Functionalized Material Surfaces And Devices For Capturing Rare Cells In The Blood

Rare cells such as circulating endothelial cells,immune cells subgroup,circulating tumor cells(CTCs),and nucleated red blood cells(NRBCs)show considerable promise for the application in clinical diagnosis.CTCs and NRBCs have attracted substantial attentions owing to their unique properties.In the previous reports,the captured CTCs have been proved as a potentially helpful strategy for the early diagnosis,personalized treatment,and tumor drug resistance evaluation.NRBCs,containing complete genetic information,have been researched frequently in the field of minimally invasive prenatal diagnosis.The capture of these rare cells remains to be a technical challenge because of the low frequency in the peripheral blood(approximately 100 rare cells while more than 109 normal blood cells per milliliter of blood).Traditional strategies to capture rare cells include physical method,immune affinity method,acoustic method,etc.However,research was still needed to be improved because of the complicated operation steps,the high costs,and the difficulty of in-situ capture.In this thesis,we eliminate the limits by means of practicing a functional UV-curable coating PMMA and introducing a piece of functionalized Nylon wire to capture rare cells The specific research results showed as follows:1.Summarized the recent studies on the enrichment of rare cells,mainly including physical separation methods(such as separation based on difference in cell size,hydrodynamic propertied,and dielectric properties);Immune affinity methods(such as chemical modification and microstructure of different materials);other methods include acoustic and optical separation.The characteristics and limitations of these methods are described systematically.2.For the capture of CTCs,UV-curable coating was employed to introducing carboxyl groups quickly and the carboxyl groups were used to immobilize antibodies The appropriate proportion of monomers was determined by water contact angle(WCA)and protein adhesion value.The structure of modified PMMA substrate was confirmed by ATR-FTIR,toluidine blue staining method.The biocompatibility was proved through platelet adhesion value,plasma calcium rehydration time,cytotoxicity methods.The capture tests were conducted in whole blood and CTCs culture medium respectively.After CFSE staining,it showed that CTCs were captured from whole blood and cell medium efficiently.These results showed the feasibility of functional UV-curable coating for circulating tumor cells capture.3.Carboxybetaine Methacrylate(CBMA)was synthesized by 3-chloropropionic acid and dimethylamine ethyl methacrylate(DMAEMA).CBMA is rich in carboxyl group which not only can provide location sites of CD71 antibody,but also can improve the hydrophilic of Nylon surface.A flowing system made by a peristaltic pump was used to mimic blood circulatory system.NRBCs capture experiments were carried out after the immobilization of CD 71 antibody on the CBMA grafted Nylon surface.Fluorescence microscope was used to observe the number of captured NRBCs after CFSE staining.Utilizing the methods of FTIR,NMR,the chemical structures of CBMA was analyzed.A series of characterization methods such as ATR-FTIR,XPS were performed to verify the structure of CBMA modified Nylon surface.The result showed that we have successfully synthesized CBMA and the carboxyl groups of CBMA were grafted onto Nylon surface.The WCA determination,BSA adsorption tests,cytotoxicity test were carried out to estimate the blood compatibility and the results show that the blood compatibility was improved obviously.The NRBCs cells were stained with CFSE and observed by fluorescence microscope after the capturing tests were conducted.It showed that NRBCs were captured both in maternal peripheral blood and fetal cord blood and FISH tests further showed that the target cells were captured.However,direct in situ capture in humans requires further study and validation.