Reactive electrophoretic activation of a microelectrode for enzyme-amplified recognition and for melting temperature determination of 105 copies of a simple oligonucleotide

The hybridization of 10(5) copies of 25-30-base single-stranded poly(deoxythymidine)-5'-phosphate [ss-pd(T)(25-30)] was detected amperometrically with a 10 mu m-diameter microelectrode. The melting of 10(5) copies of the hybrid with horseradish peroxidase (HRP)-labeled poly(deoxyadenine)-5'-phosphate [ss-pd(A)(25-30)-HRP] was also tracked by amperometry. The microelectrode was coated with its hybridization-sensing layer in a two-step process involving electrophoretic deposition, which yielded reproducible electrode coatings. In the first step, a thin film of an electron-conducting redox polymer was deposited electrophoretically at constant current on the vitreous carbon surface of the microelectrode. In the second step, carbodiimide-activated 5'-phosporylated ss-pd(T)(25-30) was reactively electrophoretically deposited and covalently attached to the redox polymer him. Subsequent hybridization led to electrical contact between the HRP label of ss-pd(A)(25-30) and the conducting redox polymer. This contact resulted in catalysis of H2O2 electroreduction to water at 0.0 V vs Ag/AgCl. The 20 +/- 2 pA current produced by 10(5) copies of hybridized pd(A)(25-30)-HRP was at least 8 times greater than the 2.5 +/- 2.5 pA current measured with noncomplementary HRP-labeled poly(deoxyguanidine)-5'-phosphate and 40 times greater than the 0.5 pA electrical background noise.