A sensitive dual colorimetric and fluorescence system for assaying the activity of alkaline phosphatase that relies on pyrophosphate inhibition of the peroxidase activity of copper ions

A novel and highly sensitive colorimetric and fluorescence assay for the accurate determination of alkaline phosphatase (ALP) activity has been developed. The assay takes advantage of the inhibition of the peroxidase activity of Cu2+ ions caused by complexation with pyrophosphate (PPi), a natural substrate for ALP. This inhibition disappears when PPi undergoes ALP catalyzed hydrolysis to generate phosphate, which does not bind to Cu2+ ions. Thus, ALP causes generation of uncomplexed Cu2+ ions, which promote multiple oxidation reactions of Amplex UltraRed in the presence of hydrogen peroxide in conjunction with the production of intense fluorescence and colorimetric signals. By employing the fluorescence and colorimetric assay strategies, ALP can be detected at respective concentrations as low as 4.3 pM and 5.4 pM, detection limits that are much lower than those associated with previously described methods. The practical diagnostic capability of the assay system has been demonstrated by its use to detect ALP in human blood serum.