Construction of an Antifouling Electrochemical Biosensor Based on Bilateral Proline-Rich Peptide Scaffolds with Enhanced Stability for Detection in Complex Biofluids

Abstract The precise detection of biomarkers in complex biofluids remains a significant challenge for electrochemical biosensors, largely due to biofouling and the susceptibility of peptide-based antifouling interfaces to enzymatic degradation. Herein, we report a bilateral proline (P)-rich peptide scaffold, CPPCEKEKEKEKCPPC (Cys-Pro-Pro-Cys-Glu-Lys-Glu-Lys-Glu-Lys-Glu-Lys-Cys-Pro-Pro-Cys), which introduces CPPC motifs at both termini of the antifouling EKEKEKEK sequence to enforce a rigid architecture across the entire antifouling sequence. Molecular dynamics (MD) simulations reveal a fundamental structural distinction: unlike the linear peptide CEKEKEKEK and its simple cyclic-like analogue CEKEKEKEKCboth of which adopt extended, flexible conformationsCPPCEKEKEKEKCPPC retains a cyclic architecture due to the bilateral proline-rich constraint. This intrinsic rigidity confers markedly enhanced resistance to proteolytic degradation and exceptional long-term antifouling stability exceeding 45 days in complex media including serum, saliva, and sweat, substantially outperforming both linear and conventional cyclic-like peptide analogues. As a proof of concept, we constructed an electrochemical aptasensor using CPPCEKEKEKEKCPPC as the antifouling self-assembled monolayer (SAM), enabling direct detection of C-reactive protein (CRP) in undiluted human saliva with a detection limit of 3.31 fg/mL. Importantly, the biosensor demonstrates reliability with commercial ELISA methods in clinical sample analysis. With its high accuracy and capacity for direct detection in complex biofluids, this bilateral constraint strategy provides a broadly applicable platform for the formation of a stable antifouling SAM toward diverse biomedical applications.