Real time monitoring of mammalian circadian rhythm by bio-sensor

蛋白质在固体基底上的组装被认为是制备高性能生物传感系统和生物电子器件的重要关键技术。本研究的目的是发展一种新的利用生物亲和结合和解离的蛋白质组装方法。作者着重研究了生物素类似物与亲和素分子的亲和结合反应。预期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体支持物上，并且所构建的蛋白质复合物通过与游离生物素分子的交换反应而分解。利用倏逝波技术研究了亲和素与生物素或生物素类似物修饰蛋白质的亲和结合和解离反应，并与生物素修饰蛋白质的亲和结合反应进行了比较，结果表明，亲和素与生物素修饰蛋白质的亲和结合反应与生物素修饰蛋白质的亲和结合反应具有良好的生物活性，生物活性与生物活性之间存在着良好的线性关系，生物活性与生物活性之间存在着良好的线性关系。 ...更多信息 激发荧光测量。解离的亲和素和生物素类似物修饰的蛋白质复合物肯定是诱导的自由生物素添加。通过解离度的测量，证明了生物素传感在1 × 10 - <-9>4 M至1 × 10 - 4 <-5>M的浓度范围内是可能的。实验结果进一步表明，诱导解离的生物素浓度依赖于生物素类似物与亲和素之间的生物亲和性。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电极表面，对NADH的氧化起着生物电催化作用。在三年的时间里，我们克隆了哺乳动物生物钟基因（mPer 1，mPer 2，mPer 3和mTim）。将mPer 1-promotor-Luciferase融合基因转染Rat 1成纤维细胞。我们用灵敏的光子探测器观察了单细胞水平发光的变化。血清休克后20 ~ 28小时内，红细胞发光开始呈现节律性变化。少

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