An electrochemical aptasensor for thrombin detection based on direct electrochemistry of glucose oxidase using a functionalized graphene hybrid for amplification

In this work, we reported a new label-free electrochemical aptasensor for highly sensitive detection of thrombin using direct electron transfer of glucose oxidase (GOD) as a redox probe and a gold nanoparticle–polyaniline–graphene (Au–PANI–Gra) hybrid for amplification. The Au–PANI–Gra hybrid with large surface area provided a biocompatible sensing platform for the immobilization of GOD. GOD was encapsulated into the three-dimensional netlike (3-mercaptopropyl)trimethoxysilane (MPTS) to form the MPTS–GOD biocomposite, which not only retained the native functions and properties, but also exhibited tunable porosity, high thermal stability, and chemical inertness. With abundant thiol tail groups on MPTS, MPTS–GOD was able to chemisorb onto the surface of the Au–PANI–Gra modified electrode through the strong affinity of the Au–S bond. The electrochemical signal originated from GOD, avoiding the addition or labeling of other redox mediators. After immobilizing the thiolated thrombin binding aptamer through gold nanoparticles (AuNPs), GOD as a blocking reagent was employed to block the remaining active sites of the AuNPs and avoid the nonspecific adsorption. The proposed method avoided the labeling process of redox probes and increased the amount of electroactive GOD. The concentration of thrombin was monitored based on the decrease of current response through cyclic voltammetry (CV) in 0.1 M PBS (pH 7.4). With the excellent direct electron transfer of double layer GOD membranes, the resulting aptasensor exhibited high sensitivity for detection of thrombin with a wide linear range from 1.0 × 10−12 to 3.0 × 10−8 M. The proposed aptasensor also showed good stability, satisfactory reproducibility and high specificity, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules.