Colorimetric Detection of Alkaline Phosphatase Activity Based on Manganese Single Atom Nanozyme

Manganese (Mn) single atom nanozyme with peroxidase-like activity was synthesized through a solvothermal method and used for the detection of alkaline phosphatase (ALP) activity. By using formamide as the carbon and nitrogen source and MnCl2 as the Mn source, formamide converted Mn and nitroren co-doped carbon (f-MnNC) could be obtained after reacting at 180 celcius for 12 h. The structure and morphology were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, X-ray diffractometer, etc. , and the peroxidase-like activity was investigated mainly using UV-visible absorption spectroscopy. In the presence of H2O2, f-MnNC could catalyze the oxidation of colorless 3, 3', 5, 5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB), with a characteristic absorption peak centered at 652 nm. ALP could catalyze the hydrolysis of L-ascorbic acid-2-phosphate (AA2P ) to produce ascorbic acid (AA), which then reduced oxTMB to TMB, resulting in faded color and decreased absorbance. The degree of colorimetric signal change was related to ALP activity, and thus ALP activity could be quantitatively detected by employing AA2P as the substrate. In the activity range of 0. 1-10 mU/mL, there was a good linear relationship between the absorbance at 652 nm and the ALP activity, and the detection limit (S/N= 3) was 0. 059 mU/ mL. Other proteins and enzymes had no obvious interference with ALP detection, indicating good selectivity for ALP activity detection. The method was successfully applied to the determination of ALP activity in serum with satisfactory results.