Naked-eye sensitive detection of alkaline phosphatase (ALP) and pyrophosphate (PPi) based on a horseradish peroxidase catalytic colorimetric system with Cu(ii)

Abstract

In this paper, a novel colorimetric method for the detection of alkaline phosphatase (ALP) and pyrophosphate (PPi) was designed based on a Cu²⁺–horseradish peroxidase (HRP)–3,3′,5,5′-tetra-methylbenzidine (TMB)–H₂O₂ system. In the presence of ALP, L-ascorbic acid-2-phosphate (AAP) could be hydrolyzed to ascorbic acid which could reduce Cu²⁺ to Cu⁺ to inhibit the enzymatic activity of HRP in the colorimetric system. The change in absorbance was found to be proportional to the ALP concentration with a linear detection range and a limit of detection of 5.4 mU mL⁻¹. In the presence of PPi, because Cu²⁺ was chelated by PPi, the conversion of Cu(II) by AA was effectively inhibited. The color of the HRP–TMB–H₂O₂ system with Cu²⁺ showed blue. The HRP–TMB–H₂O₂ system with the Cu²⁺ colorimetric system could also detect PPi with a satisfying result. In summary, this method possesses sensitivity, reproducibility, and cost-effectiveness without labelling and separation and the use of a colorimetric method is more in line with the requirements of on-site detection and green chemistry.