Self-Correction of pH-Induced Signal Variations in Methylene Blue-Labeled Aptamer Electrochemical Biosensors: Wearable Cortisol Detection in Sweat

Common electrochemical aptamer-based (E-AB) biosensors employing methylene blue (MB) redox reporters suffer from significant pH-induced signal variations. Consequently, dynamic pH fluctuations in biofluids, such as in sweat, can greatly distort the measured signal and lead to inaccurate target quantification. Here, we introduce a self-correction strategy that enables accurate analyte quantification by compensating for pH-induced signal variations in MB-based aptasensors using the Nernstian shift of the MB peak potential. The highly reproducible reversible pH dependence of the MB peak potential enables real-time monitoring of the sample pH that provides a continuously synchronized built-in compensation of the pH interference, thereby eliminating the need for external pH sensors or additional measurement steps. This precise yet simple and effective self-correction of pH effects ensures that the Square Wave Voltammetry (SWV) signal reliably reflects real-time analyte variations, as demonstrated using a cortisol MB-labeled E-AB biosensor in both in vitro and on-body settings. In vitro measurements in artificial sweat across pH 5.5-7.5 showed excellent correlation with real-time pH and cortisol changes, confirming the reliability of the peak potential-based correction. On-body measurements using an epidermal wearable patch showed cortisol changes in sweat that would have been missed without this signal-correction method. This approach is broadly applicable to other MB-labeled E-AB biosensors and biofluids, providing a robust strategy for continuous on-body monitoring.