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

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 threedimensional (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 GiiSens 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 (Aβ1-40 and Aβ1-42), and
bovine serum albumin (BSA).