SGER: Quantitating Low-Copy-Number Proteins in Individual Cells Using Microfluidics and Single-Molecule Counting

在过去的十年里，分析仪器取得了巨大的进步，彻底改变了我们研究生命系统的方式，并大大提高了我们对这些系统的理解。基因组测序和DNA微阵列技术的突破，展示了新仪器的显着力量。它们允许在有趣和相关的系统上生成大量数据。这些数据中的大部分是同时从数千个细胞中收集的。微流体技术可用于操纵单细胞以及应用于涉及微量样品的其他分析任务。还已知单分子技术可以允许精确定量和分析单个分子。最近，主要研究者开发了一种装置，该装置将单分子检测与微流体操作相结合，以获得单个蓝藻细胞内藻胆蛋白亚复合物的绝对定量，该藻胆蛋白亚复合物可以以低拷贝数存在。该SGER项目首先专注于提高单细胞分析设备的性能和通用性。然后，该装置将用于表征细胞间变异如何影响蓝藻的进化选择和生物多样性。获得的信息对于未来理解复杂的生物系统至关重要。而不是检查细胞匀浆，人们可以研究单个细胞，并了解每个细胞如何彼此不同。因此，这种仪器的开发将引起细胞生物学研究人员的普遍兴趣。这种方法的智力价值在于，它提供了一种最终的分析工具，可以对单个细胞中的稀有拷贝数蛋白质进行定量分析，并且可以揭示细胞的群体行为与细胞间变异的关系。更广泛的影响：本项目开发的设备将成为一个通用的平台，用于对单个细胞中的生物分子进行定量分析（低至单分子水平）。它将大大提高微流体和单分子方法对科学界的可及性。这项研究包括对研究生进行微流体制造，化学分析，电子学，编程和显微镜的最先进培训。

The last ten years have witnessed great advances in analytical instrumentation that have revolutionized our way of studying living systems and significantly improved our understanding of these systems. The genome sequencing and DNA microarray technology are breakthroughs that demonstrate the remarkable power of new instrumentation. They have allowed massive amounts of data to be generated on interesting and relevant systems. Much of this data is collected from thousands of cells simultaneously. Microfluidics techniques can be used to manipulate single cells as well as applied to other analysis tasks involving minute quantities of samples. It is also known that single molecule techniques can allow precise quantification and analysis of individual molecules. Recently, the principle investigator has developed a device that combines single molecule detection with microfluidic manipulation to obtain absolute quantification of phycobiliprotein subcomplexes within a single cyanobacterial cell, which can exist at low copy numbers. This SGER project first focuses on improving the performance and generality of the single cell analysis device. Then this device will be used to characterize how the cell-to-cell variation affects evolutionary selection and biological diversity of cyanobacteria. Information obtained is crucial to the future understanding of complex biological systems. Instead of examining cell homogenates, one can investigate single cells and understand how each cell differs from one another. Consequently, the development of such an instrument will be of great general interest to those who study the biology of cells. The intellectual merit of this approach is that it provides the ultimate analytical tool for quantifying rare-copy-number proteins cell by cell, and that it can reveal how the group behavior of cells are related to cell-to-cell variations.Broader Impacts:The device developed in this project will be a general platform for quantitative analysis (down to the single molecule level) of biomolecules in a single cell. It will significantly improve the accessibility of microfluidic and single molecule methodology to the scientific community. This research includes state-of-the-art training of graduate students with microfluidic fabrication, chemical analysis, electronics, programming, and microscopy.