A carbon dot-based ratiometric fluorometric and colorimetric method for determination of ascorbic acid and of the activity of ascorbic acid oxidase

Abstract

A dual-mode method was developed for the determination of ascorbic acid (AA) and of ascorbic acid oxidase (AA-Ox) activity. It combines the advantages of ratiometric fluorometry and colorimetry. The assay is based on the oxidation of o-phenylenediamine (OPDA) by permanganate (KMnO₄). A yellow substance (referred to as oxOPDA) with an absorption peak at 425 nm is rapidly produced in the presence of the oxidant. oxOPDA reduces the blue fluorescence of carbon dots (C-dots) peaking at 450 nm (upon 380-nm excitation), and a new emission peak is found at 565 nm. If AA is pesent, it consumes a certain fraction of KMnO₄, so that less OPDA will be oxidized. This is accompanied by a decrease in the intensity of the fluorescence at 565 nm and an increase in the intensity at 450 nm. In parallel, the color of the solution changes from yellow to colorless. The determination of the activity of ascorbic acid oxidase (AA-Ox) is performed as follows: AA is oxidized by AA-Ox. This causes the fluorescence and colors to change in the opposite directions compared with AA detection. The ratio of fluorescences (I₅₆₅/I₄₅₀) becomes larger if the color the solution turns from colorless to yellow. Thus, the fluorescence intensity ratio (I₅₆₅/I₄₅₀) and colorimetric "bare-eye" readout can be used for determination of both the concentration of AA and the activity of AA-Ox. The fluorometric assay for AA has a linear range that extends from 0.6 to 40 μM, and the colorimetric assay from 0.2 to 70 μM. The respective data for AA-Ox activity are 0.04 ~ 5 mU·mL^-1 and 0.04 ~ 8 mU·mL^-1, respectively. The limits of detection for AA are 9 and 40 nM, and the LODs for AA-Ox activity are 0.017 and 0.012 mU·mL^-1. Graphical abstract Schematic presentation of the assay. Permanganate (KMnO4) rapidly oxidizes ortho-phenylenediamine oxide to form a product (oxOPDA) having a yellow fluorescence peaking at 565 nm. The yellow color of oxOPDA can be detected visually. It also reduces the intensity of the blue fluorescence of carbon dots (C-dots) peaking at 450 nm. Ascorbic acid (AA) can consume permanganate, and this results less oxidation of OPDA. Ascorbic acid oxidase (AA-Ox) catalyzes the oxidization of AA by oxygen, and this - in turn - causes the changes in absorbance and fluorescence to change in the opposite directions.

Keywords: Chitosan; Dual mode; Redox reaction; o-Phenylenediamine.