Small-Molecule Interaction-Driven Luminol-Artemisinin Chemiluminescent Probe for Highly Sensitive Determination of Alkaline Phosphatase.

Alkaline phosphatase (ALP) is a pivotal enzyme in diverse physiological processes, and its activity serves as an essential biomarker in clinical diagnostics and biomedical research. The development of reliable and highly sensitive assays for monitoring ALP is therefore of great importance. Herein, we present a luminol-artemisinin (ART) chemiluminescence (CL) platform for the ultrasensitive determination of ALP activity. The assay relies on ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AA2P) to produce ascorbic acid (AA), which efficiently quenches luminol-ART CL. Systematic optimization, sensitivity, and selectivity studies revealed a strong correlation between ALP concentration and the quenching of the CL intensity, enabling quantitative analysis with high sensitivity and excellent selectivity. The assay was further validated using human serum samples from healthy donors and patients with clinically documented elevated ALP levels. In healthy diluted serum samples, the standard addition method yielded endogenous ALP activities of 0.104-0.118 U L-1 with recoveries of 97.01-107.89%. Furthermore, the ALP activities in diluted serum samples from three clinical patients were determined to be 0.347, 0.183, and 0.221 U L-1, in close agreement with the corresponding clinical reference values of 0.353, 0.171, and 0.226 U L-1, demonstrating the accuracy and practical applicability of the proposed assay. Compared with existing analytical techniques, the luminol-ART CL assay provides superior sensitivity, operational simplicity, and compatibility with complex biological matrices, offering a promising platform for clinical diagnostics and biochemical analysis.