Detection of alkaline phosphatase activity based on ATP hydrolysis and CRISPR/Cas12a

Alkaline phosphatase (ALP) activity serves as a crucial biomarker for livestock disease diagnosis, nutritional management, and the evaluation of milk pasteurization efficacy, holding significant implications for food safety and food science. In this study, we developed a novel method for detecting ALP activity in food samples, based on ATP hydrolysis coupled with a CRISPR/Cas12a system. The assay employs a DNA molecular recognition lock probe, comprising an ATP-specific aptamer and an activator strand designed to trigger the CRISPR/Cas12a trans-cleavage activity. In the absence of ALP, ATP acts as a trans-hydrolysis substrate to open the lock structure, releasing the activator strand. This subsequently activates the Cas12a protein, leading to the generation of a fluorescent signal. Conversely, when the target ALP is present, it catalyzes the hydrolysis of ATP via a dephosphorylation reaction. This prevents the opening of the "Aptamer-Activator" molecular lock, thereby inhibiting Cas12a activation and resulting in a corresponding decrease in fluorescence intensity. Under optimized conditions, the assay demonstrated a detection limit of 2.52 mU/mL for ALP activity, with a linear range of 0-18.75 mU/mL. The total detection time was 70 min. The method was successfully applied to detect ALP activity in samples from various livestock (chickens, pigs, sheep, cattle) and in milk, achieving recovery rates between 92 and 99%. In conclusion, we have developed a sensitive, cost-effective, and rapid method for ALP detection. This work provides a promising strategy for the development of point-of-care testing (POCT) devices in food safety monitoring.