Rapid electrochemical biosensor with enhanced accuracy and repeatability using Pyrene NHS ester modified 3D graphene electrodes

Abstract

A consistent and reproducible chemical modification of the transducer interface is essential to ensure effective immobilization of bioreceptors, thereby improving the sensitivity and reproducibility of biosensors. Although notable progress has been made in fabricating graphene-based biosensors, the effects of surface biofunctionalization on sensing performance remain not fully understood. This study shows that combining three-dimensional (3D) graphene foam electrodes with a straightforward, single-step surface biofunctionalization method is key to enhancing the performance of graphene-based biosensors. Specifically, it presents a single-step approach to functionalize the 3D graphene foam (Gii-Sens) with 1-pyrenebutyric acid N-hydroxysuccinimide ester (Pyrene-NHS ester). Electrochemical techniques, including cyclic voltammetry (CV) and differential pulse voltammetry (DPV), confirmed the successful attachment of Pyrene-NHS ester to the graphene foam surface, at levels sufficient to serve as a covalent linker for antibody attachment. The graphene structure was maintained through non-covalent functionalization, and the linker's functionality was verified using the functionalized Gii-Sens electrochemical sensor for detecting Tau-217 peptides. Measurements with the developed electrochemical sensor showed a linear response to Tau-217 peptides within the 1 fM to 1 nM concentration range, with a limit of detection (LoD) of 0.41 fM in both phosphate-buffered saline PBS and serum. The sensor's reproducibility and repeatability were tested using the standard addition method, resulting in a relative standard deviation (RSD) of less than 5%. Specificity was evaluated against Tau-181, Tau-441, amyloid proteins (Aβ_1-40 and Aβ_1-42), and bovine serum albumin (BSA).