Development Of New Methods For The Enrichment Of Circulating Tumor Cells And Exosomes Based On Artificial Antibodies

Circulating tumor cells(CTCs)and exosomes are important markers of liquid biopsy,and they carry abundant information such as nucleic acids and proteins.The efficient capture and detection of CTCs and exosomes are of great significance for the studies of disease occurrence and development,drug evaluation,early tumor diagnosis and prognosis.In this research,based on the artificial antibodies,new methods were developed for the capture and analysis of CTCs and exosomes,to improve capture efficiency and selectivity,to make the materials compatible with proteomic analysis,and improve the versatility.For the current limited antibody types and the incompatibility of the aptamer method with subsequent analysis,the aptamer-functionalized open column with specific recognition and capture ability for SMMC-7721 cells was prepared,based on the atomic transfer radical polymerization and biotin-avidin systems.The captured cells could be released and identificated by the proteomic analysis.To improve the capture efficiency of the CTCs capture methods,the cell-imprinted polydimethylsiloxane was prepared by cell imprinting and aptamer modification,which enabled the capture and release of SMMC-7721 cells.The capture efficiency could reach to 97.49%± 11.24%.To further improve the capture selectivity,the aptamers were modificated only into the cell-imprinted sites to form the multivalent effect,and the capture efficiency could reach to 75%even in the presence of 1000 times interfering cells.Moreover,the capture of tumor cells in the blood was achieved.To improve the versatility of the aptamer-based methods,the cell-imprinted hydrogel was prepared by introducing phenylboronic acid to bind the overexpress sialic acid on the tumor cells.Capture and undamaged release of SMMC-7721 cells were achieved by conformational matching and chemical affinity.The enrichment factor of SMMC-7721 cells in complex systems could be as high as 13.45 ± 3.20,and the release efficiency of cells was as high as 99%.Aiming at the low purity of exosome enrichment methods,based on the surface chargeability and special size of exosomes,the new exosome-enriched material was developed by dull template imprinting technology(EXO-MIP),which enabled the capture exosomes in urine and in cell culture medium.The proteomic analysis results showed that for urine,the number of Top 100 urine exosome marker proteins captured by EXO-MIP was 21.35%higher than that by the commercial kit.For Hela cell culture medium,the number of Top 100 common protein of exosomes enriched by EXO-MIP was 10%higher than that by the commercial kit.