Enzyme conjugated magnetic nanocomposite hydrogel based on chitosan as a green fluorescent cholesterol biosensor

A green fluorescent cholesterol biosensor (ChOx-MNH) was developed by immobilizing cholesterol oxidase (ChOx) within a nanomagnetic chitosan-polyacrylamide (CS-PAAm) hydrogel matrix. The structural, morphological, and magnetic properties of the biosensor were examined using FT-IR, XRD, TGA, FE-SEM, TEM, BET, MTT, and VSM analyses. The resulting ChOx-MNH acts as a metal-oxide/enzyme composite catalyst, thereby establishing a cascade-based sensing approach for cholesterol determination. ChOx catalyzes the oxidation of cholesterol, followed by the catalytic decomposition of generated hydrogen peroxide (H₂O₂) into highly reactive hydroxyl radicals (^•OH) by Fe₃O₄ nanoparticles. These radicals subsequently react with coumarin to form fluorescent 7-hydroxycoumarin, whose emission intensity increases proportionally with cholesterol concentration. During method development, the effects of coumarin concentration, reaction medium pH, and nanozyme (Fe₃O₄) content on the fluorescence response were systematically evaluated, and suitable operating conditions were selected to ensure efficient signal generation. Under these conditions, the biosensor enabled selective and quantitative cholesterol detection over a linear range of 0.0996-12.93 mM with a limit of detection of 0.4638 mM. In addition to its analytical performance, the proposed platform offers advantages such as low cost, environmental compatibility, and convenient magnetic separation. The practical applicability of the ChOx-MNH was further demonstrated by the analysis of human serum samples, which showed good agreement with a commercial diagnostic kit.