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Improvement of performance of medical biosensors based on composite enzyme membranes

基于复合酶膜的医用生物传感器性能改进

基本信息

批准号：
08458282
负责人：
ANZAI Jun-ichi
金额：
$ 4.99万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458282/
关键词：
composite membrane multilayr membrane biosensor platinum electrode enzyme membrane biotin avidin 水晶振動子

项目摘要

It is important to develop interference-free biosensors for their medical applications. In this study, the elimination of ascorbate (Vitamin C) interference of lactate biosensors was examined based on composite enzyme membranes. In order to prepare the composite membranes, lactate oxidase (LOx) and ascorbate oxidase (AOx) were first modified with biotion and the modified enzymes were built into the multilayr membranes together with avidin by depositing the enzyme and avidin alternately. The multilayr structure of the enzymes was characterized based on spectrophotometry and microgravimetry.The optimum loading of LOx and AOx and the effects of the deposition sequence were elucidated. Since ascorbate can be oxidized easily at the electrode surface (+0.6V), the lactate sensor equipped with only LOx suffered from the disturbance originating from 0.1 mM ascorbate (i.e., physiological level in blood) significantly (ca.20%). In other words, it is impossible to determine lactate in human blood with this sensor. In contrast, the lactate sensors modified with the composite membrane of LOx and AOx were not sensitive to 0.1 mM ascorbate in the concentration range of 0.1 mM lactate or higher. The ratio of LOx : AOx=10 : 1 was enough for the elimination of ascorbate interference. Because of low stability of AOx, it is realistic to use the composite membrane of LOx : AOx=1 : 1. Virtually no effect was observed for the deposition sequence of LOx and AOx. It became clear the lactate sensors modified with LOx and AOx composite membrane can be used for the determination of lactate in blood. Streptavidin and deglycosylated avidin can be also used for the construction of the composite membranes.

因此，研制无干扰的生物传感器对医学应用具有重要意义。本研究以复合酶膜为基础，考察了乳酸生物传感器抗坏血酸（维生素C）干扰的消除。为了制备复合膜，首先用生物素对乳酸氧化酶（LOx）和抗坏血酸氧化酶（AOx）进行修饰，通过交替沉积的方法将修饰后的酶与亲和素一起构建到多层膜中。利用分光光度法和微量重量法对酶的多层结构进行了表征，并对酶的最佳装载量和沉积顺序的影响进行了研究。由于抗坏血酸盐在电极表面（+0.6V）容易被氧化，因此仅配备有LOX的乳酸盐传感器遭受源自0.1mM抗坏血酸盐的干扰（即，血液中的生理水平）显著（约20%）。换句话说，用这种传感器不可能测定人体血液中的乳酸盐。相反，用LOx和AOx的复合膜修饰的乳酸传感器在0.1mM乳酸或更高的浓度范围内对0.1mM抗坏血酸不敏感。当LOX：AOx=10：1时，抗坏血酸的干扰可消除。由于AOx的稳定性较低，因此使用LOx：AOx=1：1的复合膜是现实的。几乎没有观察到对LOX和AOx的沉积顺序的影响。结果表明，用LOx和AOx复合膜修饰的乳酸传感器可用于血中乳酸的测定。链霉亲和素和去糖基化亲和素也可用于构建复合膜。