Fabrication and application of a new DNA biosensor based on on-substrate PCR and electrochemistry

DNA probes immobilized on a gold electrode (AuE) were employed as the primers of asymmetric PCR on the AuE. In the asymmetric PCR process, the DNA probes extended in the presence of target strands in the PCR solution. After PCR the dsDNAs were denaturalized and the target DNAs were eliminated and only the extended probes maintained on the AuE. At last the electrochemical indicator of methylene blue combined to the extended probes and the electrochemical signal of indicator was measured. This signal was higher than that of the AuE modified only by original probe. When there was no target in the PCR solution, the probe did not extend and the signal did not increase. The specific sequences of chitinase gene were detected successfully from four sorts of target with different length: oligonucleotide acid, PCR products, molecule cloning vector DNA and total genome DNA of transgenic capsicum, and the estimated detection limit were 7.3 × 10−12, 3.2 × 10−11, 5.4 × 10−11 and 4.1 × 10−10 mol l−1 respectively. The regeneration of the biosensor was also tested and the results indicated that its half life was 6 times.