Colorimetric detection of H₂O₂ with Fe₃O₄@Chi nanozyme modified µPADs using artificial intelligence

Peroxidase mimicking Fe₃O₄@Chitosan (Fe₃O₄@Chi) nanozyme was synthesized and used for high-sensitive enzyme-free colorimetric detection of H₂O₂. The nanozyme was characterized in comparison with  Fe₃O₄ nanoparticles (NPs) using X-ray diffraction, Fourier-transform infrared spectroscopy, dynamic light scattering, and thermogravimetric analysis. The catalytic performance of Fe₃O₄@Chi nanozyme was first evaluated by UV-Vis spectroscopy using 3,3',5,5'-tetramethylbenzidine. Unlike Fe₃O₄NPs, Fe₃O₄@Chi nanozyme exhibited an intrinsic peroxidase activity with a detection limit of 69 nM. Next, the nanozyme was applied to a microfluidic paper-based analytical device (µPAD) and colorimetric analysis was performed at varying concentrations of H₂O₂ using a machine learning-based smartphone app called "Hi-perox Sens++ ." The app with machine learning classifiers made the system user-friendly as well as more robust and adaptive against variation in illumination and camera optics. In order to train various machine learning classifiers, the images of the µPADs were taken at 30 s and 10 min by four smartphone brands under seven different illuminations. According to the results, linear discriminant analysis exhibited the highest classification accuracy (98.7%) with phone-independent repeatability at t = 30 s and the accuracy was preserved for 10 min. The proposed system also showed excellent selectivity in the presence of various interfering molecules and good detection performance in tap water.