Rapid electrochemical genosensor assay using a streptavidin carbon-polymer biocomposite electrode.
A sensor capable of detecting a specific DNA sequence was designed by bulk modification of a graphite epoxy composite electrode with streptavidin (2% w/w). Streptavidin is used to immobilise a biotinylated capture DNA probe to the surface of the electrode. Simultaneous hybridisation occurs between the biotin DNA capture probe and the target-DNA and between the target-DNA and a digoxigenin modified probe. The rapid binding kinetic of streptavidin-biotin allows a one step immobilisation/hybridisation procedure. Secondly, enzyme labelling of the DNA duplex occurs via an antigen-antibody reaction between the Dig-dsDNA and an anti-Dig-HRP. Finally, electrochemical detection is achieved through a suitable substrate (H2O2) for the enzyme-labelled duplex. Optimisation of the sensor design, the modifier content and the immobilisation and hybridisation times was attained using a simple nucleotide sequence. Regeneration of the surface is achieved with a simple polishing procedure that shows good reproducibility. The generic use of a modified streptavidin carbon-polymer biocomposite electrode capable of surface regeneration and a one step hybridisation/immobilisation procedure are the main advantages of this approach. In DNA analysis, this procedure, if combined with the polymerase chain reaction, would represent certain advantages with respect to classical techniques, which prove to be time consuming in situations where a simple and rapid detection is required. This innovative developed material may be used for the detection of any analyte that can be coupled to the biotin-streptavidin reaction, as is the case of immunoassays.
Williams
E
E
Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain.
Pividori
M I
MI
Merkoçi
A
A
Forster
R J
RJ
Alegret
S
S
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004566:Electrodes; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006108:Graphite / Q000737:chemistry*; D008420:Manufactured Materials; D009862:Online Systems; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry*
D020099:Coated Materials, Biocompatible; D015342:DNA Probes; D006108:Graphite; D004247:DNA; D019809:Streptavidin