Label-free electrochemical aptasensor for sensitive thrombin detection using layer-by-layer self-assembled multilayers with toluidine blue–graphene composites and gold nanoparticles

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.