Study on Dertermination of Vitamin C with Biosensor

食品中维生素C的测定通常采用2，4-二硝基苯肼（DNP）法。但该方法操作繁琐、耗时长，需要一种更方便、快速的测定方法。众所周知，抗坏血酸氧化酶特异性催化抗坏血酸（阿萨）氧化为脱氢抗坏血酸（D阿萨）的反应。在这项研究中，我们使用的生物传感器和测定阿萨的溶解氧（DO）在反应过程中消耗的溶液中的减少量的估计。建立了以二硫苏糖醇为还原剂还原维生素A测定维生素C的方法。通过估算反应过程中溶解氧的消耗量，确定了测定阿萨的最佳分析条件和D阿萨的还原条件。建立了高、低浓度阿萨样品的标准测定方法和灵敏测定方法。DO的吸收量与阿萨D阿萨浓度呈线性关系。阿萨和D阿萨浓度是DO吸收的两倍，因此该方法使得可以在不使用校准曲线的情况下测定阿萨和D阿萨。结果表明，该传感器系统可检测低至0.01mM的阿萨和D阿萨。本方法测定的果汁中阿萨含量与DNP法和HPLC法测定的结果具有良好的可比性。该分析方法操作简便，适用于少量样品的分析。由于所用酶的特异性，可以准确测定维生素C。该方法适用于食品中总维生素C含量的快速测定。

Generally, 2, 4 dinitrophenyl hydrazine (DNP) method is employed as a standard method for the determination of vitamin C in foods. However, the operation procedures of this method are rather complicated and time consuming, so a more convenient and rapid determination method is necessary. It is well known that ascorbic acid oxidase specifically catalyzes the oxidation reaction of ascorbic acid (AsA) to dehydroascorbic acid (DAsA). In this study, we used a biosensor and determined AsA by the estimation of the decreasing amount of dissolved oxygen (DO) in the solution consumed during the reaction. We established a method for the determination of total vitamin C by using dithiothreitol as reducing agent for the DAsA reduction. The optimum analysis conditions for the determination of AsA and reduction conditions of DAsA were established by estimating the consumption of DO during reaction. The two determination methods, the standard method and the sensitive one for high and low AsA concentration samples respectively, were established. A linear relationship was found between the DO uptake and AsA DAsA concentrations. AsA and DAsA concentrations were twice the DO uptake, thus this method makes it possible to determine AsA and DAsA without using a calibration curve. It was found that the determination level of AsA and DAsA as low as 0.01mM is possible using this biosensor system. The values of AsA contents in the juice obtained using the present method have a good comparability to those obtained by DNP and HPLC method. The procedure for this analytical method was quite simple and applicable for small amount samples. Due to the specificity of the enzyme used, it was possible to determine vitamin C accurately. This method is applicable for a rapid determination of total vitamin C contents in foods.