Droplet-based Spatially Encoded Live Cell Digital Extraction

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Abstract This proposal aims to develop a high throughput droplet-based digital subsampling tool that enables continuous extraction of intracellular molecules (>1000 cells/sec) from individual living cells and achieve digital single molecule detection. Technological advances in single cell analysis have resolved cellular heterogeneity and enabled discovery of rare cell subpopulations. They have opened up new opportunities to detect subtle molecular changes in the presence of variability in biological systems. Due to the uniqueness of individual cells in their composition, functionality, and structures, molecular analyses at the single-cell level are critical for understanding the complexity of biological processes and cellular responses to perturbations. To accurately profile cellular dynamics and behaviors, it is essential to longitudinally monitor cellular changes and responses over time. However, it has been challenging to acquire temporal molecular information from the same cell populations due to the need of keeping them alive during the course of observation while minimizing their perturbations. Recently, nanotechnology methods (e.g. nanowire, nanobiopsy, nanostraw) have been developed for longitudinal cell monitoring where nanoscale dimensions are used to penetrate cells and sample intracellular molecules while providing minimal cytotoxicity. They have successfully achieved longitudinal cell subsampling and analysis, but it has still been difficult to resolve inherent heterogeneity of individual cells and their contents due to low throughput and sensitivity. In these studies, cells were placed on a substrate that consists of different nanostructures and a scarce amount of molecules were extracted, limiting the ability to achieve high throughput sampling and comprehensive downstream analysis. To build a robust longitudinal intracellular extraction tool, there is a need to spatially barcode and profile individual cells at high throughput and measure scant molecules with ultra-high sensitivity to maintain minimal perturbations during extraction. Here, we propose a live cell digital subsampling technology that will combine a cell membrane perforator and digital detection using droplet microfluidics to achieve i) live cell subsampling via hydrodynamic stretching of individual living cells, ii) ultrasensitive digital profiling of individual molecules sampled from single cells, and iii) monitoring of phenotypic and genotypic nutrient sensing pathway associated molecules for the early diagnosis of Alzheimer's disease and the development of new therapeutics. We will advance this platform to better understand cell biology and apply it to diverse fields including neuroscience, immunology, and cancer biology.