Fluorescent nanozyme based on bimetallic Mn/Ce-doped carbon dots for dual-mode acid phosphatase activity monitoring and inhibitor screening

The development of dual-mode biosensors for the accurate detection of acid phosphatase (ACP) activity and the screening of its inhibitors is of vital importance in clinical diagnostics and environmental monitoring. In this work, we report the synthesis of Mn–Ce co-doped carbon dots (Mn&Ce@CDs) exhibiting both oxidase-mimetic catalytic activity and intrinsic fluorescence. The nanozymes efficiently catalyze the oxidation of colorless o -phenylenediamine (OPD) to produce a yellow chromogen, 2,3-diaminophenazine (DAP), characterized by a distinct absorption peak. Significantly, the excitation spectrum of DAP overlaps with the emission wavelength of Mn&Ce@CDs, resulting in an inner filter effect (IFE) that quenches the blue fluorescence of the carbon dots while simultaneously enhancing DAP's emission. This mechanism establishes a robust ratiometric fluorescence signal. The enzymatic hydrolysis of ascorbyl-2-phosphate by ACP yields ascorbic acid, which inhibits the nanozyme-catalyzed oxidation of OPD. Consequently, this leads to a decrease in DAP formation, diminishing both its characteristic absorption and the IFE-induced fluorescence quenching, thus producing a dual-signal response. Quantitative ACP detection exhibited a linear range of 0.5–15 U/L with limits of detection of 0.037 U/L in fluorescence mode and 0.49 U/L in colorimetric mode. The platform demonstrated high selectivity and reliability, showing recovery rates of 97.7–109.3 % (fluorescence) and 97.9–107.5 % (colorimetric) in spiked serum samples. Additionally, smartphone-based RGB analysis enables rapid on-site detection. The system was successfully applied for screening ACP inhibitors, with chlorpyrifos exhibiting IC 50 values of 50.45 μg/mL (fluorescence) and 43.77 μg/mL (colorimetric). This integrated dual-mode approach offers a powerful, reliable tool for clinical diagnostics and environmental assessment.