Download Electrochemical DNA Biosensors

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 thiolatedoligonucleotide 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