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

本研究的主要目的是研制一种基于多种热稳定酶的流动型高灵敏多生物探针，用于同时检测多种分析物。1）基于热稳定尿酸酶和过氧化物酶的尿酸传感器-碳毡混合流动型生物电催化检测器该传感器系统可连续检测0.1 ~ 100□M的尿酸。该尿酸传感器对高稀释度的人尿样品进行分析时灵敏度高、准确度高、无干扰，分析结果与分光光度法一致。该传感器稳定性好，保存3个月以上仍保持其原有活性。2）利用热稳定谷氨酸氧化酶和DNA-Cu（II）配合物的谷氨酸生物传感器，利用DNA-Cu（II）配合物的新型电催化活性，阴极检测谷氨酸氧化酶释放的H_2O_2，研制了新型谷氨酸生物传感器。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膜的选择透过性，即使在体液（如脑液）中也可以实现无干扰检测。在未来的工作中，如果采用其他热稳定氧化酶代替尿酸酶和谷氨酸氧化酶，将有可能建立多生物探针。

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.