| Circulating tumor cell detection technology is divided into in vitro detection and in vivo detection.The carrier materials for in vivo detection often use metal wires and chemical fiber filaments.Due to the limitation of fiber and wire loading capacity,the diameter(>500?m)is usually much larger than the captured target cell CTC(10-50?m).For the capture of CTC,the carrier wire exhibits a large surface steric hindrance,which seriously affects the capture efficiency and detection sensitivity.Therefore,in this study,we chose natural macromolecular silk fibroin as a carrier,which has the advantages of softness,less steric hindrance,easy grafting and easy degradation to achieve efficient capture of CTC.We first copolymerized vinyl monomer acrylamide(AM)and glycidyl methacrylate(GM)onto silk by free radical polymerization,and optimized the grafting concentration of GM and AM by calculating the grafting rate of silk.It was found with SEM that due to the large amount of grafting reaction and the easy self-polymerization of the monomer,even with a small grafting rate,a large amount of large particles were accumulated on the surface of the silk,which affected subsequent captures.As a macromolecular protein,silk has a large number of active groups on its surface.PEBG is reacted with silk at 50? for 6 h.After reacting with EDA at 25? for 14 h,the PAA was activated at pH?5 and 37? for 2 h,and the capture carrier is obtained..The samples were characterized by IR,ZETA potential and amino colorimetry during the grafting process.The results showed that PEBG,EDA and PAA were successfully grafted.The surface state of the grafted silk was characterized by SEM,and no obvious polymer accumulation was found on the surface,indicating that the surface graft distribution was uniform.Thereafter,the amino acid-modified nucleic acid aptamer was immobilized on the grafted PAA to realize the construction of the CTC capture device.We optimized the performance of the capture device.By changing the molecular weight of the PAA,the number of cells captured by the capture device has increased,but after Mw of PAA reaching 100,000,the number of cells capture became stable.Through specific capture of target cells and non-specific ratio discovery,cell-specific capture was highest with glycine blocking after grafting.Through experiments with different capture time,it was found that the specific capture amount increased with time,but after 30 minutes,the number of cell-specific captures no longer increased,but the specific adsorption amount was still rising,so the comprehensive specificity capture rate can set the capture time to 30 minutes.It has been proved by experiments that increases the amount of aptamer,the cell number of capture increases,but when the amount of aptamer reaches 100 pmol,the aptamer has reached saturation and the number of cell capture no longer increases.It has been found that as the number of silk increases,the specific surface area increases,and the contact sites with cells increase,hence the number of captured cells increases.Using performance-optimized capture device,we examined the capture capabilities and biocompatibility of the capture device.As the target cell density decreases,the total amount of capture decreases.When the cell density is extremely low,that is 500/ml,the amount of captured cells is still 100-200,showing the high-capacity capture ability of CTC.For capture interference,target cells are added to whole blood to maintain target cell density of 500/ml.the number of captured cells were not affected,indicating that the capture device has a strong specificity for capture the target cells and is not affected by interfering cells.In addition,we evaluated the non-capture properties of the capture device associated with in vivo capture,including carrier silk loading capacity,contact cytotoxicity,extract cytotoxicity,and hemolysis.The results showed that the good performances,indicating that the capture device can be used for in vivo experiment. |
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