New Methods For Detection Of Circulating Tumor Cells And Exosomes Based On Steric Hindrance Effect

Early screening and diagnosis of cancer is one of the significant ways to improve the cure rate and quality of daily life of patients,and even can prolong their life.Biopsy as the gold standard of cancer diagnosis cannot provide early pathological information at present.In addition,the invasive sampling method of biopsy is easy to cause secondary injury to patients with high cost and long time.In recent years,the emerging liquid biopsy technology has provided more opportunities for the early diagnosis of cancer.Liquid biopsy possesses advantages of easy sampling,simple operation,timely and rapid.Circulating tumor cells（CTCs）and exosomes are considered as primary targets of liquid biopsy and they have shown great potential in clinical applications.Compared with nucleic acid,protein and other biomarkers（for example,circulating tumor DNA,mi RNA,carcinoembryonic antigen,etc.）,cells are relatively large,which often cause lager steric effect in the detection process and increase the difficulty to establish new methods.But at the same time,the academia is also trying to make use of this feature to carry out the research of new detection methods.Based on this characteristic,we have established new methods to detect CTCs and exosomes in this dissertation,respectively.The details are summarized as follows:1.A new method for detection of circulating tumor cells based on the target-initiated steric hindranceIn this part,we have proposed an electrochemical detection method for circulating tumor cells based on the target-initiated steric hindrance.The method makes use of the steric effect caused by the large size of target cells to prevent the catalysis of terminal deoxynucleotidyl transferase（Td T）on binding deoxyribonucleoside triphosphate to the 3’hydroxy of aptamer on the electrode surface.Thus,the aptamer cannot be extended to combine with a large amount of Ru³⁺,and cannot produce strong electrochemical signal.However,when the concentration of CTCs is low,Td T contacts with the aptamer easily,the amplification reaction is easy to occur,and the stronger the electrochemical signal can be obtained.We have explored the feasibility,optimization,quantification,selectivity and anti-interference ability of the method.Taking He La cells as model,our method can achieve quantitative detection of He La cells in the range of1.6×10² to 1.6×10⁶ cells/m L with the limit of detection as 53 cells/m L.Therefore,the proposed method is sensitive and simple,which is expected to be used in clinical application of circulating tumor cells.2.A new method for exosomes detection based on steric hindrance-controlled signal amplificationIn this part,we have proposed a simple and sensitive method for exosomes detection based on steric hindrance-controlled signal amplification.Specifically,we have tried to use the steric hindrance effect of exosomes to hinder the complementary pairing process of single DNA embedded in exosomes and free signal DNA.Therefore,the signal DNA can remain single stranded and expose its 3’hydroxyl.Then,the long DNA with G-quadruplex structures can be extended by the amplification of Td T enzyme.Ultimately,strong fluorescence can be produced by the combination of Thioflavin T and G-quadruplex.The feasibility,selectivity and quantitative capability of the method are satisfactory,which can achieve quantitative analysis of exosomes in the concentration range of 1.66×10³ to 1.66×10⁶ particles/μL with the detection limit as low as480 particles/μL.Furthermore,it can maintain excellent performance in 10%human serum and distinguish the serum of cancer patients and healthy people well.Therefore,the method possesses certain application potential in clinical detection.