Microdroplet Biosensor For The Applications Of Tumor Cell Metabolism Research At The Single-cell Level

Cancer is a growing threat to human health and it has become the top fatal disease in our society nowadays.Most of the malignant tumors do not exhibit obvious symptom in their early stages.However,when the patients develop some specific symptoms,the tumors are frequently in their late stages.According to the estimation by the international allies against cancer,1/3 of cancers are probably preventable,and 1/3 of cancers can be cured assisted by accurate and early diagnosis.Therefore,it is urgent to develop new technologies for early diagnosis of malignant tumors,which has drawn great attention among scientists.Due to the limitations of sensitivity issues or instrumental availability,the traditional methods typically require the measurments of cell populations.They tend to ignore the cell-to-cell differences,thus unable to reveal the cell heterogeneity at the single cell level.There has been an increasing number of reports which suggest the importance of single-cell analysis of cell heterogeneity for tumor biology and the development of targeted therapies.In this thesis,I have developed a microfluidic approach to generate microdroplets with a high throughput performance.The microdroplets are featured with an ultra-small volume,little sample consumption,and potentially high sensitivity.They can isolate a single cell in the closed micro-reactors,thus preventing cross contamination between the cells in comparsion to the conventional methods.Our apporach provides an important platform to explore the cell heterogeneity at the single cell level.My thesis includes the contents in the framework as follows:The first part:brief instruction of the characteristics of microfluidic technologies,the widely-used methods of microfabrication,and representative applications of microfluidic chips.Microdroplets have also been introduced in this chapter as an important branch of microfluidic technology,including various methods and working principles,microdroplet manipulations,and the applications of microdroplets.Reactive oxygen species（ROS）and lactic acid as representative cell metabolites are also introduced in the chapter,because of their biological importance and relevance to my research projects.The second part:glass capillary microfluidic devices have been assembled to generate microdroplets in a high throughput manner.We have synthesized HRP-AuNCs successfully by biomineralization,and then loaded them into the microdroplets.HRP-AuNC can react with H₂O₂,resulting the fluorescence quench of HRP-AuNCs.Controlling the concentration of cell suspension and the flow rate ratio of continuous phase and dispersed phase allow us to encapsulate 1-2 cells per droplet.The fluorescence change in each microdroplet enables sensitive detection of single cell secreted H₂O₂.We have demonstrated high sensitivity and specificity for our assay.We have attempted to quantitatively analyze the difference between tumor cells and normal cells in secreting H₂O₂ at the single cell level.The third part:due to the“Warburg effect”and the tendency of lactic acid secretion featured by the tumor cells,tumors are typically in an acidic environment（pH:6.2-6.9）.I have developed a microdroplet-based assay by taking advantage of the reaction to lactic acid under the assistance of enzymes:LA+NAD⁺?PA+NADH+H⁺.The product of NADH can emit blue-fluorescence,while the reactants or other products are not fluorescent,which allows us to detect lactic acid secreted by a single cell in the microdroplets.We have further attempted to synthesize Ca²⁺-Alginate microgel in our microdroplets,and immobilize the enzymes into microgel to achieve sensitive detection of single tumor cell secreted lactic acid.In a short summary,I have developed a microdroplet approach to study the difference between tumor cell and normal tissue cell in cell secreted metabolites,and successfully detect H₂O₂ and lactic acids secreted by a single cell.Further investigation using the single-cell analtyical tools promise a new platform for early diagnosis and prevention of tumor diseases.