Colorimetric "off-on" sensing of penicillamine via copper-mediated modulation of laccase-mimetic activity of trimanganese tetroxide nanoflowers.

D-Penicillamine (D-PA) is widely prescribed for Wilson's disease, rheumatoid arthritis, and cystinuria; however, its narrow therapeutic window and risk of severe renal, hematological, and autoimmune complications necessitate precise therapeutic monitoring. Traditional methods such as HPLC provide accurate quantification but are costly, time-consuming, and require sophisticated instrumentation, limiting their routine use. Nanozyme-based colorimetry has emerged as a rapid and cost-effective alternative, yet most reported systems suffer from limited selectivity and reliance on unstable oxidants. Herein, we report a novel "off-on" colorimetric sensor exploiting Cu2+-regulated laccase-mimic activity of flower-like Mn₃O₄ nanoparticles. In this system, Cu2+ ions suppress the catalytic oxidation of 2, 4-DP, while D-PA binding effectively sequesters Cu2+, restoring the catalytic activity and generating a measurable chromogenic shift. The sensor achieved an ultralow detection limit of 4.8 nM, exhibited excellent selectivity even in complex matrices, and demonstrated reliable performance for D-PA quantification in pharmaceutical formulations, serum, and urine. This strategy integrates sensitivity, operational simplicity, and eco-friendliness, positioning it as a practical alternative to conventional chromatographic assays for clinical and pharmaceutical monitoring.