Real time monitoring of mammalian circadian rhythm by bio-sensor

利用生物传感器实时监测哺乳动物昼夜节律

基本信息

批准号：
08557007
负责人：
OKAMURA Hitoshi
金额：
$ 4.86万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

It is considered that protein assembly on solid substrates is the important key technology to fabricate high performance biosensing systems and bioelectronics devices. The purpose of this study is to develop novel protein assembling method using bioaffinity binding and dissociation. The author paid attention to bioaffinity binding reaction between biotin analogues and avidin molecule. It is expected that biotin analogue-modified protein molecules are assembled on the solid supports by using avidin molecules as binders and that the constructed protein complexes are decomposed by the exchange reaction with free biotin molecules. This assembling technique might be available to fabricate recycle usable biosensing system and intelligent bioreactor system in which biocatalysis is controlled by the addition of ligand molecules as stimulation signal.Bioaffinity binding and dissociation reactions of avidin and biotin- or biotin analogue-modified proteins were characterized with evanescent wave- … More exciting fluorescence measurement. Dissociation of avidin and biotin analogue-modified protein complexes was certainly induced by the free biotin addition. It was demonstrated that biotin sensing is possible in the concentration range from 1x10^<-9> M to 1x10^<-5> M by the measurement of dissociation degree. The data further suggested that the biotin concentration for dissociation induction is dependent on the bioaffinity between the biotin analogue and avidin.At second, It was demonstrated that redox coenzyme-modified avidin molecules were assembled on the biotin-modified electrode surface and functioned as bioelecfrocatalyst for NADH oxidation. Dissociation of themodified avidin from the biotin-modified electrode surface was also induced by biotin addition.During three years we cloned putative mammalian clock genes (mPer1, mPer2, mPer3 and mTim). mPer1-promotor-Luciferasefusion gene was transfected to the Rat1 fibroblast cells. We observed the change of luminescence at single cell level by a sensitive photon detector. After serum shock, the luciferin luminescence begins to show the rhythm in a period length between 20 to 28 hours. Less

固体基质上的蛋白质组装被认为是制造高性能生物传感系统和生物电子器件的重要关键技术，本研究的目的是开发利用生物亲和力结合和解离的新型蛋白质组装方法。生物素类似物和抗生物素蛋白分子之间的相互作用预计生物素类似物修饰的蛋白质分子通过使用抗生物素蛋白分子作为结合剂组装在固相支持物上，并且构建的蛋白质复合物是通过与游离生物素分子的交换反应分解。这种组装技术可用于制造可循环利用的生物传感系统和智能生物反应器系统，其中生物催化是通过添加配体分子作为刺激信号来控制的。亲和素和生物素的生物亲和性结合和解离反应。 - 或生物素类似物修饰的蛋白质通过倏逝波进行表征 - ...更令人兴奋的荧光测量肯定是亲和素和生物素类似物修饰的蛋白质复合物的解离。通过游离生物素添加诱导的生物素感应在1x10^-9M至1x10^-5M的浓度范围内是可能的。数据进一步表明生物素浓度。解离诱导取决于生物素类似物和亲和素之间的生物亲和力。其次，证明了氧化还原辅酶修饰的亲和素分子组装在生物素修饰的电极表面上，并且作为 NADH 氧化的生物电催化剂，添加生物素也可以诱导修饰的亲和素从生物素修饰的电极表面解离。在三年内，我们克隆了假定的哺乳动物时钟基因（mPer1、mPer2、mPer3 和 mPer1-启动子-荧光素酶融合）。将基因转染至Rat1成纤维细胞后，我们通过敏感的细胞观察了单细胞水平的发光变化。血清休克后，荧光素发光开始在20至28小时之间出现节律。