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Methods and devices for comprehensive molecular analysis of single cells


Typical analytical methods for evaluating the protein and gene expression of cells rely on the molecular analysis of analytes from a large number of cells. These methods, however, do not reveal insight into the heterogeneous nature of cells and instead only provide average results of the bulk population. Moreover, these methods are insensitive to low copy number proteins or mRNA that may in fact play a significant role in a cell's phenotypic state. The ability to discern the true distributions of protein and gene expression in a cell population will give rise to the effective identification of unique subpopulations. This allows for a deeper understanding of disease progression, molecular pathways, and cellular differentiation by revealing previously undetectable states of cells. Therefore, it is essential that technologies capable of isolating and comprehensively analyzing many single cells in parallel be developed. In this dissertation, I present new methods and technologies for integrating the capture and isolation of single cells with comprehensive, quantitative on-chip molecular analysis within a single microfluidic platform. I first present the design, development, and function of polymethyldisiloxane (PDMS) microfluidic devices capable of trapping and fully isolating many single cells along a series of compartments. Additionally, I discuss various methods for trapping (dielectrophoretic or hydrodynamic) and lysing (electric-field or chemical) of the cells directly in individual, nanoliter-sized compartments. Subsequently, I present methods for the fabrication, assembly, and application of high-density antibody- conjugated microbead arrays for single-cell proteome analysis. I then discuss how such arrays can be integrated directly into the PDMS devices using a protective patterning scheme to overcome current challenges in producing patterned, modified surfaces in PDMS devices. I also present methods we have developed which could enable analysis of single-cell gene expression by directly sequencing the mRNA transcripts captured from each single cell on the device. Finally, I present my work on electric field-directed assembly of enzyme-nanoparticle layers for biosensor applications. Together, these methods and devices constitute key advancements towards the development of a technology capable of fully integrating single-cell capture and comprehensive molecular analysis

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