Detection of ∼103 Copies of DNA by an Electrochemical Enzyme-Amplified Sandwich Assay with Ambient O2 as the Substrate

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Abstract
The electrochemical sandwich-type, enzyme-amplified assay of Zhang, Kim, 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 O2 to water and obviates the need for adding H2O2. Femtomolar (10-15 M) concentrations of DNA were detected at a 10-μm-diameter tip of a carbon fiber electrode. Correspondingly, a few thousand copies of DNA were detected in ∼5-μL 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 O2 to water at +0.12 V (vs Ag/AgCl) (Figure 1). In an exemplary assay, ∼3000 copies of the iron transporting sequence of the sit gene of Shigella flexneri were detected without PCR amplification.