Microfluidic Droplets Coupled With Spectroscopy And Imaging Technologies For Single-Cell Cytokine Analysis

Single-cell technology is essential for elucidating cellular heterogeneity and chemical diversity.Single-cell analysis can facilitate the discovery of low-abundance cell subtypes,diagnose the source and progression of diseases,and explore the new mechanisms of tissue differentiation.All these are difficult to explore by means of population cell-based measurement.Single-cell analysis facilitates understanding cellular functions at the single-cell and subcellular levels that are extremely useful for clinical diagnosis and evaluation.So far,various highly sensitive single-cell manipulation techniques are involved in single-cell analyses,including flow cytometry,electrochemical analysis techniques,capillary electrophoresis,microfluidic droplet techniques,etc.Among them,microfluidic chip laboratories have many advantages in precise fluid control,cell manipulation,and rapid high-throughput signal output.In addition,the miniaturized microfluidic chips are able to integrate various detection techniques,such as mass spectrometry,surface-enhanced Raman spectroscopy(SERS),and various imaging techniques(fluorescence imaging,SERS imaging,four-wave mixing imaging),working for efficient and highly sensitive single-cell analyses.In short,microfluidics provides strong support for single-cell analysis and the studies of which have gained great attention.Single-cell secreted cytokines,such as vascular endothelial growth factor(VEGF)and interleukin-8(IL-8),are in a very small amount.In order to analyze the single-cell cytokines,highly sensitive detection techniques are needed.SERS is a technique by enhancing the Raman signals of analytes to a fold of over million times using metal or semiconductor nanostructures,which can remarkably increase the detection sensitivity of analytes.Combining microfluidic droplets with SERS can overcome many limitations of SERS,to improve SERS reproducibility and detection capability.SERS has become a powerful tool for single-cell analysis.Besides,the microfluidic chip can integrate various multidimensional and high spatial-resolved imaging techniques,to reveal the chemical distributions of probed systems,which provides an efficient analytical platform for visualizing and studying the cytokines secreted by single cells.Currently,tracking single-cell cytokines remains challenging.Previous publications showed that they manipulated droplets by encapsulating both single cells and individual carriers(additional cell or polymer bead)for analyte enrichment in one drop.This way causes the valid droplets to be a limited number.Also,once the droplets are broken,the carriers will separate from the cells,which causes the cell sorting to fail because their spatial correlation is destroyed.To address these problems,this thesis proposes a cell surface-bioconjugation strategy,and we take advantage of the microfluidic droplets and the spectroscopy/imaging technologies to establish two single-cell analysis platforms,a microdroplet-fluorescence imaging platform and a microdroplet-SERS platform.These platforms worked for the investigations of cellular heterogeneity and single-cell secreted cytokine tracking during oxidative stress.The main contents of this thesis include three parts:(1)This study reported a microfluidic droplets-fluorescence imaging platform for analyzing vascular endothelial growth factor(VEGF)secreted by single cells.Two silica nanoparticle-based immunoprobes were developed,which were attached to membrane proteins on the surface of the cells,with the secreted VEGF as a bridge.Thus,an immunosandwich structure was created on the surface of the probed cell.The fluorescence imaging analysis of single cells was employed to determine the secreted VEGF.This analytical platform was used to compare the single-cell VEGF secretory capacity of three cell lines(MCF-7,He La,and H8),demonstrating the heterogeneity of cells in terms of secreted cytokines.This single-cell analysis platform can be applied to identify other cytokines secreted at the single-cell level by altering other immune targets,which would be a useful tool to explore single-cell proteomics.(2)A microfluidic droplet-SERS platform was developed to analyze cytokines in single cells according to many instinct advantages of SERS,e.g.,in situ,nondestructive and highly sensitive.Two designed immune nanoprobes(capture probe and SERS detection probe)were encapsulated in microdroplets along with a single cell.Using a cell surface-bioconjugation strategy,the detection probes were immobilized on the cell membrane surface by cell-secreted cytokines to form an immunosandwich structure above the probed cell,thus enabling the enrichment of cytokines on the cell membrane surface and the amplification of SERS detection signals.The platform was used to track VEGF secreted by specific cells in different cell lines(MCF-7,SGC,and T24).Next,the SERS data were explored using chemometric methods(principal component analysis and random neighborhood embedding of t-distribution)to parse SERS data and build support vector machine(SVM)discriminant models.These chemometric approaches successfully identified three cancer cell lines according to secreted cytokines.In addition,the behavior of single cells in secreting VEGF was monitored temporally,demonstrating that the level of secreted VEGF increases over time.This study demonstrates that bioconjugation strategies constructed on the cell membrane surface can be used for cytokines assays,which provides physiological clues to the use of single-cell secreted cytokines as biomarkers for early tumor diagnosis.(3)Based on the former developed cell surface bioconjugation strategy,we extended these methods to track two cytokines secreted by single cells simultaneously.Three immune nanoprobes were developed,in which the capture probe has the dual function of targeting the cell membrane and capturing VEGF and interleukin-8(IL-8),while the two detection probes provide fluorescent output signals for two cytokines(VEGF and IL-8).In the presence of two cytokines,two detection probes would be linked to the capture probes to form immune sandwich structures.Thus,two cytokines secreted by single cells can be realized.In a hypoxic microenvironment,two cytokines(VEGF and IL-8)secreted by a single tumor cell were assessed.Moreover,multiple chemometric methods were used to distinguish the variability of the secretion of two cytokines at different hypoxia-induced times.The results reveal cellular heterogeneity and also demonstrate a positive correlation between two cytokines and the hypoxia-induced factors.This work demonstrates again the feasibility and viability of the droplet-based technology for tracking multiple cytokines secreted by single cells.In summary,this thesis established three methods for the analyses of single-cell cytokines,and they are valuable and significant for early cancer diagnosis and cancer prognosis assessment,etc.