Detection of ∼10<SUP>3</SUP> copies of DNA by an electrochemical enzyme-amplified sandwich assay with ambient O2 as the substrate

초록

The electrochemical sandwich-type, enzyme-amplified assay of Zhang, Mm, and Heller (Anal. Chem. 2003, 75, 3267-3269) was simplified by replacing the amplifying horseradish peroxidase with bilirubin oxidase (BOD). BOD catalyzes the reduction of ambient O-2 to water and obviates the need for adding H2O2. Femtomolar (10(-15) M) concentrations of DNA were detected at a 10-mum diameter tip of a carbon fiber electrode. Correspondingly, a few thousand copies of DNA were detected in similar to5-muL samples. The sandwich is formed in an electron-conducting redox hydrogel, to the polymer of which a DNA capture sequence is bound. Capture of the analyte DNA and its hybridization with a BOD-labeled complementary DNA sequence, electrically connects the BOD label to the electron-conducting redox polymer, which is in electrical contact with the electrode. Placing the BOD in contact with the redox polymer thus converts the noncatalytic base layer into a catalyst for the electroreduction of O-2 to water at + 0.12 V (vs Ag/AgCl) (Figure 1). In an exemplary assay, similar to3000 copies of the iron transporting sequence of the sit gene of Shigella flexneri were detected without PCR amplification.

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