A Glucose Nanosensor for Continuous Glucose Monitoring within Living Cells

用于活细胞内连续血糖监测的葡萄糖纳米传感器

基本信息

批准号：
7127525
负责人：
KAIMING YE
金额：
$ 20.82万
依托单位：
UNIVERSITY OF ARKANSAS AT FAYETTEVILLE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-05 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7127525
关键词：
blood glucose glucose microscopy patient monitoring device

项目摘要

DESCRIPTION (provided by applicant): One of the key issues to assist the understanding of the pathophysiology of diabetes and obesity is the transport and metabolism of glucose in various cells and tissues. Both of these processes are dependent upon the local concentration of glucose. A variety of methods for measuring blood glucose are readily available to researchers, particularly for animal studies. Nevertheless, the continuous monitoring of glucose within living cells has been a challenge in diabetes researches due to the lack of a methodology that is nondestructive to the cells. We herein propose to establish a novel technology to visualize glucose inside the cells using a fluorescence nanosensor and further to demonstrate the utility of this technology in determining the glucose uptake in skeletal muscle cells. Impaired insulin-stimulated glucose disposal in skeletal muscle precedes and contributes to the development of type 2 diabetes as well as the obesity. The establishing of this new technology will not only allow quantifying the two initial steps of muscular glucose metabolism: glucose transport and phosphorylation; but also help understand the development of obesity and type 2 diabetes in insulin-resistant subjects. We have isolated a glucose binding protein (GBP) from E. coli. With this protein, we engineered a "glucose indicator protein" (GIP) that displays a change in fluorescence intensity as a function of glucose concentration. We have also developed an approach to construct a combinatorial library so that we can screen for GBPs with varied affinities for the glucose. We hypothesize that a class of GIPs that possess different glucose response ranges can be engineered using various GBP's mutants that are selected from a combinatorial library and have varied affinities for the glucose. The gene of these GIPs will be introduced into cells so that an intracellular fluorescence nanosensors can be biosynthesized and remain inside the cells for visualizing the glucose through the lifetime FRET (Forster resonance energy transfer) microscopy. Three specific aims are proposed including: i) to visualize glucose within living cells through the lifetime FRET microscopy; ii) to manipulate the glucose binding affinity of GBP by mutagenesis; iii) to demonstrate the utility of GIP in determining the glucose uptake in skeletal muscle cells. This new technology will make it possible to continuously monitor glucose concentrations inside living cells, providing the key data for studying the development of type 2 diabetes and obesity in insulin resistant subjects. A long-term goal is to introduce this technology for continuous monitoring glucose and developing a close loop controlled insulin delivery system for a better blood glucose control in diabetics.

描述（由申请人提供）：有助于理解糖尿病和肥胖症病理生理学的关键问题之一是各种细胞和组织中葡萄糖的运输和代谢。这两个过程都取决于葡萄糖的局部浓度。研究人员很容易获得多种测量血糖的方法，特别是对于动物研究。然而，由于缺乏对细胞无损的方法，在活细胞内对葡萄糖的持续监测一直是糖尿病研究的挑战。我们在这里建议使用荧光纳米传感器建立一种新型技术来可视化细胞内的葡萄糖，并进一步证明该技术在确定骨骼肌细胞中葡萄糖摄取时的实用性。胰岛素刺激的葡萄糖处置受损在骨骼肌之前，并有助于2型糖尿病和肥胖症的发展。建立这项新技术不仅将允许量化肌肉葡萄糖代谢的两个初始步骤：葡萄糖的转运和磷酸化；但也有助于了解抗胰岛素耐药性受试者中肥胖和2型糖尿病的发展。我们已经从大肠杆菌中分离出一种葡萄糖结合蛋白（GBP）。使用该蛋白质，我们设计了一种“葡萄糖指示剂蛋白”（GIP），该蛋白显示出随葡萄糖浓度的函数而显示荧光强度的变化。我们还开发了一种构建组合库的方法，以便我们可以筛选具有多种亲和力的GBP。我们假设一类具有不同葡萄糖反应范围的GIP可以使用从组合文库中选择的各种GBP突变体进行设计，并且对葡萄糖具有不同的亲和力。这些GIP的基因将被引入细胞中，以便可以将细胞内荧光纳米传感器生成生物合成，并保留在细胞内，以通过生命周期FRET（Forster Resonance Enermance Transforcopicy）可视化葡萄糖。提出了三个特定的目标，包括：i）在终生的弗雷特显微镜中可视化活细胞内的葡萄糖； ii）通过诱变操纵GBP的葡萄糖结合亲和力； iii）证明了GIP在确定骨骼肌细胞中葡萄糖摄取的实用性。这项新技术将使能够连续监测活细胞内的葡萄糖浓度，从而为研究2型糖尿病的发展和抗胰岛素耐药受试者的肥胖症提供关键数据。一个长期的目标是引入该技术，用于连续监测葡萄糖并开发近环控制的胰岛素输送系统，以在糖尿病患者中获得更好的血糖控制。