Nanoelectrochemistry and Single Cell Metabolomics

项目总结： 在单细胞水平上对人类疾病的详细了解构成了重要的 生物医学科学的前沿。细胞间的异质性存在于组织内，并且 从代谢物分布的角度理解这些差异的本质具有广泛的意义 不管是什么类型的疾病。使用荧光传感器的美丽实验已经被 用来量化细胞内的小分子代谢物；然而，发光的行为 已经被证明对细胞有有害的影响。此外，这些传感器并不容易 一概而论。我们努力对细胞代谢物进行最准确的测量 使用纳米电化学测量，对细胞动态平衡的扰动最小。 这些测量有可能为亚细胞中未实现的敏感性打开大门 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，并量化细胞内的活性物种。然而，它需要的时间为一个 纳米电极提供足够的电荷，以在安培和 伏安实验约为100毫秒。因此，新的技术必须是 开发最大限度地减少对细胞动态平衡的扰动，以确保准确 对自然细胞过程的测量。我们小组最近研究了断路 电位法，之所以选择它，是因为进行测量时可以忽略不计 电流。我们发现，这种技术与电极大小无关。这一发现 表示测量的灵敏度不会随时间发生明显变化，并且 可以在单细胞内进行纵向实验。我们建议开发 在开路条件下运行的代谢物特定纳米电极。我们将从 我们制造纳米电极和研究单细胞内氧含量的经验 开发传感器创建单细胞代谢组学通用平台的经验 测量。特异性是通过捕捉到的氧化还原酶来获得的 电极表面用一种水凝胶。代谢物浓度是通过酶的周转来获得的 速率，这取决于底物浓度。通过这笔赠款开发的方法 Period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依赖于 一种相关的氧化还原酶的可用性。

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.