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Nanoelectrochemistry and Single Cell Metabolomics

纳米电化学和单细胞代谢组学

基本信息

批准号：
10710410
负责人：
Jeffrey E Dick
金额：
$ 37.25万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2025-07-31
项目状态：
未结题

项目摘要

Project Summary: A detailed understanding of human disease at the single cell level constitutes an important frontier of biomedical science. Cell-to-cell heterogeneities exist within tissues, and understanding the nature of these differences in terms of metabolite profile has vast implications regardless the type of disease. Beautiful experiments using fluorescent sensors have been developed to quantify small molecule metabolites within cells; however, the act of shining light on a cell has been shown to have deleterious effects. Further, these sensors are not easily generalizable. We endeavor to make the most accurate measurements of cellular metabolites with minimal perturbation to cellular homeostasis using nanoelectrochemical measurements. These measurements have the potential to open the door to unrealized sensitivity in sub-cellular metabolite quantification. Electrochemistry at nanoelectrodes has been used to interrogate cellular processes and quantify reactive species within cells. However, the time it takes for a nanoelectrode to deliver enough charge to convert a cell's contents during amperometric and voltammetric experiments is on the order of 100 ms. Therefore, novel techniques must be developed to minimize the perturbation to cellular homeostasis to ensure accurate measurements of natural cellular processes. Our group has recently investigated open circuit potentiometry, which was chosen because the measurement is carried out with negligible current. We have discovered that this technique is independent of electrode size. This finding indicates the sensitivity of the measurement will not change with time appreciably, and longitudinal experiments within single cells can be carried out. We propose to develop metabolite-specific nanoelectrodes that operate under open circuit conditions. We will draw from our experience fabricating nanoelectrodes and studying oxygen content within single cells and experience developing sensors to create a generalized platform for single cell metabolomics measurements. Specificity is gained via oxidoreductase enzymes that are trapped on top of an electrode surface by a hydrogel. Metabolite concentration is obtained by the enzymatic turnover rate, which is dependent on substrate concentration. Methodology developed through this grant period has the potential to forge a foundation for generalized metabolomics studies with nanoelectrode sensors, where the library of metabolite of interest depends only on the availability of an associated oxidoreductase enzyme.

项目概要：在单细胞水平上对人类疾病的详细了解，生物医学科学的前沿组织内存在细胞间异质性，了解这些代谢物特征差异的本质具有巨大的意义无论疾病的类型。使用荧光传感器的美丽实验已经被开发用于量化细胞内的小分子代谢物;然而，已经显示出有害的影响。此外，这些传感器不容易实现。可概括的我们奋进于对细胞代谢物进行最精确的测量使用纳米电化学测量对细胞内稳态的干扰最小。这些测量有可能打开亚细胞中未实现的敏感性的大门。代谢物定量。纳米电极上的电化学已经被用来询问细胞过程和量化细胞内的活性物质。然而，纳米电极，以提供足够的电荷，以在电流分析和伏安法实验是在100毫秒的数量级。因此，新的技术必须是开发用于最小化对细胞内稳态的干扰，以确保准确的自然细胞过程的测量。我们小组最近研究了开路电位测定法，选择该电位测定法是因为测量在可忽略的情况下进行。电流我们已经发现这种技术与电极尺寸无关。这一发现表示测量的灵敏度不会随时间发生明显变化，可以在单电池内进行纵向实验。我们建议发展在开路条件下操作的代谢物特异性纳米电极。我们将从我们在制造纳米电极和研究单细胞内氧含量方面的经验，开发传感器以创建单细胞代谢组学通用平台的经验测量.特异性是通过氧化还原酶获得的，氧化还原酶被捕获在电极表面的水凝胶。代谢物浓度通过酶促周转获得速率，其取决于底物浓度。通过该赠款制定的方法这一时期有可能为广义代谢组学研究奠定基础，纳米电极传感器，其中感兴趣的代谢物库仅取决于纳米电极的浓度。相关氧化还原酶的可用性。