Different strategies to develop an electrochemical thrombin aptasensor

Three configurations of an electrochemical aptasensor for thrombin detection are reported. In the most straightforward configuration the thrombin interaction with an aptamer selective for thrombin was detected by the quantification of p-nitroaniline produced by the thrombin’s enzymatic reaction. While the detection of p-nitroaniline can be accomplished either optically or electrochemically, electrochemical detection offers benefits in terms of sensitivity and speed.Thrombin was also detected using an enzyme labeled sandwich format. Peroxidase labeled thrombin was incubated with the aptamer and the interaction was measured electrochemically by detection of a diffusional mediator generated in a peroxidase catalyzed reaction. The catalytic current achieved for the sandwich interaction was 2.32 μA which was 0.7 μA above the controls. The limit of detection was 80 nM.In a third strategy also employing an enzyme label, thrombin was immobilized on the sensor surface and incubated with a biotin labeled aptamer. The sensor was subsequently incubated with streptavidin-HRP (horseradish peroxidase), which bound to the biotin on the aptamer. The aptamer was again quantified by the electrochemical detection of a peroxidase catalyzed reaction. In this case the limit of detection was 3.5 nM. This strategy is applicable to competitive assays for detection of unlabeled thrombin.