The traditional nanozymes-based ratiometric fluorescence sensing platforms usually necessitate the supplementary addition of fluorescent probes, therefore greatly restricting its convenient and broad application. In this study, a highly sensitive and selective ratiometric fluorescence platform for alkaline phosphatase (ALP) detection was established, only employing Prussian blue (PB) nanozymes and a commercially available chromogen of o-phenylenediamine (OPD). PB nanozymes with remarkable peroxidase-like (POD-like) activity can effectively catalyze OPD chromogen to yield 2,3-diaminophenazine (OPDox) with an intense yellow fluorescence at 573 nm emission peak. Target ALP can facilitate ascorbic acid 2-phosphate (AAP) dephosphorylation to generate phosphate and ascorbic acid (AA). Significantly, both these two resultant hydrolysis products could effectively decrease the OPDox generation via a dual-path based inhibition on the PB nanozymes POD-like activity. On the other hand, the generated dehydroascorbic acid (DHAA) from AA oxidation would exclusively react with OPD chromogen to yield 3-(dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ) with a strong blue fluorescent signal at 434 nm, which further providing a significant enhancement on the sensing selectivity of ALP detection. As a result, an increased yellow fluorescence of OPDox and decreased blue fluorescence of DFQ could be clearly observed with different ALP addition. A robust linear relationship between the fluorescence ratio of F434/F573 and ALP activity ranging from 0.25 U/L to 6 U/L was obtained, with a low detection limit of 0.112 U/L. This proposed method demonstrates high sensitivity, excellent selectivity, cost-effectiveness, and operational simplicity, yet enabling an effective detection of ALP levels in human serum.
Read full abstract