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- All Subjects: Single cell proteins--Analysis.
- Creators: Guo, Jia
- Creators: Gupta, Aakriti
Description
Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF) has been a well-established protein analysis platform. To gain comprehensive insights into cell biology and diagnostic pathology, a crucial direction would be to increase the multiplexity of current single cell protein analysis technologies.
An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.
This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus.
An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.
This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus.
ContributorsLiao, Renjie (Author) / Guo, Jia (Thesis advisor) / Borges, Chad (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2019
Description
The ability to profile proteins allows us to gain a deeper understanding of organization, regulation, and function of different biological systems. Many technologies are currently being used in order to accurately perform the protein profiling. Some of these technologies include mass spectrometry, microarray based analysis, and fluorescence microscopy. Deeper analysis of these technologies have demonstrated limitations which have taken away from either the efficiency or the accuracy of the results. The objective of this project was to develop a technology in which highly multiplexed single cell in situ protein analysis can be completed in a comprehensive manner without the loss of the protein targets. This was accomplished in the span of 3 steps which is referred to as the immunofluorescence cycle. Antibodies with attached fluorophores with the help of novel azide-based cleavable linker are used to detect protein targets. Fluorescence imaging and data storage procedures are done on the targets and then the fluorophores are cleaved from the antibodies without the loss of the protein targets. Continuous cycles of the immunofluorescence procedure can help create a comprehensive and quantitative profile of the protein. The development of such a technique will not only help us understand biological systems such as solid tumor, brain tissues, and developing embryos. But it will also play a role in real-world applications such as signaling network analysis, molecular diagnosis and cellular targeted therapies.
ContributorsGupta, Aakriti (Author) / Guo, Jia (Thesis director) / Liang, Jianming (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12