hence the selectivity of DNA biosensors Watterson, J. H., Piunno, P. A. E., Wust, C. C., Krull, U. J. (2000) Langmuir 16, 4984–4992.

Immobilization Schemes self-assembly of organized monolayers of thiol functionalized probes onto gold transducers Carbodiimide covalent binding to an activated surface Attachment of biotin-functionalized probes to avidin-coated surfaces Adsorptive accumulation onto carbon-paste or disposable strip electrodes.

The Hybridization Event Variables affecting the hybridization event: – salt concentration – Temperature – The presence of accelerating agents – Viscosity – Contacting time – Base composition (%G + C) – Length of probe sequence.

significantly enhanced selectivity can be achieved by the use of peptide nucleic acid (PNA) probes.

Attention should be given also to the reusability of the DNA biosensors Thermal and chemical (urea, sodium hydroxide) regeneration schemes have been shown useful for “removing” the bound target in connection with different DNA biosensor formats. Even more elegant is the use of controlled electric fields for facilitating the denaturation of the duplex

Electrochemical Transduction of DNA Hybridization Indicator-Based Detection – small redox-active DNA-intercalating or groovebinding substances that possess a much higher affinity for the resulting duplex compared with the single-stranded probe. – linear-scan or square-wave voltammetric modes or constant-current chronopotentiometry

Redox Indicators Anthracycline antibiotics such as daunomycin Bisbenzimide dyes such as Hoecht Cobalt Phenanthroline Methylene Blue Nile Blue Ferrocenyl naphthalene diimide (offering greater discrimination between ss- and ds-DNA are being developed for attaining higher sensitivity) ….

Use of Enzyme Labels for Detecting DNA Hybridization Heller’s group Willner’s group

Label-Free Electrochemical Biosensing of DNA Hybridization exploit changes in the intrinsic electroactivity of DNA accrued from the hybridization event monitoring changes in the conductivity of conducting polymer molecular interfaces, e.g., using DNA-substituted or doped polypyrrole films changes in the capacitance of a thiolated- oligonucleotide modified gold electrode, provoked by hybridization to the complementary strand (and the corresponding displacement of solvent molecules from the surface), can be used for rapid and sensitive detection of DNA sequences