Enzyme-Amplified Amperometric Sandwich Test for RNA and DNA

Abstract

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.

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

“…Incorporation of avidin into the electron-conducting hydrogel provided a platform to which biotinylated oligonucleotides could be promptly and simply attached (Campbell et al, 2002). This immobilisation procedure resulted in an higher probe hybridisation efficiency, in comparison with that of previously reported covalently bound probes.…”

“…About 34 000 copies of SBP-labelled hybrid could be detected in a 10 min assay. Heller's group also reported an enzyme-amplified amperometric sandwich test for RNA and DNA (Campbell et al, 2002). DNA or RNA sequences were simultaneously allowed to hybridise with a HRP-labelled detection sequence and the surface-confined probe.…”

Electrochemical DNA sensors

“…Heller and colleagues 36 have reported an enzyme-amplified DNA sensing technique involving the electropolymerization of a polycationic redox polymer, upon which amineterminated oligonucleotide single strands are electrodeposited by ligand exchange with bipyridyl osmium complexes impregnated within the polymer gel. Target DNA is captured at the electrode surface and subsequently hybridized to a reporter strand modified with HRP.…”

Label-free electrochemical detection of human methyltransferase from tumors

“…However, electrochemical detection schemes have typically been restricted to measurements of highly purified samples because of the increased congestion and decreased accessibility of surface (vs. solution) platforms. Electrochemistry has been used to detect nucleic acids with high sensitivity and without the need for PCR amplification in bacterial lysate and serum (18)(19)(20)(21)(22), but protein detection remains a challenge (23)(24)(25)(26). In fact, although protein detection from simple serum has been accomplished (27,28), to date no reported electrochemical systems have effectively detected active protein, of any kind, from crude cell lysate.…”