Highly Sensitive Detection Of Lactate And Luciferase Secreted By A Single Cell With A Microdroplet Biosensor

Cancers and bacterial infections are major diseases threatening human health.Due to the high risks of tumor recurrence and metastasis,there is an urgent need to develop new methods for the early diagnosis,effective treatment,and prognosis.As for bacterial infections,although many bacteria can be cleared by the human immune system,some species can cheat the immune system and hide themselves,then eventually harm the immune cells.At present,most of the techniques in these fields are based on the assays of testing large populations of cells.Those traditional methods suffer from the limitations of masking the cell-to-cell difference or tending to produce false negative results when only low numbers of cells are available.The methodology development for single-cell analysis can provide the key information between the individual cells,thus becoming the hot spot in the field of biomedical research.The advances in the microfluidic technologies provides a variety of platforms for single-cell experiments,with the advantages of high sensitivity,low sample consumption,point-of-care diagnosis,and high cost-effectiveness This thesis describes my efforts of integrating single-cell encapsulated microdroplets and luminescent assays for sensitive detection of important biomolecules,and my preliminary investigation on tumor cell heterogeneity at the single cell level.My thesis includes four chapters as follows:Chapter 1:A brief overview of the development of microfluidic technologies,including fabrication procedures,advantages,and applications of microchips.Then I introduce the technique of microdroplets as a representative approach,and review the principle,classification,methods,and applications of this specific technique.Considering the background of my projects in this thesis,I briefly go over the biology of lactic acid secretion by tumor cells and polarization of macrophages under bacterial stimulation.Chapter 2:In order to develop tools for understanding the metabolic processes in tumor biology and "Warburg effect",here I develop a highly sensitive and specific method to detect lactic acid secreted by a single tumor cell.My approach features isolating single tumor cells individually in the droplets of nano liters,and taking advantage of the enzymatic reaction involving lactate dehydrogenase,lactic acid,and the oxidizing coenzyme I of NAD+.The product of reducing coenzyme I(NADH)can emit blue fluorescence under the excitation at 360 nm.Monitoring the fluorescence changes during this redox reaction provides a highly sensitive method to quantify lactic acid secretion by single tumor cells encapsulated inside the microdroplets.I have demonstrated that this approach can distinguish the secretion of lactic acid between normal mammalian cells and different tumor cell lines.Chapter 3:Investigation of the effects on lactic acid secretion of tumor cells with glycolytic inhibitors.Based on the platform developed in Chapter 2,here I attempt to encapsulate single tumor cells,inhibitors,LDH and NAD+ together in the microdroplets,so that I can study tumor cell secretion and metabolic changes induced by a small panel of glycolytic inhibitors.This approach allows for revealing the heterogeneous behaviors of single tumor cells under the effects of the inhibitors,demonstrating the proof-of-concept for tumor therapeutic drug screening.Chapter 4:A pilot project of a new application based on the microdroplet biosensor.Macrophages can be polarized into two types under the stimulation of external pathogens.In this chapter,I design the plasmids to transfect the fused modules,including an extracellular secreted luciferase expression gene and an arginase promoter,onto the macrophages.Upon bacterial stimulation,macrophages can take the polarized transformation accompanying the metabolic changes of arginine.This process will activate the expression of arginase and secretion of luciferase extracellularly,which enables the chemiluminescent assay with the substrate of luciferin.Integration of the microdroplet biosensor with this assay principle promises a new tool for the research on polarization of macrophages and antibacterial drug screening at the single cell level.In summary,microdroplet biosensors have been developed to investigate the heterogeneous behaviors in lactic acid secretion of single tumor cells induced by glycolytic inhibitors,as well as the interactions between macrophages and bacteria.This technique offers a useful platform for the studies on the glycolytic processes of cancers,macrophage/bacteria interacting mechanisms,and screening for new therapeutic drugs.