-DNA aptamers were heated to 85 °C for 5 min in physiological buffer and slowly cooled to 25 °C over 10 min for annealing, with native human thrombin added to a final concentration of 10 nM. The mixture was incubated in physiological buffer at room temperature for 30 min, followed by addition of 100 μM fluorogenic substrate, benzoyl-Phe-Val-Arg-AMC. Relative fluorescence was measured by the Varioskan Flash system with excitation wavelength at 350 nm and emission wavelength at 450 nm.
Relative thrombin enzymatic activity was determined by change of relative fluorescence unit , with relative fluorescence unit measured at 0 min set to 0 and RFU of the negative control in physiological buffer alone set to 100%, and ΔRFU measured at 16 min was used to calculate the relative thrombin enzymatic activity . ICwas calculated by fitting the relative thrombin enzymatic activity to the sigmoidal equation using the KaleidaGraph software.
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