A single base pair mismatch in an 18-base oligonucleotide was detected amperometrically with a 7-mu m-diameter carbon microelectrode. The hybridization was followed directly and in real time by steady-state amperometry. The microelectrode was coated with a hybridization-sensing layer in a two-step electrophoretic process, which yielded microelectrodes with reproducible dimensions. In the first step, a thin film of an electron-conducting redox polymer was deposited electrophoretically at constant potential in a low ionic strength solution. In the second step, a carbodiimide-activated single-stranded probe was reactively electrophoretically deposited and covalently attached to the redox polymer film. The labeling enzyme, thermostable soybean peroxidase (SBP), was covalently bound to the 5＇-end of the target single-stranded oligonucleotide. When the redox polymer and the enzyme were brought to close proximity by hybridization of the target and probe oligonucleotides, the film on the electrode switched from being a noncatalyst to a catalyst for H2O2 electroreduction at -0.06 V vs Ag/AgCl. The current observed corresponded to that generated by similar to 40 000 surface-bound and electrically connected SEP molecules.