In Situ Regenerable Molecularly Imprinted Polymer Biosensor for Electrochemical Detection of Nonelectroactive Branched-Chain Amino Acids in Human Sweat

The significant challenge in achieving in situ regeneration for conventional molecularly imprinted polymers (MIPs) restricts their promising application in continuous monitoring of biochemical molecules closely related to human health, especially nonelectroactive molecules. This is because they are either limited to a single use or require removal of imprinted templates through chemical washing steps, which is clearly impractical for sustainable monitoring. Here, a class of in situ regenerable MIP biosensors, taking nonelectroactive branched-chain amino acids (BCAAs) as templates and methyldopa as a functional monomer, was engineered to achieve repeatable in situ regeneration and in situ target recognition. The in situ regeneration was realized through an amperometric i–t technique with a negative voltage (−0.9 V) according to intrinsic isoelectric points of analytes instead of conventional wash steps. This electrochemical extraction process not only maximally repelled the imprinted templates, creating a large number of cavities (recognition sites) and significantly enhancing sensitivity, but also ensured the successful in situ regeneration of developed biosensing interfaces. The template extraction was evaluated by examining changes in the surface morphology, elemental composition, distribution, content, and interfacial properties. The developed BCAA MIP biosensors achieved sensitive target detection with the linear range from 0.001 to 10.0 μg/mL and limits of detection down to 0.45 (Leu), 0.47 (Ile), and 0.31 (Val) ng/mL. Beyond that, the biosensors demonstrated an excellent ability in decreasing biofouling, realizing repeatable in situ target detection in human sweat, and the obtained results were highly consistent with those of the enzyme-linked immunosorbent assay, indicating high feasibility, reliability, and accuracy in practical application. Meanwhile, the biosensors showed excellent specificity, selectivity, and stability.