High Through-put Generation Of Single Cell Encapsulated Microdroplets And Application In Detecting Tumor MMP9 Enzymatic Activities

Cancers have been a serious threat to human health mainly because of malignant tumor metastasis.Metastasis is the process of spreading of malignant tumor cells throughout the body and forming of secondary tumors by invasion into different organs/tissues,which causes the majority of deaths(nearly 90%)associated with cancers.It is critical to develop sensitive methods for the detection of malignant cells invasiveness.Recently the advances in microfluidic technologies have provided new tools for varieties of biochemical assays.The typical advantages of microfluidic platforms include small volume to save expensive reagents,high sensitivity,high throughput performance,and ease for large scale integration.Therefore,microfluidic devices promise a broad range of applications in molecular biology,chemical analysis,clinical diagnostics and other research areas.In this thesis,I describe my efforts to develop a new “off-the-shelf” capillary microfluidic device for the generation of microdroplets and test tumor invasiveness using single cell-encapsulated microdroplets.The device is assembled by two polyetheretherketone(PEEK)chromatography T-shaped connectors and a set of coaxially aligned glass capillaries.Compared microfluidic chip processing technology,our devices greatly reduce the experimental cost,extremely easy to assemble,easily cleaning,and can be repeated to generate stable uniform size droplets.We have demonstrated single cell encapsulation in the microdroplets with our devices,and detect MMP9 enzymatic activities very sensitively.The main contents of this thesis are organized as follows:Chapter 1: Introduction of the development and progress of microfluidic technologies,the characteristics and advantages of micro total analysis system or microfluidics.Then this chapter focuses on the microdroplet technology,including various methods for microdroplet generation,distinct features,and their applications in the literature.Chapter 2:We use commercial PEEK and some glass capillaries to assemble a new glass microfluidic device.Different types of microdroplets have successfully been prepared using this device,including water-in-oil,oil-in-water and gel-in-oil droplets.The reproducibility and stability of the microdroplets have been investigated in this chapter.Chapter 3: This microfluidic device allows us to encapsulate single cell in microhydrogel droplets.I have attempted to explore the enzymatic activities of matrix metalloproteinase(MMP9)in different types of tumor cells using our approach.The principle of the detection,calibration of enzyme activities,and comparison of different cell lines are included in the discussion of this chapter.In summary,this thesis has successfully demonstrated a sensitive method for cancer cell invasiveness assay using the microdroplet technology.We have achieved single-cell encapsulation with microdroplets and demonstrated detection of MMP9 enzymatic activities at the single cell level for the first time.It will provide a powerful tool for early diagnosis/prognosis of cancers,and hopefully facilitate personalized treatment.