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An ultrasensitive label-free bioelectrochemical method for rapid determination of thrombin has been developed by directly detecting the redox activity of adenine (A) nucleobases of anti-thrombin aptamer using a pyrolytic graphite electrode. The bioelectrochemical protocol involves a sandwich format. Thrombin, captured by immobilzed anti-thrombin antibody on microtiter plates, is detected by anti-thrombin aptamer-Au nanoparticle bio bar codes. A systematic optimization of the parameters has been carried out via ELAA (enzyme linked aptamer assay) performance. The adenine nucleobases were released by acid or nuclease from Au nanoparticles bound on microtiter plates. Differential pulse voltammetry was employed to investigate the electrochemical behaviors of the purine nucleobases. Well-defined adenine signal was observed at about 0.85 V in pH 5.9 acetate buffer. Because the nanoparticle carries a large number of aptamers per thrombin binding event, there is substantial amplification and thrombin can be detected at a very low level of detection (0.1 ng/mL). This method has been used to detect thrombin in complex matrix such as fetal calf serum with minimum background interference.

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