CTC Capture With Silk As A Carrier And Screening Of Aptamers Based On Capillary Electrophoresis

In the first part of this paper,we have used the natural silk as a carrier to built a catcher and then studied the capture performance by using it to capture circulating tumor cells(CTC).We try to combine the use of silk and chitosan based on their good biocompatibility and different reactivity.The chitosan was coated on the silk surface by the method of drop injection,so that the surface will have a large number of active amino groups.Polyacrylic acid(PAA)and aptamer(Apt)were then grafted by carbodiimide method in order to construct a trap.It is fixed in the venous indwelling needle,and 2 cm silk extended outside of the front end was used for capture.The capture efficiency was investigated by a circulatory system constructed with a peristaltic pump to simulate the blood circulation in the body.Because of the nonuniform distribution of chitosan film obtained by this method,the cells accumulated in the chitosan coated part,while there are few cells in the membrane free part,which leads to the low overall capture efficiency.To improve the capture efficiency,1,4-Butanediol diglycidyl ether(MSDS)was grafted on the silk through the ring-opening reaction of epoxy bonds and amino groups or carboxyl groups at the side chain of silk protein.A uniform layer of active epoxy bond on the silk surface were then reacted with the carboxyl groups of PAA and Apt were finally immobilized through carbodiimide method.Each step of reaction were characterized by Fourier transform infrared spectroscopy(FTIR)and scanning electron microscopy(SEM).We have built a new circulatory capture system,and studied the capture efficiency and specificity of the modified filament.This trap device has better capture efficiency and good specificity.The trapping effect of different silk quantity was also studied.It was found that the more the silk quantity,the better the trapping effect.When the silk quantity is enough,the trapping ability will be excellent.The cytotoxicity and biocompatibility of the capture device was then tested.It shows only minimal cytotoxicity and the biocompatibility meets the requirements,which means it has the potential for in vivo capture of cancer cells.In the second part,we studied the combination of polyacrylamide gel electrophoresis technology and Cell-Systematic evolution of ligands by exponential enrichment technology(Cell-SELEX),trying to screen the aptamers of the target cells target by cell gel electrophoresis.The capture of CTC was established on the basis of aptamer identified and combined with cancer cells.An aptamer only specifically identifies one specific cancer cells.There's still a large number of cancer cells remain to be screened for their specific aptamers.The traditional selection is time-consuming and complex,so a new method is proposed in this paper.A preliminary feasibility study is completed for the new and fast screening technology,which provides a good basis for further research.Briefly,the target cells were mixed with polyacrylamide gel and then injected and polymerized in a coated capillary column.The FAM labeled original library was separated by this cell gel capillary electrophoresis.The signal was detected by the embedded fluorescence excitation mode.The influence of gel type,concentration,electrophoresis voltage and injection time were studied and optimized as follows:10%non crosslinking polyacrylamide gel,electrophoresis voltage 400V/cm,20 seconds electric injection time.Due to the abundance and quantity of different type of proteins on the cell surface,the same kind of ssDNA contacted different types and amounts of proteins in this electrophoresis process,resulting in signals at each retention time,Therefore,we can not simply judge the affinity of ssDNA to cell according to the retention time of signal.Which means ssDNA with different affinity cannot be separated.