Improvement of performance of medical biosensors based on composite enzyme membranes

开发无干扰的生物传感器对其医学应用具有重要意义。本研究以复合酶膜为基础，研究了乳酸生物传感器对维生素C干扰的消除。为了制备复合膜，首先对乳酸氧化酶(LOX)和抗坏血酸氧化酶(AOX)进行生物修饰，然后通过酶和亲和素的交替沉淀将修饰后的酶与亲和素一起构建成多层膜。用分光光度法和微重法对酶的多层结构进行了表征，阐明了LOX和AOX的最佳负载量以及沉积顺序的影响。由于抗坏血酸在电极表面(+0.6V)容易被氧化，因此仅配备LOX的乳酸传感器受到来自0.1 mM抗坏血酸(即血液中的生理水平)的干扰(约20%)。换句话说，用这个传感器是不可能测定人体血液中的乳酸的。相反，用LOX和AOX复合膜修饰的乳酸传感器在0.1 mM或更高的乳酸浓度范围内对0.1 mM抗坏血酸不敏感。LOX：AOX=10：1足以消除抗坏血酸干扰。由于AOX的稳定性较低，采用LOX：AOX=1：1的复合膜是可行的。对LOX和AOX的沉积顺序几乎没有影响。研究结果表明，经修饰后的乳酸传感器可以用于血液中乳酸的测定。链霉亲和素和脱糖亲和素也可用于构建复合膜。

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.