A ratiometric fluorescent assay for evaluation of alkaline phosphatase activity based on ionic liquid-functionalized...

A ratiometric fluorescent assay is fabricated for the evaluation of alkaline phosphatase (ALP) activity. This assay is composed of ionic liquid-functionalized carbon dots (IL-CDs) with blue fluorescence signal at 470 nm and 2,3-diaminophenazine (DAP) with yellow fluorescence signal at 570 nm. IL-CDs were synthesized via electrochemical method by using ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate) and ultrapure water as precursors. DAP is produced by the oxidation reaction between o-phenylenediamine and H₂O₂ under the catalysis of horseradish peroxidase. H₂O₂ is reduced by ascorbic acid which is the hydrolysis product of ascorbic acid 2-phosphate under the catalysis of ALP, finally reducing the amount of DAP. The activity of ALP is evaluated through the ratiometric fluorescent signal between IL-CDs and DAP via Förster resonance energy transfer. Under optimal experimental conditions, this ratiometric fluorescent assay has a response that covers the 0.04 to 3.2 U L^-1 (12 to 960 pM) ALP activity. This assay possesses ultralow detection limit of 0.012 U L^-1 (3.6 pM) for ALP and high selectivity for ALP among several enzymes. The method was used to measure ALP activity in human serum samples with satisfying results. Graphical abstract Schematic presentation of IL-CDs-based ratiometric fluorescent assay for ALP activity evaluation via FRET strategy between IL-CDs and DAP. This ratiometric fluorescent assay possessed low detection limit of ALP activity (0.012 U L^-1) and high selectivity among several enzymes.