Enzyme-amplified amperometric detection of 3000 copies of DNA in a 10-μL droplet at 0.5 fM concentration

We reported earlier the detection of a 38-base DNA strand at 20 pM concentration by an enzyme-amplified sandwich-type amperometric assay. The assay utilized a carbon electrode on which a redox polymer, comprising a DNA capture sequence, was electrodeposited. When present in the tested solution, part of the probed sequence hybridized with the capture probe. Hybridization of its remaining part with a horseradish peroxidase (HRP)labeled sequence resulted in the flow of an H2O2 electroreduction current, the redox polymer wired HRP forming an electrocatalyst. Here we report a > 10(4)-fold improvement in the detection limit of the assay. DNA was detected at 0.5 fM concentration when the earlier used 3.6-mm-diameter carbon electrode was replaced by a 10-mum-diameter microelectrode. The radial diffusion of electrons through the film on the microelectrode allowed the electrodeposition of a thicker film of the redox polymer, an increase in the loading of the capture sequence, and increased the collection efficiency of the electron vacancies originating in the electroreduced H2O2. When the volume probed by the microelectrode was 10 muL, as few as 3000 copies of DNA were detected.