[Determination of human thrombin by an aptamer based on affinity capillary electrophoresis-laser induced fluorescence...

The method for the determination of human thrombin by an aptamer was developed based on capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The concentration of thrombin was calculated through the peak area of the thrombin-aptamer complex, which was separated and detected by CE-LIF. Because of the binding favorable G-quartet conformation potentially involved in the specific aptamer, it was assumed that monovalent and bivalent cations promoting the formation of a stable G quadruplex conformation in the aptamer may enhance the binding of the aptamer and thrombin. Therefore, the effects of various metal cations on the binding of human thrombin and the aptamer were investigated. The results showed that the cations like K+ and Mg2+ could not stabilize the affinity complex. The linear range, detection limit and reproducibility were measured. The linear range was 0.25 - 10 nmol/L (r2 = 0.991), and the detection limit of thrombin was 55.6 pmol/L. Regarding the advantages of high efficiency and rapid separation, low sample consumption, and high sensitivity, CE-LIF is a potential and powerful alternative to conventional immunoaffinity assays in clinical diagnostics.