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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

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Published on: April 11, 2014

Fluorescent reaction between ascorbic acid and DAN and its analytical application.

J Yang¹, C Tong, N Jie

¹Department of Chemistry, Shandong University, Jinan 250100, China.

|May 1, 1997

Summary

This summary is machine-generated.

Ascorbic acid (AA) reacts with 2,3-diamino-naphthalene (DAN) to form fluorescent products. This reaction enables sensitive detection of AA, with a detection limit of 0.4 µg/mL.

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Area of Science:

Analytical Chemistry
Organic Chemistry
Biochemistry

Background:

Ascorbic acid (vitamin C) is a vital antioxidant.
2,3-diamino-naphthalene (DAN) is a known fluorophore precursor.
Developing sensitive analytical methods for ascorbic acid is important.

Purpose of the Study:

To investigate the fluorescent reaction between ascorbic acid (AA) and 2,3-diamino-naphthalene (DAN).
To establish a quantitative method for AA determination based on this fluorescence.
To determine the optimal reaction conditions and analytical performance.

Main Methods:

Studied the reaction between AA and DAN under varying pH conditions.
Utilized spectrofluorometry to measure fluorescence intensity.
Determined excitation (400 nm) and emission (520 nm) wavelengths.
Established a calibration curve for AA concentration (2-300 µg/mL).

Main Results:

AA and DAN react at pH 10.2-10.5 to form fluorescent heterocyclic condensation products.
A strong linear correlation (R²=0.9993) was observed between fluorescence intensity and AA concentration.
The method achieved a low detection limit of 0.4 µg/mL (S/N=2).

Conclusions:

The reaction between AA and DAN provides a sensitive and reliable method for AA quantification.
This fluorescence-based assay is suitable for determining AA concentrations in relevant samples.
The established method offers excellent linearity and a low detection limit.