Gene expression in human cells is the process that translates the information embedded in agene into the synthesis of a cellular product. It is a critical aspect of both normal and pathological development of cells and tissues. Current bulk gene expression assays rely on molecules extracted from multiple cells or tissue samples, therefore containing various degrees of cellular heterogeneity. As a result, it is difficult to determine the regulatory relationship of genes in different phases of development from these 'cell population averaging' measurements. The investigators have previously demonstrated that a microfluidic technology can be used for extracting total messenger Ribonucleic acid (mRNA) from single-cells and synthesizing complementary Deoxyribonucleic acid (cDNA) on the same chip for high efficiency single-cell gene expression profiling and reduces the minimum detectable number of mRNA molecules. However, one of the current challenges of them microfluidic device is the difficulty of using pneumatic pumping and valving mechanism for single cell addressing, which is a time-consuming and labor intensive task. In this project a microfluidic platform will be developed for massively parallel single cell mRNA analysis for gene profiling applications. The proposed device integrates three functional regions on a single Polydimethylsiloxane (PDMS) microfluidic chip: a high speed microscale fluorescence activated cell sorter (µFACS), optoelectronic tweezers (OET) for massively parallel single cell manipulation, and 1000 microfluidic wells for single cell mRNA extractions and cDNA conversion. This device will solve the technical issues in integrating OET with microfluidic devices to enable massively parallel single cell manipulation. One thousand cells will be individually trapped and transported into microfluidic wells where cells are split, or lysed, for gene profiling analysis. Integrating OET allows eliminating the multiplexed microfluidic control network that has been proven extremely inefficient in single cell manipulation and replacing it with dynamic optical images that can be reconfigured in real-time. Success in this proposal will realize a low-cost, fully integrated microfluidic chip capable of conducting massively parallel gene profiling on 1000 single cells. Knowledge developed during the course of this project will be incorporated into the investigators? teaching activities at both the undergraduate and graduate levels. Results of this proposal will be published in international conferences and peer-reviewed journals and information will also be available on the investigator website. Minority graduate and undergraduate students will participate in these projects through independent research courses. Students involved in this project will be exposed to an excellent multidisciplinary training environment between the USC Medical School and UCLA Engineering School. The PI will also be involved with the outstanding outreach program (CEED) in UCLA to recruit underrepresented college students for constructing a 'Virtual Chemical Lab' allowing everyone in the world to control cells in investigators' lab through Internet.
The aim of this grant is to develop an integrated microscale fluidic system for high throughput single cell gene expression analysis. It is a biomedical instrument that permits users to quickly identify rare cells from a population, such as circulating tumor cells in blood, and select them using a high speed laser induced cavitation bubbles. It is also a system that allows light beams to trap and transport selected cells individually into isolated microfluidic wells in a parallel fashion on a stamp size chip. These cells can then be taken out from the small chip for downstream single cell gene expression profiling analysis. Under the funding of this grant, the PI has successfully demonstrated the world fastest microfluidic fluorescence activated cell sorter, called Pulse Laser Activated Cell Sorter (PLACS), that can acheive high purity (>90%), high cell viability (>90%), cell sorting at a throughput of 23,000 cells/sec in a single microfluidic channel. Since PLACS sorting requires only few microfluidic channels for sample introduction, sheath flow focusing, and sample collection, it can be readily integrated with another novel device called Microfluidic Integrated Optoelectronic Tweezers (MIOET) that is also supported under this grant. MIOET is a technology allowing users to select single cells using light beams under a microscope for further purification to 100% purity. It also allows multistep microfluidic operations such as cell lysing, liquid plug generation, fluid mixing, and delivery to outside the chip. Integration of PLACS and MIOET allow users to quickly isolate rare cells from a large population with 100% purity for high throughput single cell gene expression analysis or other molecular biology analysis.
