Alkaline phosphatase (ALP) is a phosphomonoester hydrolase and serves as a biomarker in various diseases. However, current detection methods for ALP rely on bulky instruments, extended time, and complex operations, which are particularly challenging in resource-limited regions. Herein, we synthesized a MOF-derived Fe-N-C nanozyme to create biosensors for the coulometric and visual detection of ALP. Specifically, we found the Fe-N-C nanozyme can efficiently oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue-colored tetramethyl benzidine (TMBox) without the need for H2O2. To construct the biosensor, we incorporated the ALP enzymatic catalytic reaction to inhibit the oxidation of TMB by Fe-N-C oxidase nanozyme. This biosensor showed rapid and highly sensitive detection of ALP in both buffer and clinical samples. The limit of detection (LOD) of our approach could be achieved at 3.38 U L-1, and the linear range was from 5 to 60 U L-1. Moreover, we also developed a visual detection for ALP by using a smartphone-based assay and facilitated practical and accessible point-and-care testing (POCT) in resource-limited areas. The visual detection method also achieved a similar LOD of 2.12 U L-1 and a linear range of 5-60 U L-1. Our approach presents potential applications for other biomarker detections by using ALP-based ELISA methods.
Keywords: alkaline phosphatase; metal-organic framework; nanozyme; oxidase-like enzyme; visual detection.