Design of Histidine-Based Antifouling Zwitterionic Peptides for the Reliable Electrochemical Detection of Cortisol in High-Salinity Sweat

The reliable detection of cortisol in sweat presents a promising, noninvasive approach for stress monitoring. However, in demanding scenarios such as firefighting and mining, rapid sweat evaporation concentrates salts to high levels that destabilize hydration layers and compromise electrochemical biosensors based on conventional zwitterionic peptides. In this work, we developed a salt-tolerant antifouling layer using the polypeptide CPPPP(HE) 6, which features alternating histidine and glutamic acid residues, and integrated it with cortisol aptamers to construct a wearable electrochemical biosensor. Compared with conventional zwitterionic peptides of CPPPP(EK) 6, the CPPPP(HE) 6 peptide was designed by replacing the charge-localized ammonium cation (e.g., lysine-derived NH 3 + ) with a charge-delocalized imidazolium cation from histidine. Combined computational and experimental studies reveal that both systems undergo electron redistribution with Cl – (Cl – is the most abundant anion in sweat). In contrast to the single-site polarization in NH 3 +, imidazolium + features multicenter rearrangement coupled with proton migration, which reinforces the hydrogen-bonding network and fosters an exceptionally stable hydration layer under high salinity. As a result, the CPPPP(HE) 6 coating exhibits superior fouling resistance compared to CPPPP(EK) 6 . The resulting antifouling electrochemical biosensor, functionalized with CPPPP(HE) 6 and cortisol aptamers, enabled accurate and reliable detection of cortisol in human sweat and high-salinity conditions, with results validated against the standard ELISA method. This work provides a molecular-level design strategy for creating robust antifouling materials, advancing the development of high-fidelity sensors for complex biofluids.