electrochemical dna biosensors lecture04. introduction sequence-specific dna detection: –...

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 33258

• 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