Microchimica Acta

, 186:246 | Cite as

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

  • Yanying Wang
  • Yan Yang
  • Wei Liu
  • Fang Ding
  • Ping Zou
  • Xianxiang Wang
  • Qingbiao ZhaoEmail author
  • Hanbing RaoEmail author
Original Paper


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.


Dual mode  o-Phenylenediamine Redox reaction Chitosan 



This work was supported by a grant from the Two-Way Support Programs of Sichuan Agricultural University (Project No. 03572228, 03572827, 03572826), the Education Department of Sichuan Provincial, PR China (Grant No. 16ZA0039).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2019_3341_MOESM1_ESM.doc (968 kb)
ESM 1 (DOC 968 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Yanying Wang
    • 1
  • Yan Yang
    • 1
  • Wei Liu
    • 1
  • Fang Ding
    • 2
  • Ping Zou
    • 1
  • Xianxiang Wang
    • 1
  • Qingbiao Zhao
    • 3
    Email author
  • Hanbing Rao
    • 1
    Email author
  1. 1.College of ScienceSichuan Agricultural UniversityYucheng DistrictPeople’s Republic of China
  2. 2.Nanshan District Key Lab for Biopolymers and Safety Evaluation, Shenzhen Key Laboratory of Polymer Science and Technology, Guangdong Research Center for Interfacial Engineering of Functional Materials, College of Materials Science and EngineeringShenzhen UniversityShenzhenPeople’s Republic of China
  3. 3.Key Laboratory of Polar Materials and Devices (MOE), Department of OptoelectronicsEast China Normal UniversityShanghaiPeople’s Republic of China

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