Study On Fluorescence Detection Methods Of Cellular Sialylation-Related Substances

Glycosylation is an important post-translational modification of proteins in biological processes,which plays an important role in cell growth and differentiation,cell-cell communication,immune response,cell adhesion and intracellular signaling.Abnormal glycosylation is closely related to the development and metastasis of various malignant tumors.As a type of glycosylation,sialylation has been attracted much attention.Sialic acids(SAs),usually located at the end of the glycan chains,are involved in glycoproteins or glycolipids to regulate diverse physiological and pathological processes.Cancer cells usually aberrantly display an increase in sialylation,leading to excessive secretion of sialylation-related substances.Thus,the detection of sialylation-related substances can provide a comprehensive understanding of cell status with abnormal cell metabolism and malignant tumor progression.In this dissertation,based on cytology,biochemistry and molecular biology and combined with the fluorescence signal amplification technology,the quantitative detection of secreted sialoglycoconjugates(SiaGCs)and gangliosides on cell surface were developed by chemoselective recognition of boronic acid toward SA.The dissertation contains the following two parts:1.Fluorescent visual quantitation of cell-secreted sialoglycoconjugates by chemoselective recognition combined with hybridization chain reactionIn this part,we developed a fluorescent visual method for the quantitative detectionof metabolically labeled SiaGCs secreted from living cells by chemoselective recognition combined with hybridization chain reaction.The cell-secreted SiaGCs,which have been labeled with azide group through metabolic glycan labeling technique,were captured by the chip through chemoselective recognition of boronic acid towards SA.After further conjugating the azide group with alkyne modified DNA probe,the captured SiaGCs could be conveniently endowed with the amplified fluorescent signal through a hybridization chain reaction of dye-labeled DNA hairpins.The quantitative detection of secreted metabolically labeled SiaGCs was achieved combined with a calibration curve of azide-modified sialic acid.This method can be used to distinguish the cancer cells from normal cells,and monitor the variation of the secreted SiaGCs during drug treatment,providing a useful tool for investigating the glycosylation and glycan-related biological processes.2.Quantitative Screening of Gangliosides on Cell Surfaces by Nondestructive Extraction and Hydrophobic CollectionIn this part,we achieved nondestructive quantitation of gangliosides on cell surfaces by designing functionalized extractors and collectors.The extractors were obtained by combining maleimide silica bubbles with a DNA probe.The DNA probe contains an endonuclease cleavage site and a boronic acid end,which can extract cell-surface sialic acid-containing compounds.The collectors were obtained by modified silica bubbles with trichloro(octadecyl)silane(OTS).The extractors can capture sialylation-related compounds on cell surfaces through chemoselective recognition of boronic acid towards SA.After the extractors were incubated with endonuclease,the collectors can selectively collect the released oligonucleotide-gangliosides through hydrophobic interactions.The fluorescent signal from the collectors amplified by HCR were used for the quantitation of gangliosides on cell surface.This method can be extended to other biological molecules on cell surface by changing the corresponding functional groups of extractors and collectors,demonstrating its practicality for the detection of molecules on cell surfaces and the potential in probing related biological processes.