Dual-signal amplification strategy for electrochemiluminescence sandwich biosensor for detection of thrombin

In the present study, the authors report a novel sandwich biosensor for detection of thrombin using electrochemiluminescence method based on dual-signal amplification strategy. Firstly, the gold nanoparticles graphene composite (GNPs-graphene) was obtained by simultaneously depositing the chloroauric (III) acid tetrahydrate [HAuCl 4 ·4H 2 O] and graphene solution with a cyclic voltammetry. Secondly, one thiol-modified thrombin aptamer (TBA1) was immobilized the glassy carbon electrode (GCE) via Au-S bonding. And then the other thiol-modified biotinylated thrombin aptamer (TBA2) that were modified with Ru(bpy) 3 2+ were anchored on the surface of the gold nanoparticles in solution. This proposed assay took dual signal amplification strategies. On the one hand, a single gold nanoparticle can connect many TBA2 to come into being a signal cluster (Ru-TBA2-AuNPs), thus when a little thrombin were present the system the ECL intensity will have a strong signal response; On the other hand, the GNPs-graphene can promote the electron transfer efficiency and increase the signal response. The ECL signal had a good linear relationship with thrombin concentration in range of 0.01–10   nM, and the detection limit for thrombin was determined to be as low as 6.3 pM. This biosensor also showed good selectivity for thrombin without being affected by some other proteins, such as BSA and lysozyme and so on. Moreover, this proposed method was successfully applied to thrombin analysis in diluted human serum samples.