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Abstract

In the wake of heterogeneous nature of most biological systems, single-cell analysis has gained enormous importance and popularity in the field of biology, medicine and even microbiology. Traditional population-averaged analysis offers good information on the system level, however at the price of missing out cell-to-cell variations, which are critical information for building disease models, understanding drug resistance, or identifying cancer stem cells. Though single-cell transcriptomic technologies are well established, single-cell proteomic platforms seem to be lagging behind. This is mostly due to the low-level protein quantities present in single cells and the difficulty to amplify protein molecules for end-point detection. In this thesis, I will report microfluidic solutions for ultra-sensitive single-cell endogenous protein and enzymatic activities analysis. I will focus on the design principles of the microfluidic platforms, working pipeline validation and biological application demonstrations. Firstly, I will demonstrate an ultrasensitive single-cell protein quantification platform based on a valve-based microfluidic chip designed and fabricated in house. I will show how the microfluidic platform improves the digital proximity ligation assay (digital PLA) performance, thus enabling quantification of rare protein species in single cells. Then, the assay will be extended to simultaneously quantify protein and mRNA copy numbers from the same single cell. Then, the same microfluidic platform has been adapted to profile multiplexed enzymatic activities in single cells. By combining family-wide chemical probes and PLA, the assay - named single-cell activity-dependent proximity ligation (scADPL) – quantifies active enzymatic molecules of targeted protein species (instead of total molecules) in single cells. The proof-of-concept is then demonstrated with 6-plex measurements in cancer cell lines, and the assay has been applied to characterize cancer organoids derived from various patient samples.

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