In Situ Single Cell Laser Lysis and Downstream qRT-PCR Profiling

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DESCRIPTION (provided by applicant): Current RT-PCR analysis of single-cells has been strictly limited to analysis of disassociated cells, preventing its compatibility with in-situ analsis of cell states and loses information of initial cell location and morphology within the native tissue. We propose to develop an innovative microfluidic based tool for clinical and scientific users to analyze gene expression heterogeneity, in situ, using single-cell mRNA expression analysis. The device uses a two-photon laser to serially lyse individual cells at known coordinates within a 3D tissue. Differing from conventional single-photon laser lysis, two-photon laser lysis relies on the nonlinear interaction between an ultrafast pulsed light source and the biological material to achieve an energy transfer to the cell precisely within the nanometer-scale focal volume. The lysate is immediately transported to an emulsion-based (oil-droplet) qRT-PCR module to profile mRNA expression. Carryover contamination between sequentially lysed cells is minimized by optimizing laser power and by using hydrodynamic flow focusing with precise flow rate control. Because of the small scale of the microfluidic channels, the total volume flux for sample processing is reduced to microliters, the elapsed time interval between cell lysing and lysate encapsulation is on the order of seconds, and completion of qRT-PCR is on the order of one hour. This technology is well suited to basic biomedical research and clinical applications such as assessing tumor cell population heterogeneity in single-cell gene expression. Additionally, the technology is also amenable to future developments to increase the number of genes that can be quantified. The ultimate implementation would be a highly multiplexed platform capable of detecting dozens of mRNA sequences for each initial droplet eluted from the sample. PUBLIC HEALTH RELEVANCE: We propose to develop an innovative integration of nanometer-resolution laser lysis and microfluidic tool to analyze single-cell heterogeneity in situ using qRT-PCR. In its ultimate implementation, users can simply load a tissue slice in the device, select the target cells based on their location and morphology, and then the system delivers mRNA expression of dozens of genes of individual cells in one hour.