A ratiometric fluorescent assay for evaluation of alkaline phosphatase activity based on ionic liquid–functionalized carbon dots

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.