Electrochemical Detection of Asymmetric PCR Products by Labeling with Osmium Tetroxide

Single stranded DNA-targets from asymmetric polymerase chain reaction (PCR) of a sequence of the gram positive, spore forming bacterium Clostridium acetobutylicum were detected by square-wave voltammetry after labeling with osmium tetroxide bipyridine and hybridization with DNA capture probes immobilized on gold electrodes. The asymmetric PCR, performed with a 10-fold excess of the forward-primer, was used without any further purification for hybridization with protective strands and covalent labeling with osmium tetroxide bipyridine. Square-wave voltammetric signals of 20 nmol/L targets were significantly higher at 50 degrees C compared with 23 degrees C hybridization temperature. A fully noncomplementary protective strand yielded thoroughly modified targets unable for further hybridization. Coupling this with thermal discrimination opens new opportunities for sequence specific DNA detection.