Chitosan-Modified Gold Nanoparticle-Based Electrochemical Immunosensor for C-Reactive Protein Detection

C-reactive protein (CRP) is one of the most essential biomarkers for the early detection of inflammation and infection. In this study, we developed a sensitive and selective electrochemical immunosensor for CRP detection, leveraging the unique properties of gold nanoparticles (AuNPs). A nanostructured layer of AuNPs was deposited onto a screen-printed carbon electrode (SPCE), followed by the formation of a self-assembled monolayer (SAM) of L-cysteine and EDC/sulfo-NHS chemistry. The antibody was covalently immobilized onto the modified electrode through optimized dual-crosslinking chemistry. Detection conditions were systematically optimized, with pH 8.0 in Tris buffer providing the best electrochemical response. Electrochemical characterization was performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) in a 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] redox probe solution containing 0.1 M KCl. CRP detection was achieved by monitoring the increase in charge transfer resistance (Rct) upon specific binding of the target CRP antigen to the immobilized antibody. Spiked recovery experiments showed spiked recovery rates ranging from 98.01% to 107.14%, with a standard deviation below 4%. Regeneration studies demonstrated high efficiency, confirming the suitability of the sensor interface for repeated and reliable measurements. Under optimized conditions, the immunosensor exhibited excellent analytical performance, including a low limit of detection (LOD) of 0.16 µg/mL, a wide linear detection range of 5-100 µg/mL, high selectivity against 13 potential interferents (including inflammatory cytokines), and good reproducibility with a relative standard deviation (RSD) of 3.69%. The sensor also showed strong stability, retaining more than 95% of its signal after 15 days, and high regeneration efficiency of 97% over seven cycles. These results highlight the strong potential of the proposed immunosensor for point-of-care (POC) applications due to its simple fabrication, cost-effectiveness, user accessibility, and robust analytical performance.