Amperometric DNA sensing using wired enzyme based electrodes
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A water soluble copolymer of acrylamide and 4-vinylpyridine complexed with [Os(bpy)2Cl]+/2+ (bpy = 2,2’-bipyridine), was synthesized. An electrodeposition method of making redox polymer films on electrodes was developed. This method was also shown to be effective in incorporating enzymes and amine-terminated DNA sequences in the redox polymer film. A 38-base DNA sequence was detected at 20 pM concentration in 15-35 μL droplets by an electrochemical enzyme-amplified sandwich-type assay on a mass-manufacturable screen printed carbon electrode with a diameter of 3.5 mm. A DNA-capturing oligonucleotide was attached to the pre-deposited redox polymer film using the electrodeposition method. The electrode was exposed to the droplet containing the tested DNA sample, and was then treated with a droplet containing horseradish peroxidase-labeled detection sequence. Formation of the capture-target-detection sandwich brought the horseradish peroxidase-label of the detection sequence in electrical contact with the redox polymer, making the sandwich an electrocatalyst for the reduction of hydrogen peroxide to water at + 0.2 V (Ag/AgCl). The radial diffusion of electrons through the redox polymer 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. With a 10-μm diameter carbon fiber microelectrode, as few as 3000 copies of the 38-basse DNA sequence were detected at 0.5 fM concentration in a 10 μL sample. A biofuel cell operating at a power density of 50 μW cm–2 at 0.5 V under physiological conditions (air saturated, pH 7.4, 0.14 M NaCl, 37.5°C, 15 mM glucose) was developed. The cell had a glucose electro-oxidizing anode and an O2 electro-reducing cathode. The anode and the cathode were 7 μm diameter, 2 cm long carbon fibers, on which the catalytic enzyme-redox polymer adducts were cross-linked. When the miniature cell operated at 0.5 V, the power output dropped to about 60% of its initial value after 2 days of continuous operation at 37.5°C.