<i>In\nSitu</i> Regenerable Molecularly Imprinted\nPolymer Biosensor for Electrochemical Detection of Nonelectroactive\nBranched-Chain Amino Acids in Human Sweat

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