"Turn-on" fluorometric probe for α-glucosidase activity using red fluorescent carbon dots and 3,3',5,5'-tetramethylbenzidine

Abstract

A turn-on method for determining α-glucosidase activity is described using a chemical redox strategy in which the fluorescence of red fluorescent carbon dots (CDs) is modulated. The red fluorescent CDs were prepared using a solvothermal method with p-phenylenediamine and sodium citrate. The excitation and emission maxima of the CDs were 490 and 618 nm, respectively. Ce⁴⁺ ions catalyze the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) to give a blue oxidized TMB product (oxTMB). Absorption by oxTMB overlaps with the red light emitted by the CDs because of the fluorescence inner filter effect; therefore the presence of oxTMB decreases the intensity of fluorescence emission by the CDs. However, hydrolysis of L-ascorbic acid-2-O-α-D-glucopyranosyl by the enzyme α-glucosidase causes formation of ascorbic acid . Ascorbic acid reduces oxTMB to TMB, so that the inner filter effect disappeared and the fluorescence recovered. The strategy allows α-glucosidase activity to be successfully determined down to 0.02 U mL^-1 and gives a dynamic linear range of 0-5.5 U mL^-1. The strategy is very selective for α-glucosidase activity in the presence of potentially interfering substances. The method has been successfully applied to the determination of α-glucosidase activity in spiked human serum samples and gave satisfactory results. Graphical Abstract Schematic of the method used to prepare the carbon dots and the mechanisms involved in determining α-glucosidase activity.

Keywords: 3,3′,5,5′-Tetramethylbenzidine; Carbon dots; Ce4+ ions; Fluorescence detection; L-ascorbic acid-2-O-α-D-glucopyranosyl; p-Phenylenediamine; α-Glucosidase activity.