Protein Resistance Driven by Crosslinked Biorecognition Layer for a Win-Win Analysis of the Immunoassay in Urine

Currently, biosensor antifouling and biorecognition layer designs are considered apart. The antifouling layer unavoidably impairs the electrochemical link between the biorecognition layer and base electrodes, resulting in a double-edged antifouling strategy. Here, we propose an approach guided by the concept of the multiplicative sensor, which transforms the biorecognition layer into an antifouling coating. For example, we fabricate a multiplicative sensor integrated with antifouling, hydrogen peroxide detection, and tetramethylbenzidine (TMB) detection in a 2 mm² working electrode. The antifouling function was obtained by controlling the crosslinking time of bovine serum albumin (BSA) and glucose oxidase (GOx) and remained effective for a week, even in simulations of contamination with plasma and 1 wt % BSA. By leveraging the capacity of the Prussian blue-containing working electrode to detect hydrogen peroxide and TMB and the numerous active functional groups (e.g., -NH₃, -COOH) on BSA@GOx, we have successfully constructed both a glucose biosensor and a human chorionic gonadotropin (HCG) immunosensor using the BSA@GOx layer. The multiplicative sensor exhibited detection limits of glucose, and HCG assays were measured as 0.036 mM and 0.341 ng mL^-1 with dynamic ranges of 0-0.8 mM and 0.1-100 ng mL^-1 (1.24-124 mIU mL^-1), respectively. Integrating enzymatic glucose detection into the immunosensor detection process enhances its sensitivity, thereby facilitating the detection of low-concentration enzymatic substrates. We anticipate that this methodology can be easily applied in special electrochemical biosensors featuring simplicity, cost-effectiveness, and high sensitivity.