A regenerable non-invasive salivary glucose biosensor enabling less-painful monitoring

A regenerable amperometric biosensor for non-invasive glucose detection in human saliva is presented. The platform employs a microfabricated Pt electrode modified with a conductive PANI:PSS/C-SWCNT nanocomposite, followed by immobilization of glucose oxidase within a chitosan hydrogel containing ferrocene as redox mediator. Spectroscopic (UV–VIS, FTIR, Raman) and SEM analyses confirmed structural integrity and preservation of enzymatic activity. Electrochemical characterization demonstrated high fabrication reproducibility (RSD < 12%, n = 40) and efficient Fc/Fc + redox mediation. Cyclic voltammetry and chronoamperometry revealed apparent diffusion coefficients on the order of 10 −5 cm 2 s −1 and fast heterogeneous electron transfer kinetics (~10 −2 cm s −1 ), consistent with immobilized enzyme-mediated systems. As glucose is not electroactive, the measured current arises exclusively from ferrocene-mediated enzymatic oxidation. Under steady-state conditions, the biosensor followed Michaelis–Menten kinetics with an apparent Kₘ of 0.50 mM, indicating high enzyme affinity. A linear response was obtained in the physiologically relevant salivary range (10–1000 μM), with a limit of detection of 3.0 μM and limit of quantification of 10.0 μM. Stable performance was maintained between pH 6.0–8.5, with repeatability better than 20% at 0.6 V. Validation using 80 saliva samples showed excellent agreement with ELISA (Pearson's r = 0.996). The demonstrated regenerability and stability support the suitability of this biosensor for in vitro point-of-care monitoring and future wearable applications.