Development Of An Automated Cell-selex Instrument For Tumor Cells Based On The Enclosed Cartridge

The malignant tumor is one of the most important factors that endanger the human health,because of the treatment difficulty and the low postoperative survival rate.The early diagnosis of tumor lesions and the timely targeted therapy will help to improve the cure effect of tumor patients.Using the difference between the surface proteins of normal cells and tumor cells to find specific markers is of great benefit to the tumor diagnosis and treatments.The aptamer is the ss DNA or RNA selected from artificially synthesized random singlestranded DNA or RNA libraries,and it binds to the target with a high affinity and a high specificity.The cell-SELEX targets intact cells,and finds aptamers that specifically bind to the target through the interaction between single-stranded nucleic acids and molecules on the cell surface from the nucleic acid library.In practical applications,the selection process of aptamers is complicated and cumbersome,which requires a lot of manpower and money.What is more,because the nucleic acid in the library easily escapes to cause the aerosol pollution,the reliability of the selection results cannot be guaranteed.In order to solve the problem of the nucleic acid aerosol pollution generated during the aptamer selection,an enclosed cartridge was designed and produced in this thesis to ensure the reliability of results in each selection round.At the same time,in order to improve the selection efficiency,this paper has developed an automated cell-SELEX instrument and verified its performance.Finally,this paper used the cartridge and the instrument to complete the aptamer selection of Hep G2 cells within 150 hours,and successfully obtained one aptamer with the high specificity.Specific contents include:1.The design and implementation of the automated cell-SELEX instrument based on the enclosed cartridgeBased on the enclosed cartridge,the design requirements of the automated cell-SELEX instrument were put forward according to the principle of aptamer selections of tumor cells.Then,according to the operation process of the cartridge,the corresponding module design was completed,and the design and assembly of the instrument function partition and the overall structure were completed.The research had been conducted on the control system of the automated cell-SELEX instrument,and the design and developments of the hardware circuit and main control programs had been completed to ensure the stable operation of the automated aptamer selection process based on the enclosed cartridge.2.The method optimization of the aptamer selection and the design of the enclosed cartridgeThe method of selecting aptamers of tumor cells was optimized for the operation in the cartridge,which simplified the step of collecting nucleic acids bound to the surface of target cells and directly used the ultrapure water to obtain and amplify nucleic acids.Based on the improved selection method,a separate structure of the cartridge and its internal functional modules were proposed and designed.The maximum volume of the pipette tip in the cartridge is 1 m L,the volume of the cleaning solution chamber is 8 m L,and the volume of the flat square reagent chamber and the PCR amplification chamber is 500 ?L.Then,all parts were manufactured by 3D printing technologies and the cartridge was assembled.After three versions of improvements,the actual cartridge structure and all functions met needs of the cellSELEX.Finally,the air tightness,the drainage performance,the thermal conductivity of the reagent chamber and the moving shaft in the cartridge were tested,and the results showed that the functions of each part met the application requirements.3.The research on liquid transfer controlIn order to cooperate with the liquid transfer in the cartridge and realize the operation of the large volume PCR amplification,cell cleaning and ultra pure water transfer,the control technology of the liquid transfer was studied in this paper,and a liquid transfer system including the liquid gas path module and the motion positioning module was established.The40PC100G2 A pressure sensor was added to the liquid gas path module to realize the real-time measurement of the gas pressure.The normal gas path and abnormal gas path pressure models were established to judge whether there was any abnormal situation in the process of the liquid transfer,so as to ensure the accuracy and reliability of the liquid transfer volume.In the motion positioning module,the S-type equal frequency acceleration and deceleration algorithm was introduced to make the start and stop of the stepping motor smoother and reduce the out of step situation.The test results showed that the absolute value of the single horizontal positioning error of the tip was 0.25 mm,and the average cumulative positioning error was 0.2 mm.4.The research on large-volume PCR amplification moduleIn order to solve the problem of the nucleic acid aerosol caused by the frequent transfer of multi tube PCR products in the process of tumor cell aptamer selection,this paper proposed and designed a large-volume PCR module,which provided the heat source for the thin-walled square PCR amplification chamber(the maximum amplification volume is 500 ?L)by the vertical heat tank.In order to solve the problem of the temperature lag between the heat tank and the PCR amplification chamber,the traditional control method was improved,and the fuzzy adaptive control method was introduced to improve the temperature rising and falling performance.At the same time,the temperature of the heat tank and the amplification chamber was taken as the controlled object to solve the temperature lag problem and ensure that the reaction liquid temperature in the amplification chamber was consistent with the set temperature.The test results showed that the temperature difference between the PCR amplification chamber and the heat tank was small after the introduction of the cascade fuzzy PID.The temperature of the PCR amplification chamber can reach the set value quickly with the small overshoot and good temperature control effects,which meets the requirements of the temperature rise and fall of the PCR amplification.5.The performance test experiment of the automated cell-SELEX instrumentFirstly,the performance of the automated cell-SELEX instrument was tested,including the library transfer and module matching test,the liquid transfer performance and the large-volume PCR amplification.The automatic library transfer between different rounds of selection cartridges requires the cooperation of multiple modules.After 6 cartridge tests,the results showed that the modules and structures of the instrument cooperated well,and the library automatic transfer process can be completed within 60 s.The results of liquid transfer performance tests showed that when the liquid volume was between 100 ?L and 800 ?L,the pipetting mean error of two pipette tips was less than 7.2 %,and the CV value of the same pipetting volume was less than 6.2 %.Finally,the cartridges and the instrument were applied to select the aptamer for Hep G2 cells,and the control cells were L02 cells.The results showed that one aptamer was obtained through 16 rounds of the selection within 150 hours,and the sequence and the secondary structure of the aptamer were obtained.The binding experiment of the aptamer to the cells showed that the aptamer has a high affinity and specificity.