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 μm-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 film. 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 H 2 O 2 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.