Study On Detection And Analysis Of High-throughput Single Cell Bioinformation Based On Microfluidic Chips

In response to the major needs of single-cell information analysis,this thesis builds a high-throughput single-cell secreted proteins detection microfluidic chips with high sensitivity,reproducibility,and reliabil ity through the combination of microfabrication technology,new electronic sensing materials and biotechnology.Based on the detection information,a high-accuracy cell clustering model was established.Cells contain a variety of complex biological information.Among them,the secreted proteins produced by cells play an important role in the process of differentiation,migration and cell-tocell communication.Due to the widespread existence of cell heterogeneity,the results of traditional cell secreted protein analysis are at the cell population level,which will mask the differences between cells,Separating the population of cells and analyzing their secreted proteins at the single-cell level can provide insight into the heterogeneity of cells mediated by secreted proteins.However,single cell isolation and subsequent single-cell secreted protein analysis face the following major technical challenges:(1)Under the premise of ensuring cell viability,single cells can be quickly and highthroughput separated from the population of cells and cultured for a long time.(2)Under the premise of ensuring high-sensitivity detection,real-time,multiple,in-situ detection of secreted proteins from real and reliable living single cell secretion products.Based on the above challenges,this thesis conducts research on high-throughput singlecell biological information detection and analysis based on microfluidic chips,as follows:The first chapter is based on the literature research on the current traditional and microfluidic technology-based single-cell separation and single-cell secreted protein analysis research status,and puts forward the main technical challenges faced by the current single-cell separation and subsequent single-cell secreted protein analysis.Leads out the main research content and innovations of this article.The second chapter mainly studies the single-cell distribution analysis based on microfluidic chips,through micro-nano processing technology,single-cell microchamber array chips are designed and prepared independently,graphene oxide quantum dot nanomaterials are modified on the surface,and single-cell printing methods are used to quickly separate single cells.Plates up to 7021 high-throughput single cell isolation and normal suspension/adherent culture for up to 3 days.The third chapter mainly studies the high-throughput detection of single-cell secreted proteins based on microfluidic chips,through nanotechnology and micro-nano processing technology,antibody array chips are designed and prepared independently.Based on the principle of protein adsorption by graphene oxide quantum dot nanomaterials on the antibody array chip,an antibody array glass substrate that captures up to 20 secreted proteins is made,Integrated with a single-cell microchamber array chip,capture and detect single-cell secreted proteins by immunosandwich method,can achieve a minimum detection concentration of 100 fg/mL,multiple,high sensitivity,high specificity in situ real-time detection and analysis of up to 20 secreted proteins.Using this method,we have achieved the analysis of 12 main secreted proteins in HEY,MDA-MB-231,K562,293T and SH-SY5Y cell lines,and verified the reliability of the established clustering model and the more than 90 percent accuracy of cell identification in repeated experiments.The four cell lines of HEY,MDA-MB-231,K562,and 293T were further subpopulated,and 26 cell subpopulations with different expression levels were separated.Finally,it summarized the paper as a whole and looked forward to the future research direction.Compared with other single-cell separation and secreted protein analysis technologies,the single-cell analysis technology based on micro-processing technology and new electronic sensing materials studied in this article has the advantages of fast speed,high throughput,good cell activity,and a wide range of applicable cell types(suspension,adherent cells,rare cells),and the detection of secreted proteins has the advantages of high throughput,high specificity,high sensitivity,and real-time in situ detection,and has broad application prospects.