In Situ Formation of 2,3-Diaminophenazine for Evaluation of Alkaline Phosphatase Activity via the Inner Filter Effect

We report a fluorescence assay for alkaline phosphatase (ALP) detection using in situ generation of 2,3-diaminophenazine (OPDox) through an inner filter effect (IFE). AgNO3 can oxidize o-phenylenediamine in a short time to obtain fluorescent OPDox. p-nitrophenol is obtained from ALP-catalyzed hydrolysis of p-nitrophenylphosphate, which can result in the fluorescence quenching of OPDox via the IFE. Consequently, in situ generation of OPDox can be utilized for evaluating the activity of ALP. The fluorescence of OPDox decreases linearly with increasing concentration of ALP in the range from 0.1 to 8.0 mU mL-1, and the detection limit is calculated to be 0.05 mU mL-1. More importantly, this assay has been used to construct a logic gate. The IFE-based fluorescence assay exhibits several distinctive advantages, including high sensitivity, good selectivity, and simple operation. Consequently, it may pave the way for the detection of ALP by utilization of the in situ generation of fluorophores.