A novel modification-free thrombin electrochemical sensing based homogeneous strategy

Abstract In this study, a novel modification-free electrochemical sensing approach for thrombin that utilizes both electrically assisted bond and bisferrocene to enhance the signal output in a homogeneous solution was reported. First, the selected aptamer probe with a stem–loop structure was labeled with bisferrocene and thiol at the 3′ and 5′ terminals, respectively, and the closed–loop structure of the probe prevented thiol from reaching the electrode surface. However, the stem–loop structure opened after treatment with the target thrombin protein; hence, the thiol can rapidly attach to the electrode to form a Au–S self-assembly by potential-assisted, followed by the measurement of the bisferrocene electrochemical signal. Binding was directly performed in the solution, and it did not require the probe to be fixed in advance; meanwhile, necessary electrode modification was prevented. This modification-free homogeneous strategy exhibited a detection limit as low as 0.8 × 10−12 M of thrombin. Moreover, advanced specificity against common interfering proteins was observed.