A turn-on ratiometric fluorescent assay is described for the determination of the activity of enzymes participating in ascorbic acid-forming reactions. Blue-emitting carbon dots (bCDs; with excitation/emission wavelength at 380/450 nm) serve as fluorescent indicator. Their fluorescence is reduced by Fe3+ ions via an inner filter effect. Yellow-emitting CDs (yCDs; with excitation/emission wavelength at 380/550 nm) serve as internal reference because their fluorescence is insensitive to Fe3+. Upon exposure to ascorbic acid (AA), Fe3+ is reduced to Fe2+. Hence, the fluorescence of the bCDs is restored. Thus, enzymes participating in AA-related reactions such as α-glucosidase (α-Glu) and alkaline phosphatase (ALP) can be determined. α-Glu activity was quantified in the range from 0.13 to 6.70 U mL-1, and ALP activity was determined between 2.0 and 130 U L-1. Endowed with excellent sensitivity, selectivity and low background signals, the method may also be used to screen the inhibitors of α-Glu and ALP. Graphical abstractSchematic representation of a redox modulated ratiometric fluorometric method based on the use of dual-color carbon dots for determination of the activity of enzymes participating in ascorbic acid-related reactions. Blue-emitting carbon dots (bCDs) serve as fluorescent indicator while yellow-emitting CDs (yCDs) serve as internal reference.
Keywords: Alkaline phosphatase; Ascorbic acid-related; Dual-color carbon dots; Human serum; Inhibitors screening; Inner filter effect; Ratiometric fluorometry; α-Glucosidase.