MnO2 nanosheets as oxidase mimics for colorimetric detection of alkaline phosphatase activity

Abstract

A sensitive colorimetric method is described for the determination of the activity of alkaline phosphatase (ALP). It is based on the regulation of the oxidase-mimicking activity of MnO₂ nanosheets. In the absence of ALP, MnO₂ nanosheets are capable of catalyzing the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to form a blue oxidized product (TMB Ox) with an absorption peak at 652 nm. In the presence of ALP and its substrate ascorbic acid-2-phosphate, the latter is hydrolyzed to form ascorbic acid (AA). AA triggers the decomposition of MnO₂ nanosheets by reducing MnO₂ to Mn²⁺, thereby weakening the enzyme mimicking activity of the MnO₂ nanosheets and causing a drop in absorbance. The drop in absorbance at 652 nm is related to the ALP activity in the range from 0.05-10 m-units per mL (mU·mL^-1), and the detection limit is 0.05 mU·mL^-1. The method was applied to the determination of ALP in spiked calf serum samples and gave satisfactory results. Graphical abstract Schematic presentation of a facile and sensitive colorimetric method for detecting the activity of alkaline phosphatase (ALP) based on enzymatic regulation of the oxidase-mimicking activity of MnO₂ nanosheets.

Keywords: 3,3′,5,5′-Tetramethylbenzidine; Ascorbic acid; Ascorbic acid-2-phosphate; Enzyme activity assay; Nanozyme; Visual detection.