In the present study, toluidine blue-graphene (Tb-Gra) nanocomposites were prepared to design a Lable-free electrochemical aptasensor for highly sensitive detection of thrombin based on layer-by-layer (LBL) technology. The nanocomposites with excellent redox electrochemical activities were first immobilized on the gold nanoparticles (nano-Au) modified glassy carbon electrodes (GCE). Then, the LBL structure was performed by electrostatic adsorption between the positively charged Tb-Gra and negatively charged nano-Au, which formed {Tb-Gra/nano-Au}(n) multilayer films for electroactive species enrichment and biomolecule immobilization. Subsequently, the thiolated thrombin binding aptamer (TBA) was assembled on the nano-Au surface through Au-S bond. In the presence of target thrombin (TB), the TBA on the multilayer could catch the thrombin onto the electrode surface, which resulted in a barrier for electro-transfer, leading to the decrease of the electrochemical signal of Tb-Gra nanocomposites. Under the optimal conditions, a wide detection range from 0.001 nM to 80 nM and a low detection limit of 0.33 pM (defined as S/N=3) for thrombin were obtained. In addition, the sensor exhibited excellent selectivity against other proteins.
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