Development And Application Of A Novel Persistent Luminescence Nanoprobe-based Strategy For Cell Surface Glycans Analysis

Cell surface glycans,one of critical structural and functional molecules of cells,play very important roles in many biological activities,such as cell adhesion,cell recognition,signal transduction and so on.The expression of the glycans on cell surface and the change of specific glycans are closely related to the physiological state and function of the cell.Aberration of surface glycans is closely related to the occurrence and development of many major diseases?such as cancer?.Therefore,development of highly sensitive and specific cell surface glycan analysis method is of great significance for understanding the relationship between abnormal glycosylation on cell surface and tumor,further elaborating the role of glycosylation in the development of malignant tumors,and developing the new treatment methods and drug screening.In this work,we have developed a novel analytical platform based on persistent luminescence nanoprobe,and further applied this platform to analyze surface glycans profiling on tumor cells and NGF-treated nerve-like pc-12 cells.The experimental works and results are as follows:?1?Persistent luminescence nanoprobe development and its application in prostate cancer cell surface glycans profilingPersistent luminescence ZnGa₂O₄:Cr³⁺?ZGC?was synthesized by a hydrothermal method.ZGC was first silanized by 3-Aminopropyl-triethoxysilane?APTES?,followed by PEGylation with NHS-PEG-Biotin,which not only introduces biotin,but significantly improves the dispersibility and stability of the nanoparticles.Neutral-avidin was then coupled on ZGC surface through the specific biotin-avidin interaction,producing ZGC-PEG-avidin nanoprobe.As for cell surface glycan detection,different surface glycans are recognized with their corresponding biotinylated lectins,which are then traced by ZGC-PEG-avidin.The persistent luminescence signal is recorded by a microtiter plate reader in time-resolved fluorescence mode.Based on the constructed analytical platform,three biotinylated lectins?ConA-biotin,WGA-biotin,PNA-biotin?that identify the end of the cell surface sugar chain?-mannose??-Man?,N-acetylglucosamine?N-GlcNAc?,and acetylgalactosamine?N-GalNAc?were used as models to investigate the species and expression levels of the surface sugar chains in normal prostate epithelial cells?RWPE-1cells?and prostate cancer cells?DU145 cells?.The experimental results show that the expression level of?-Man,N-GlcNAc and N-Gal NAc on cancer cell DU145 compared with normal cell RWPE-1.In addition,the proportion of three sugar chains on these two cells is also significantly different.The results clearly indicate the difference of glycans expression between tumor and normal cell.This work provides a powerful analytical platform for study on tumor cell surface glycans and cancer diagnosis.?2?Study on cell surface glycans changes during differentiation of NGF-treated pc-12based on persistent luminescence nanoprobeBased on the above persistent luminescence analysis platform,change of surface glycan types and expression level during cell differentiation has been investigated using rat pheochromocytoma pc-12 that could differentiate under NGF treatment as a cell model.This work first used nerve growth factor?NGF?to induce differentiation of pc-12 cells in order to obtain nerve-like cells at different differentiation stages.Biotinylated lectins?ConA-biotin,WGA-biotin,PNA-biotin?were then incubated with GNF-treated pc-12cells at different differentiation stages.Finally,persistent luminescence nanoprobe ZGC-PEG-avidin was introduced onto pc-12 cells through the specific biotin-avidin interaction.The persistent luminescent signal was acquired by a microplate reader in the time-resolved mode,eliminating the background signal from samples and the substrate.Experimental results show that the morphology of pc-12 cells changed significantly after NGF treatment for 1,3 and 5 days.Sugar moieties?-Man and N-GalNAc level decreases gradually while the moiety GlcNAc increases slightly with the cell differentiation.This phenomenon indicates that the expression level of three sugar moieties on the cell surface is closely related to cell differentiation,which provides experimental evidence regarding the relationship between cell differentiation and surface glycans profile.The proposed simple and rapid analytical platform demonstrates a great application potential in profiling cell surface glycans in the process of cell differentiation.Traditional confocal microscope fluorescence imaging and HRP-TMB colorimetric system were used to verify the detection results of surface glycans of tumor cells and pc-12 cells during differentiation.The results are well consistent with the one observed from the persistent luminescence analysis platform,proving its good reliability and accuracy.Based on the persistent luminescence characteristics of ZGC,the platform effectively eliminates the background signals from cells and detection substrates,and achieves high signal-to-noise ratio and detection sensitivity.This thesis provides a promising method for early tumor detection,which is of great significance for the study of tumorigenesis and the diagnosis of diseases.Meanwhile,the relationship between cell surface glycans expression profile and cell differentiation is preliminarily explored,which provides a reliable strategy for the study and further understanding of cell differentiation.