Development of highly sensitive multi-biotip by using thermostable enzymes of thermophilic microorganism.

本研究的主要目的是开发一种利用几种热稳定酶同时检测多种分析物的流动型高灵敏度多生物探针。1)尿酸传感器使用热稳定型尿酸酶和过氧化物酶混合碳毡为基础的流动生物电催化检测器该传感器系统允许在0.1至100□M的浓度范围内持续检测尿酸。该尿酸传感器对高度稀释的人尿样品进行了高灵敏度、高准确度的分析，无干扰，分析结果与分光光度法一致。该传感器在3个多月的储存过程中保持稳定，并保持其原有的活性。2)利用热稳定型谷氨酸氧化酶和DNA-Cu（II）复合物制备谷氨酸生物传感器。研制了一种新型谷氨酸生物传感器，利用DNA-Cu（II）配合物的新型电催化活性，对谷氨酸氧化酶释放的H_2O_2进行阴极检测。该传感器允许在0.1至50□M的浓度范围内对谷氨酸进行安培检测。基于dna膜的准选择性，即使在脑液等体液中也可以进行无干扰检测。在今后的工作中，如果采用其他耐热氧化酶代替尿酸酶和谷氨酸氧化酶，将有可能建立多生物尖。

Primary aim of this study is a development of flow-type highly sensitive multi-biotip using several thermo-stable enzymes to detect simultaneously several kinds of analytes.1) Uric acid sensor using thermo-stable uricase and peroxidase-hybrid carbon felt-based flow-through bioelecterocatalytic detectorThis sensor system allowed continues detection of uric acid over the concentration range from 0.1 to 100□M. Moreover this uric acid sensor allowed highly sensitive and accurate analysis of highly diluted human urine sample without any interference, and analytical results is well consistent with that of spectrophotometry. The sensor is much stable and kept its original activity during more than 3 month storage.2) Glutamate biosensor using thermo-stable glutamate oxidase and DNA-Cu(II) complex.Novel type glutamate biosensor was developed, in which H_2O_2 liberated during the glutamate oxidase was cathodically detected by the novel electrocatalytic activity of DNA-Cu(II) complex. This sensor allowed amperometric detection of glutamate over the concentration range from 0.1 to 50□M. Based on the permselectivity of DNA-film, interference-free detection was possible even in the case of body fluid such as brain liquids.On future work, if other thermostable oxidases are adopted in place of uricase and glutamate oxidase, the establishment of multi-biotip would be possible.