Koga Takeru, Yoshida Aina, Tomisawa Mayuko, Nakamura Mitsuhiro, Tai Akihiro
Graduate School of Technology, Industrial and Social Sciences, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8513, Japan.
Graduate School of Sciences and Technology for Innovation, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8513, Japan.
Analyst. 2025 Jun 23;150(13):2865-2871. doi: 10.1039/d5an00319a.
A highly sensitive, highly selective, and high-throughput method for determination of intracellular total ascorbic acid (AA) was developed by measuring the fluorescence intensity of 1,2-diamino-4,5-methylenedioxibenzene (MDB)-labeled AA. First, the reaction buffer, temperature, pH, and time were investigated to establish optimal reaction conditions, and it was found that AA was detected with high sensitivity by reacting oxidized AA and MDB in 100 mM citrate buffer (pH 5.0) for 10 min at room temperature. Next, a scale-up synthesis was performed to determine the chemical structure of the MDB-labeled AA, and its excitation and emission wavelengths were optimized to 375 and 450 nm, respectively. HPLC analysis revealed that MDB reacts with AA with high selectivity under the established conditions. It was also found that AA could be determined under optimal reaction conditions, regardless of the presence or absence of cellular components. In conclusion, the established method for determination of AA with MDB can quantify AA in the range of 25 nM to 20 μM in a microplate with a detection limit of 10 nM. Furthermore, the method was confirmed to be able to quantify intracellular AA in a cell line by comparison with a standard HPLC method. This method is applicable to highly sensitive, highly selective, and high-throughput analysis and could be applied to the determination of AA in various cells used in the evaluation of AA activity.
通过测量1,2 - 二氨基 - 4,5 - 亚甲基二氧苯（MDB）标记的抗坏血酸（AA）的荧光强度，开发了一种用于测定细胞内总抗坏血酸（AA）的高灵敏度、高选择性和高通量方法。首先，研究了反应缓冲液、温度、pH值和时间以建立最佳反应条件，发现通过在100 mM柠檬酸盐缓冲液（pH 5.0）中于室温下使氧化型AA与MDB反应10分钟，可以高灵敏度地检测AA。接下来，进行了放大合成以确定MDB标记的AA的化学结构，并将其激发和发射波长分别优化为375和450 nm。HPLC分析表明，在既定条件下MDB与AA具有高选择性反应。还发现，无论细胞成分是否存在，在最佳反应条件下都可以测定AA。总之，所建立的用MDB测定AA的方法能够在微孔板中定量25 nM至20 μM范围内的AA，检测限为10 nM。此外，通过与标准HPLC方法比较，证实该方法能够定量细胞系中的细胞内AA。该方法适用于高灵敏度、高选择性和高通量分析，可用于评估AA活性的各种细胞中AA的测定。
