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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
13.6K
Development of a highly sensitive, highly selective and high-throughput method for determination of total ascorbic
Takeru Koga1, Aina Yoshida2, Mayuko Tomisawa3
1Graduate School of Technology, Industrial and Social Sciences, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8513, Japan. atai@tokushima-u.ac.jp.
The Analyst
|May 30, 2025
Summary
A new method uses 1,2-diamino-4,5-methylenedioxibenzene (MDB) to accurately measure total ascorbic acid (AA) in cells. This highly sensitive and high-throughput assay is ideal for evaluating AA activity in various cellular studies.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Cell Biology
Background:
- Ascorbic acid (AA) is a vital nutrient with antioxidant properties.
- Accurate intracellular quantification of AA is crucial for understanding cellular health and disease.
- Existing methods for AA determination may lack sensitivity, selectivity, or throughput for comprehensive cellular analysis.
Purpose of the Study:
- To develop a highly sensitive, selective, and high-throughput method for determining intracellular total ascorbic acid (AA).
- To optimize reaction conditions and characterize the fluorescent properties of the AA-MDB conjugate.
- To validate the method's applicability for quantifying intracellular AA in cell lines.
Main Methods:
- Development of a fluorescence-based assay using 1,2-diamino-4,5-methylenedioxibenzene (MDB) to label AA.
- Optimization of reaction parameters including buffer, temperature, pH, and time.
- Scale-up synthesis and structural determination of the MDB-labeled AA conjugate.
- High-Performance Liquid Chromatography (HPLC) analysis for selectivity assessment.
- Validation of the method using a standard HPLC method in a cell line.
Main Results:
- Optimal conditions for AA detection involved reacting oxidized AA with MDB in 100 mM citrate buffer (pH 5.0) for 10 min at room temperature.
- The MDB-labeled AA conjugate exhibited optimal excitation and emission wavelengths at 375 nm and 450 nm, respectively.
- The method demonstrated high selectivity for AA, even in the presence of cellular components.
- Quantification of AA was achieved in the range of 25 nM to 20 μM with a detection limit of 10 nM.
- The method successfully quantified intracellular AA in a cell line, comparable to standard HPLC methods.
Conclusions:
- The developed MDB-based fluorescence assay provides a highly sensitive, selective, and high-throughput method for intracellular total ascorbic acid determination.
- This method is suitable for microplate-based analysis and has a low detection limit.
- The assay is robust and applicable to various cell types for evaluating ascorbic acid activity, offering a valuable tool for biochemical and cell biology research.
