Amperometric Detection of DNA by Electroreduction of O2 in an Enzyme-Amplified Two-Component Assay

Abstract

The two-component type enzyme amplified amperometric DNA assay is described to use an ambient O2of the substrate of the DNA labeling enzyme. Although the assay detects DNA only at . 0.5 nM concentration,a concentration ~106 fold higher than the sandwich-type enzyme amplified amperometric DNA assay, it can be runwith an always available substrate. The assay utilizes screen-printed carbon electrodes (SPEs) which were pre-coatedby a co-electrodeposited film of an electron conducting redox hydrogel and a 37-base long single-stranded DNAsequence. The DNA in the electron conducting film hybridizes and captures, when present, the 37-base long detection-DNA, which is labeled with bilirubin oxidase (BOD), an enzyme catalyzing the four-electron reduction of O2 to water.Because the redox hydrogel electrically conects the BOD reaction centers to the electrode, completion of the sandwichconverts the film from non-electrocatalytic to electrocatalytic for the reduction of O2 to water when the electrode ispoised at 200 mV vs. Ag/AgCl. The advantage of the assay over the earlier reported sandwich type enzyme amplifiedamperometric DNA assay, in which the amplifying enzyme was horseradish peroxidase, is that it utilizes ambientO2 instead of the less stable and naturaly unavailable H2O2 .