Imaging Detection And Analysis Of Glycans On Cell Surface

As an important structural and functional molecule,glycan on cell surface plays crucial roles in almost all the biological processes including cell adhesion,signal transduction,immune recognition and molecular transport.Moreover,the cell physiological states and many major diseases,such as infection,inflammation and cancer,have been demonstrated to be closely relevant to the change of the specific glycan expression and structure on cell surface,especially the transformations of protein-specific glycan expression and glycan chain structure.Therefore,it is very important to develop high sensitivity and specific methods for the analysis glycans on cell surface to reveal the relationship between the cell surface glycosylation and pathological process,developing the new methods for clinical diagnosis and treatment.In this thesis,based on the metabolic labelling approaches and bioorthogonal chemistry,imaging methods for specific glycans on cell surface have been developed with the biological probe.The main sections of this dissertation are as follows:1.Silver nanoparticles(AgNPs)plasmonic enhanced Fo?rster resonance energy transfer(FRET)imaging of protein-specific sialylation on cell surface.In this strategy,the metabolic labelled target monosaccharide on cell surface was modified with the FRET acceptor Cy5 via biorthogonal chemistry.In addition,aptamer linked AgNPs was combined with Cy3 fluorophore by DNA hybridization as the FRET donor probe,which could be conjugated to the target glycoprotein based on the specific aptamer-protein recognition.The Cy5 fluorescence signal was obtained under the excitation of Cy3 exciting wavelength via FRET.Moreover,the FRET fluorescence signal was obviously enhanced owing to the plasmonic effect of AgNPs in a proper distance with Cy3 on cell surface.Hence the protein-specific sialic acids on cell surface were detected with high contrast.The results showed that the AgNPs plasmonic enhanced FERT method was not only superior to the bare FRET method but also can be used to evaluate the expression of sialoglycoprotein in different cell types under pharmacological treatments.The AgNPs plasmonic enhanced FERT method provides a valuable way in the research of glycan metabolism biological processes,active sites of glycoprotein and drug screening.2.Excimer-based imaging detection of the monomer and dimer sialic acids on cell surface.Owing to the transition from monomer to excimer,the fluorescence emission spectrum of pyrene and perylene showed obvious red shift.In this strategy,the alkynyl modified pyrene and perylene were used as the fluorescent probe,which possess high quantum yield and monomer-excimer fluorescence interconversion.On cell surface,the metabolic labelled target sialic acid was modified with fluorophore via bioorthogonal chemistry,and the dimer sialic acid would shorten the distance of two fluorophores conjugated on every sialic acid by click chemistry,forming the stacked pyrene or perylene excimer structure.Due to the difference of the characteristic emission spectrum between the monomer and excimer fluorophores,the monomer and dimer sialic acids on cell surface can be simultaneously imaged.This strategy provides a promising tool for detecting glycan chain with different structure,revealing the relationship between the specific glycosylation and disease progresses.