Study on Dertermination of Vitamin C with Biosensor

一般采用2,4二硝基苯肼（DNP）法作为食品中维生素C测定的标准方法。但该方法操作步骤复杂，耗时长，需要一种更方便快捷的测定方法。众所周知，抗坏血酸氧化酶特异性地催化抗坏血酸（AsA）氧化反应为脱氢抗坏血酸（DAsA）。在这项研究中，我们使用了一个生物传感器，并通过估计在反应过程中消耗的溶液中溶解氧（DO）的减少量来确定AsA。建立了以二硫苏糖醇为还原剂还原DAsA测定总维生素C的方法。通过估算反应过程中DO的消耗，确定了测定AsA的最佳分析条件和DAsA的还原条件。建立了高、低AsA浓度样品的标准法和灵敏法两种测定方法。在DO摄取和AsA DAsA浓度之间发现了线性关系。AsA和DAsA浓度是DO吸收量的两倍，因此该方法可以在不使用校准曲线的情况下测定AsA和DAsA。结果表明，该系统可实现低至0.01mM的AsA和DAsA的测定。本方法与DNP法和HPLC法测定的果汁中AsA含量值具有良好的可比性。该分析方法操作简便，适用于少量样品。由于所使用的酶的特异性，可以准确地测定维生素C。本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.