Highly Sensitive Detection Of Nitric Oxide By Microdroplets For Investigation Of The Interactions Between Single Macrophage And Bacteria

Bacterial infections can induce macrophage polarization and trigger inflammation in the body,causing sepsis in the blood,inflammatory bowel disease(IBD)in the intestine,ulcerative inflammation,and Crohn’s disease(CD).When the intestine is infected with foreign pathogens,such as Gram-negative bacteria like E.coli and H.pylori,the secretion of endotoxins by the pathogens can induce polarization of macrophages towards an antiinflammatory direction.The ROS and RNS secreted by macrophages also suppress bacteria by affecting their normal physiological activities.However,there are still many technical difficulties in the in vitro methods for the detection of NO,such as low sensitivity and short half-life.In addition,NO affects the physiological state of the cell population and interferes with the assay results.Most current assays can only indirectly provide the averaged NO levels based on a large number of cells.The cell-to-cell communications and heterogeneity between macrophage populations poses a challenge for the traditional methods of NO detection.Therefore,it is important to develop singlecell analysis techniques for investigation of macrophages during the inflammation.Microdroplet technology is a highly efficient method for single-cell analysis,by generating microdroplets(tens to hundreds of microns)as the biological reactors.It has the advantages of miniaturization,modularization,and high-throughput performance.Microdroplet technology provides a method to overcome the difficulties of conventional techniques,by requiring only a small amount of reagents and a relatively short period of time.In this thesis,I have developed a new platform by integrating microdroplet technology and the fluorescence detection technique for monitoring NO secretion of single macrophages under external stimuli.This platform has been tested to probe the heterogeneity of macrophages,and the interactions between single macrophages and E.coli.My thesis includes four chapters as follows:Chapter 1:The development history of microfluidic technology,the advantages of microfluidic technology,and fabrication and typical applications of microfluidic chips.Then I focuses on the introduction of the microdroplet technology derived from microfluidics,the methods and principles of microdroplet generation and manipulation.The biological significance of individual macrophages during inflammation is briefly described as the background justification of my thesis.Chapter 2:According to the biological characteristics of nitric oxide released by macrophages after M1 polarization,I have developed a new method to sensitively detect the extracellular NO secreted by a single macrophage.By encapsulating and isolating single macrophages in droplets(picoliter volume),interferon-γ and lipopolysaccharide can stimulate macrophages to secret NO extracellularly towards M1 polarization.The reaction between NO and the fluorescence probe molecules(DAF-2)generates green fluorescence under the exciation at 485 nm.Using the microdroplets,the concentrations of extracellular NO can quickly be determined with high sensitivity,allowing for realtime monitoring of NO secretion by a single macrophage.The arrays of microdroplets can be applied to study the heterogeneity of macrophages.In this chapter,the changes of NO secretion by single macrophages have been compared under different levels of the external stimuli.Chapter 3:Based on the platform by Chapter 2,the interactions between E.coli and single macrophage have been investigated within the microdroplets.The experiments have been performed by co-encapsulating a single macrophage and E.coli in gradient quantities in the same microdroplet,followed by the detection of NO secretion by single macrophages,and subsequent cell/bacteria activity assays.The fluorescent intensity evaluation of the microdroplets supports the possibility of heterogeneity M1 polarization during the inflammation.This method can be used to study the mechanisms of macrophages responding to bacterial infection in intestinal inflammation.It provides an efficient and sensitive platform to study macrophage heterogeneity at the single cell level.Chapter 4:This chapter summarizes our approach and the major experimental results,providing a brief outlook of the technical development and applications in the future.In summary,we have developled a new microdroplet platform for highly sensitive detection of NO secretion by individual macrophages during inflammation.I have performed a pilot research on E.coli-macrophage interactions using this platform.My thesis offers a new research tool for early diagnosis and prevention of intestinal inflammatory diseases.