In situ reaction-based ratiometric fluorescent assay for alkaline phosphatase activity and bioimaging

Alkaline phosphatase (ALP) is an important biomarker, it is of great significance to develop a sensitive and efficient analytical method for ALP. In this study, an in situ reaction based ratiometric fluorescence assay for ALP was proposed. l-ascorbic acid-2-phosphate (AA2P) was used as a substrate for ALP, and Cu²⁺/o-phenylenediamine (OPD) were involved in this system. Cu²⁺ can oxidize OPD to 2,3-diaminophenazine (OPDox) with an emission centered at 566 nm. The presence of ALP can catalyze the hydrolysis of AA2P to ascorbic acid (AA), which will inhibit the production of OPDox and reduce the corresponding fluorescence intensity, and AA will react with OPD to generate 3-(dihydroxyethyl)furan[3,4-b]quinoxalin-1-one (DFQ) with an emission peak at 447 nm. The fluorescence ratio of F447/F566 has a linear relationship with ALP activity. The proposed method is highly sensitive, finely selective, cost efficiency and easy to operate, it exhibits good linearity in the range of 0.5-22 and 22-40 mU·mL^-1, with a detection limit as low as 0.06 mU·mL^-1. The excellent applicability of this strategy in human serum samples and MCF-7 cells imaging suggests that this method has promising prospects for biomedical research.