A CoOOH nanosheet-based colorimetric method for sensitive detection of alkaline phosphatase in biological samples

Alkaline phosphatase (ALP) is an important clinical biomarker, and rapid activity quantification is needed in routine diagnostics. Here, we report a CoOOH nanosheet-based colorimetric assay for ALP that uses a H₂O₂-free TMB chromogenic module. CoOOH nanosheets catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to produce the blue oxidized product, while ALP hydrolyzes ascorbic acid 2-phosphate (AAP) to generate ascorbic acid (AA), which reduces/decomposes CoOOH and suppresses the TMB signal, resulting in a distinct blue-to-colorless readout. Under optimized conditions, the absorbance response is linear with ALP activity from 0.01 to 25 mU mL^-1 with a detection limit of 0.01 mU mL^-1. The assay shows good robustness against common matrix components (e.g., urea, glucose, proteins, and major inorganic ions), whereas thiol-containing reductants can attenuate the signal at elevated levels. Serum recovery tests gave recoveries of 94.30-97.50% with RSDs of 1.9-4.9%, confirming reliability in complex matrices. This method provides a simple, cost-effective, and sensitive approach for ALP activity measurement. In addition, the CoOOH-TMB pair serves as a general H₂O₂-free TMB signal module, suggesting potential adaptability to microplate-based ALP-linked immunoassay/ELISA formats.