Electric Cell-substrate Impedance Sensing Of Cell Alignment And Electrochemiluminescence For Exosomes Detection

Cells are accompanied by various behaviors during growth.Among them,cell adhesion is the basis for studying the behavior of other cells.Only when cells adhere to the material first,can other migration,differentiation and proliferation be carried out.The extracellular matrix?ECM?consists of a large number of nanoscale fiber-like structures with strong cell behavioral induction.In recent years,researchers have discovered that three-dimensional micro-nano substrates can mimic extracellular matrices and have a strong"contact-guided"effect on cells,which allows cells to produce directional behavior along the direction of the nanostructures,and thus can be used to study cell adhesion.However,the current methods for studying cell behaviors on various three-dimensional structures are still based on optical methods.Such methods rely on endpoint detection and cannot feed back dynamic information in time.Electric cell-substrate impedance technique is a real-time and in situ method for monitoring cell behavior.It can obtain important dynamic information of cell growth in the state of unmarked and non-damaged cells,and has certain application prospects.In multicellular organisms,the communication between cells is also an important behavior.Through intercellular communication,the organism ensures the coordination of different types of cells in the tissue.Exosomes are a type of nanovesicles secreted by cells that contain various active ingredients,such as DNA,RNA and proteins derived from mother cells,and are important cellular signaling tools.In recent years,exosomes have been considered to be involved in the occurrence and development of various diseases,especially in the occurrence and development of tumors,and can affect the therapeutic effects of cancer patients.The analysis of exosomes can directly obtain basic information of cancer cells.Breast cancer is one of the major diseases that threaten women's health.The researchers found that the exosomes released by cancer cells are usually higher than the exosomes of normal cells.The exosomes secreted by cancer cells distinguish from other exosomes by characteristic molecules and can be used as molecular markers for cancer diagnosis.Currently,molecular detection markers commonly found in exosomes useful for cancer diagnosis include proteins and nucleic acids.Electrochemiluminescence analysis technology has the advantages of high sensitivity,good controllability and fast detection speed,and has become a popular technology in the field of analysis and detection.Electrochemiluminescence analysis technology is used to detect exosomes as a powerful tool for the study of exosome surface protein expression and physiological functions.In this paper,a nano-grooved substrate was prepared by nanoimprinting to simulate the extracellular matrix,and an electric cell-substrate impedance sensing?ECIS?was constructed.Adhesion and spreading behavior on the nanogrooves of human foreskin fibroblasts?HFF?and human immortal keratinocyte cells?HaCaT?were analyzed.In addition,Ru?bpy?₃²⁺-SiO₂ nanoparticles were synthesized and a sandwich electrochemiluminescence sensor was constructed by binding nucleic acid aptamers.After quantitative analysis of MCF-7 breast cancer cells to test the performance of the sensor,the sensor was used for quantitative analysis of MCF-7breast cancer cell derived exosomes.Chapter One:IntroductionThis chapter one introduces the classification of three-dimensional micro-nanostructures and the effects of different surface structures on cell behavior,as well as the introduction of electric cell-substrate impedance sensing?ECIS?and its application in cell behavior research.Secondly,this chapter introduces the formation,secretion and biological functions of exosomes,focusing on the existing techniques of separation and quantitative analysis of exosomes.Among them,ultracentrifugation is the method used to isolate exosomes in this paper,and electrochemiluminescence is the quantitative method.Next,this chapter describes the association between breast cancer cells and exosomes and the early diagnosis of breast cancer.At the end of the chapter,the purpose and significance of this paper are presented.Chapter Two:Real-time Monitoring Skin Cell Alignment on Nano-grooves Using Electric Cell-substrate Impedance Sensing?ECIS?In this chapter,the nano-grooves with 200 nm in width,400 nm in period and 75nm in depth were fabricated on gold substrate with the technique of nanoimprint to simulate the extracellular matrix?ECM?,and the adhesion and spreading of human foreskin fibroblasts?HFF?and human immortal keratinocyte cells?HaCaT?cells on nano-grooves were analyzed.In addition,we used electric cell-substrate impedance sensing?ECIS?to monitor the adhesion and spreading behavior of the two cells on the nano-grooves.The results displayed that the nano-grooves could induce the alignment of HFF cells,in which,the cell arrangement along the direction of nano-grooves occurred prior to the cell elongation.While the nano-grooves couldn't influence the morphology of HaCaT cells,their adhesion and spread were delayed.Moreover,there was a good linear correlation between the impedance signal generated by HFF cells on the nano-grooves and the percentage of cells arranging along the direction of nano-grooves.Compared to the flat electrodes,the nano-grooves were less suitable for the adhesion and spread of HaCaT cells with almost no change of cell morphology,and the cell adhesion could cause more obvious variation of NI values than cell spread.Chapter Three:Detection of MCF-7 cells by electrochemiluminescent aptamer sensor based on Ru?bpy?₃²⁺-SiO₂ NPsIn this chapter,Ru?bpy?₃²⁺-doped SiO₂ nanopaticles synthesized by reverse microemulsion methods as signal were combined with two nucleic acid aptamers from two different proteins on MCF-7 cells,and a sensitive electrochemiluminescent sensor for detection of MCF-7 cells were developed.The sensor quantified MCF-7 cells with good linearity in the range of 3.63×10⁴-3.63×10⁶ cells/mL with a detection limit of4000 cells/mL.The sensor has a simple preparation process and good selectivity,and is expected to be applied to many different biological systems.Chapter Four:Electrochemiluminescence aptamer sensor based on Ru?bpy?₃²⁺-SiO₂ NPs for the detection of breast cancer exosomesIn this chapter,an electrochemiluminescent aptamer sensor based on Ru?bpy?₃²⁺-SiO₂ NPs was constructed.The exosomes were specifically quantified by the MUC1protein on the surface of MCF-7 breast cancer cell exosomes.In this experiment,the specific protein CD63 and MUC1 corresponding to MCF-7 exosomes were used to design the sandwich structure,and the core-shell structure of Ru?bpy?₃²⁺-SiO₂ NPs was prepared to amplify the luminescence signal.As a result,the sensor analyzed exosomes with a wide detection range of 3.3×10²-1.6×10⁸ particles/?L and detection limit was280 particles/?L,which was much lower than that of common ELISA method.The sensor can be flexibly used for the detection of different breast cancer exosomes,and has high sensitivity and good reproducibility,and provides a new idea for clinical diagnosis of early breast cancer.