A Nanostructured CuO--Enhanced Chemiluminescent Hybrid Microfluidic Platform for Total Cholesterol Biosensing

Total Cholesterol (TC) monitoring in blood serum is critically important for assessing cardiovascular risk and metabolic health. In this work, we report a nano-bioscience-enabled cholesterol-sensing portable platform that integrates copper oxide (CuO) nanoparticles and a hybrid microfluidic chip, along with smartphone-based chemiluminescence (CL) imaging and computational analysis. The hybrid microfluidic miniaturized chip, fabricated using PDMS material and Whatman filter paper, enables efficient reagent transport, on chip micromixing, and localized signal generation. CuO nanoparticles act as catalytic enhancers for the luminol-H₂O₂ chemiluminescence reaction initiated by cholesterol oxidase, resulting in strengthened and stable photon emission. Chemiluminescence signals were captured using a smartphone camera placed in a 3D-printed dark box and analyzed through a customized mobile application, Intensity Tracker application, for intensity evaluation proportional to cholesterol concentration. The proposed work gives a linear detection range for the cholesterol over a concentration of 0.06-1.5 mM, with a limit of detection of 0.05 mM and a limit of quantification of 0.191 mM, demonstrating strong analytical performance R ${}^{{2}}~\approx ~0.98$ . Validation using human serum samples yielded recovery values ranging from 92 to 101%, confirming high accuracy and minimal matrix interference when compared with results from a standard biochemistry analyzer. The developed hybrid microfluidic chemiluminescence analyzer features a compact design, low cost, and user-friendly operation, presenting a promising solution for rapid, accurate, and decentralized cholesterol monitoring in point-of-care and resource-limited settings. Future work will focus on multiplexed biomarker detection, and integration with AI-based analysis for real-time clinical decision support.