Classical dot-blot format implemented as an amperometric hybridisation genosensor.
A new electrochemical hybridisation genosensor has been designed. This genosensor is based on a concept adapted from classical dot-blot DNA analysis, but implemented in an electrochemical biosensor configuration. The use of amperometric transduction and the enzyme label method--that increases the genosensor sensitivity--are the main features of this new approach. The analytical procedure consists of five steps: DNA target immobilisation by adsorption onto a nylon membrane, hybridisation between DNA target and biotin-DNA probe, complexation reaction between biotin-DNA probe and an enzyme (horseradish peroxidase) streptavidin conjugate; integration of the modified membrane onto an electrochemical transducer; and finally, amperometric detection using a suitable substrate for the enzyme labelled duplex. Besides the adapted dot-blot format, a competitive assay in which the target is in solution is reported as well. This procedure, based on amperometric transduction, represents certain advantages with respect to dot-blot analysis: labelled hybrid detection is far simpler, quicker and requires more ordinary or simple reactives; the response obtained is a direct analytical signal via low-cost instrumentation, a nonisotopic labelling is used, and the membranes can be reused. These characteristics are ideal in implementing the procedure developed in kit form.
Pividori
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MI
Grup de Sensors & Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain.
Merkoçi
A
A
Alegret
S
S
D000327:Adsorption; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004277:DNA, Single-Stranded; D004563:Electrochemistry; D005821:Genetic Techniques; D006735:Horseradish Peroxidase; D015151:Immunoblotting / Q000379:methods*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D019809:Streptavidin
D004277:DNA, Single-Stranded; D015345:Oligonucleotide Probes; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D006735:Horseradish Peroxidase