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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）：帮助理解糖尿病和肥胖病理生理的关键问题之一是葡萄糖在各种细胞和组织中的运输和代谢。这两个过程都取决于局部的葡萄糖浓度。对于研究人员来说，测量血糖的方法多种多样，尤其是在动物实验中。然而，由于缺乏一种对细胞无损的方法，对活细胞内葡萄糖的连续监测一直是糖尿病研究中的一个挑战。我们在此建议建立一种使用荧光纳米传感器来可视化细胞内葡萄糖的新技术，并进一步证明该技术在确定骨骼肌细胞中葡萄糖摄取方面的实用性。胰岛素刺激下的骨骼肌葡萄糖处理受损是导致2型糖尿病和肥胖的原因之一。这项新技术的建立不仅可以量化肌肉葡萄糖代谢的两个初始步骤：葡萄糖转运和磷酸化；但也有助于了解胰岛素抵抗受试者的肥胖和2型糖尿病的发展。我们从大肠杆菌中分离出葡萄糖结合蛋白（GBP）。利用这种蛋白，我们设计了一种“葡萄糖指示蛋白”（GIP），它可以显示荧光强度随葡萄糖浓度的变化。我们还开发了一种构建组合文库的方法，以便我们可以筛选对葡萄糖具有不同亲和力的GBPs。我们假设一类具有不同葡萄糖反应范围的GIPs可以使用从组合文库中选择的各种GBP突变体来设计，这些突变体对葡萄糖具有不同的亲和力。这些GIPs的基因将被引入细胞中，这样细胞内的荧光纳米传感器就可以被生物合成，并留在细胞内，通过终身FRET（福斯特共振能量转移）显微镜观察葡萄糖。提出了三个具体目标，包括：i)通过终身FRET显微镜观察活细胞内的葡萄糖；ii)通过诱变控制GBP的葡萄糖结合亲和力；iii)证明GIP在测定骨骼肌细胞葡萄糖摄取方面的效用。这项新技术将使持续监测活细胞内的葡萄糖浓度成为可能，为研究胰岛素抵抗受试者的2型糖尿病和肥胖症的发展提供关键数据。长期目标是将该技术用于持续监测血糖，并开发一种闭环控制的胰岛素输送系统，以更好地控制糖尿病患者的血糖。