Application of the Phosphomolybdate Blue Colorimetric Method to the Detection of Alkaline Phosphatase Activity

In this article, a new colorimetric method is proposed to detect the activity of alkaline phosphatase, which is based on the principle that alkaline phosphatase catalyzes the hydrolysis of L-ascorbic acid-2-phosphoric acid trisodium salt, generating ascorbic acid and phosphate. The phosphate then reacts with ammonium molybdate to produce a phosphomolybdenum heteropolyacid, which is further reduced to phosphomolybdic acid blue by SnCl₂ with an absorption at 710 nm. Thus, the quantitative detection of alkaline phosphatase (ALP) activity can be achieved by absorbance. The effects of catalytic conditions and test conditions based on the detection results were investigated, and the optimized conditions were determined as Tris-HCl solution at pH 9.5 at room temperature, 150 μmol/L of substrate, 2 mg/L of SnCl₂ solution, and 2 mg/L ammonium molybdate-sulfuric acid solution. Under the optimized conditions, the linear range of the method was 2–100 U/L, with a limit of detection of 0.18 U/L. This method was used for the detection of ALP in human serum, with recoveries close to 100%, indicating that this method has potential application prospects. Due to the fast and convenient nature of the test strip sensor technique, the performance of this method on different test strips was studied. It was found that using a polyvinylidene fluoride filter membrane as the test strip enabled rapid and qualitative detection of alkaline phosphatase with high sensitivity.