Enzyme-amplified amperometric sandwich test for RNA and DNA

A one-step enzyme-amplified amperometric sandwich hybridization test for RNA and DNA is described. The test utilizes a carbon electrode, modified with a film of co-electrodeposited avidin and redox polymer; the redox polymer electrically "wiring" horseradish peroxidase (HRP) reaction centers upon contact. The film is made specific for the particular RNA or DNA sequence tested by conjugating its avidin with a biotinylated oligonucleotide, complementary to the assayed sequence. This oligonucleotide-modified redox polymer film, prepared prior to the test, forms the base of the sandwich. The center layer of the sandwich, added in the test, is the analyte RNA or DNA; its top is a second complemetary oligonucleotide, which is HRP-labeled, and is cohybridized in the test. The test consists of mixing the analyte DNA or RNA solution, the HRP-labeled oligonucleotide solution, and a hydrogen peroxide solution, immersing the base-layer carrying electrode applying a potential of 0 V versus Ag/AgCl, and measuring the H2O2 electroreduction current. Completion of the sandwich brings the HRP label into electrical contact with the redox polymer, converting the nonelectrocatalytic base layer into an electrocatalyst for the electroreduction of H2O2 to water. Flow of H2O2 electroreduction current when the electrode is poised near Ag/AgCl potential indicates the presence of the analyte RNA or DNA. The current density for the maximally sandwich-covered electrode was 250 microA cm(-2), exceeding more than a 100-fold the current density flowing upon nonspecific binding of the HRP-labeled oligonucleotide. High concentrations of irrelevant DNA and diluted serum did not interfere with the assay. When the electrodes were rotated in order to make the solution-phase mass transport rapid, the test was completed in approximately 30 min. The test was applied in probing for the presence of a 60-base E. coli mRNA sequence.