No abstract

We reported earlier the detection of a 38-base DNA strand at 20 pM concentration by an enzyme-amplified sandwich-type amperometric assay. The assay utilized a carbon electrode on which a redox polymer, comprising a DNA capture sequence, was electrodeposited. When present in the tested solution, part of the probed sequence hybridized with the capture probe. Hybridization of its remaining part with a horseradish peroxidase (HRP)-labeled sequence resulted in the flow of an H2O2 electroreduction current, the redox polymer wired HRP forming an electrocatalyst. Here we report a > 10(4)-fold improvement in the detection limit of the assay. DNA was detected at 0.5 fM concentration when the earlier used 3.6-mm-diameter carbon electrode was replaced by a 10-microm-diameter microelectrode. The radial diffusion of electrons through the film on the microelectrode allowed the electrodeposition of a thicker film of the redox polymer, an increase in the loading of the capture sequence, and increased the collection efficiency of the electron vacancies originating in the electroreduced H2O2. When the volume probed by the microelectrode was 10 microL, as few as 3000 copies of DNA were detected.

No 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 O(2) to water and obviates the need for adding H(2)O(2). Femtomolar (10(-)(15) M) concentrations of DNA were detected at a 10-microm-diameter tip of a carbon fiber electrode. Correspondingly, a few thousand copies of DNA were detected in approximately 5-microL 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, approximately 3000 copies of the iron transporting sequence of the sit gene of Shigella flexneri were detected without PCR amplification.

A 38-base DNA sequence has been detected at 20 pmol L(-1) concentration in 15-35- microL droplets by means of an electrochemical enzyme-amplified sandwich-type assay on a mass-manufacturable screen-printed carbon electrode. Formation of the sandwich brought the horseradish peroxidase-label of the detection sequence into electrical contact with a pre-electrodeposited redox polymer, making the sandwich an electrocatalyst for the reduction of hydrogen peroxide to water at +0.2 V (Ag/AgCl). Sensitivity twenty times better than that of a related system resulted from: 1. fivefold reduction of the noise by substituting the formerly used poly( N-vinyl imidazole)-co-acrylamide comprising redox co-polymer with poly(4-vinyl pyridine)-co-acrylamide comprising redox polymer, enabling use of the electrodes at a more oxidizing potential at which noise (the rate of non-enzyme catalyzed electroreduction currents of dissolved oxygen and hydrogen peroxide) was lower; 2. doubling of the catalytic electroreduction current upon electrodeposition of a second layer of the redox polymer on the capture sequence-containing film; and 3. doubling of the current by increasing the coverage by the capture sequence.