Ligand-engineered hollow Fe-NC nanozymes with enhanced peroxidase activity for smartphone-based point-of-care testing of isoniazid

Therapeutic drug monitoring of isoniazid (INH), a first-line antitubercular agent, is critical due to its narrow therapeutic window and risk of toxicity or resistance at nonoptimal concentrations. Current methods lack the simplicity and accessibility required for point-of-care testing (POCT), especially in resource-limited settings. Herein, we report a paper-based colorimetric biosensing platform that integrates hollow-structured Fe-NC (Iron-Nitrogen-Carbon) nanozymes with smartphone-assisted readout for rapid, sensitive, and selective detection of INH in human biofluids. The nanozyme is synthesized via controlled pyrolysis of a dual-linker zeolitic imidazolate/tetrazolate framework (ZTF), where 2-methylimidazole (2-MIM) and thermally labile 5-aminotetrazole (5-AT) direct the formation of a hollow architecture that maximizes accessible Fe-N 4 active sites and enhances mass transport. This design confers superior peroxidase-like activity, enabling catalytic oxidation of chromogenic substrates in the presence of H 2 O 2 with high specificity toward INH. The resulting sensor achieves a detection limit of 0.24 μM and maintains high accuracy in human serum and urine, with minimal matrix interference. The entire assay is completed using a portable, low-cost device and a smartphone application for colorimetric analysis. Validated on human urine and serum, the platform demonstrates excellent reproducibility, stability, and field adaptability. This work presents a rational nanozyme design strategy through ligand-mediated structural control and establishes a scalable, equipment-free sensing framework for real-time therapeutic drug monitoring in POCT.