Flow injection analysis by use of a biosensor based on a co-enzyme regeneration system

使用基于辅酶再生系统的生物传感器进行流动注射分析

• 批准号: 61560149
• 负责人: MATSUMOTO Kiyoshi
• 金额: $ 0.9万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61560149/
• 关键词: Regeneration of co-enzyme; Immobilized co-enzyme; Mediator; Immobilized dehydrogenase; フローインジェクション分析

Co-enzyme NAD and dehydrogenases were immobilized on insoluble Sepharose,and the regeneration system of the co-enzyme was established. The application of the regeneration system to flow injection analysis (FIA) was performed.1. A direct immobilization method of NAD onto Sepharose was developed by use of the bifunctional reagent, glutaraldehyde (GA). The immobilization mechanism depended on the pH of the reaction mixture of NAD and the GA-activated Sepharose. It was suggested that the structure of methylol type was formed between <alpha> (beta)-unsaturated aldehyde on teh GA-activated Sepharose and N^6-amino group on the adenine moiety of NAD in the acidic region (pH 3). In the neutral or weak alkaline region (pH 8), it was supposed that the immobilization was achieved by the force of ionic interaction between the phosphate moiety of NAD and the positive charge on the support. The immobilized NAD, which was prepared at pH 8, maintained a high co-factor activity and was applicable to co immobilization with alcohol dehydrogenase (ADH).2. The modified gel (R_3-Seph) was prepared by the reaction of CNBr-activated Sepharose with hexamethylenediamine and GA, followed by the same reaction for three times. The produced modified gel had a relatively long size of spacer, and it was applied for co-immobilization of ADH and NAD. The re-usability of the co-immobilized gel mainly depended on the reaction time of GA with Sepharose. The co-immobilized gel, which was prepared by the optimum condition (GA 5%, 2 hr), proposed the linear responses for 0 -0.8 mM ethanol by the batch system.3. The co-immobilized gel of ADH, NAD and diaphorase was prepared by the nearly same method described in section 2. The optimum condition for the R_3-Seph preparation was as follows: GA concentration 20%, reaction time 1 hr. The co-immobilized gel was applied to FIA method to determine ethanol. The FIA method was applicable to ethanol determination (0.1 - 0.5 M).

将辅酶NAD和脱氢酶固定在不溶性Sepharose上，建立了辅酶的再生体系。对再生系统在流动注射分析（FIA）中的应用进行了研究。研究了以双功能试剂戊二醛（GA）为载体，将NAD直接固定在Sepharose上的方法。固定化机制取决于NAD与ga活化的Sepharose反应混合物的pH值。表明甲基型结构是在ga活化的Sepharose上的< α > (β)-不饱和醛和NAD的腺嘌呤部分的N^6-氨基之间形成的。在中性或弱碱性区域（pH 8）， NAD的磷酸基团与载体上的正电荷通过离子相互作用实现了固定化。在pH为8的条件下制备的固定化NAD保持了较高的辅助因子活性，适用于乙醇脱氢酶（ADH）的共固定化。将cnbr活化的Sepharose与己二胺和GA反应，经过3次相同的反应，制备了改性凝胶（R_3-Seph）。制备的改性凝胶具有较长的间隔段，可用于ADH和NAD的共固定。共固定化凝胶的重复使用主要取决于GA与Sepharose的反应时间。以最佳工艺条件（GA 5%, 2 h）制备的共固定化凝胶在0 ~ 0.8 mM乙醇的条件下，具有良好的线性反应。ADH、NAD和diaphorase共固定化凝胶的制备方法与第2节描述的方法几乎相同。制备R_3-Seph的最佳工艺条件为：GA浓度20%，反应时间1小时。将共固定化凝胶应用于FIA法测定乙醇。FIA法适用于0.1 ~ 0.5 M乙醇的测定。