Research On The Quantitative Detection Of Multiple MiRNAs In Single Cells Based On Microfluidic Droplets And Their Use In The Diagnosis Of Related Diseases

Cells are the basic unit of life structure and function.Cells with diverse structures and functions interact to form complex living organisms.Because of the diversification of functions,cells have obvious heterogeneity in living bodies.The heterogeneity between cells often plays a crucial role in the growth,differentiation,and development processes of living organisms.Similarly,in the development of cancer,tumor cells of different types or states have great genetic material and life-active substances in tumor cells due to the differences in their physiological environment and the differences in the development process of tumors.miRNA,as an important bioactive molecule,is widely involved in the process of life activities.Especially in the development of tumors,the corresponding content of the amount of miRNA involved in the development of tumors will change.The results of existing studies show that the classification and identification of tumor cells can be performed by the distribution of the content of miRNAs.Especially in the early stage of disease detection and timely implementation of the correct treatment program can achieve early diagnosis and prevention with very important clinical application prospects.However,there are very few diagnostic methods that can be performed early in cancer for the early diagnosis.Circulating tumor cells are important indicators for the early assessment of cancer,but the blood is extremely low,making it difficult to detect and analyze population cells based on traditional methods.Single cell analysis is a very suitable method for the detection and analysis of circulating tumor cells.Due to the extremely low content of miRNAs in single cell cells,the traditional protocol is difficult to achieve highly sensitive detection of miRNAs in single cells due to various limitations.Therefore,we proposed a high-throughput and high-sensitivity detection protocol for miRNAs in single-cell based on microfluidic droplets.And using this protocol to achieve simultaneous detection of multiple miRNAs within a single cell,and this method Applied to the classification of tumor cells.This paper consists of three parts,including:The first chapter is introduction.It first introduces the importance of single cell analysis in the life sciences,and provides a brief overview of the five main types of detection currently available in single cell detection;then the function and function of miRNA in life sciences.The application and the currently existing strategies and methods for the analysis of miRNAs in single cells are described.Finally,we propose a solution for miRNAs detection in single cells.The second chapter is the design and construction of a microdroplet system for quantitative detection of single cell miRNAs.In this chapter,we design highthroughput droplet generation using a high-throughput droplet generation chip,and perform high-throughput single-cell manipulations through an optimized micro-droplet system.The constructed micro-droplet system provides the basis for high-throughput detection of single cells.The third chapter is the quantitative analysis of two kinds of mi RNAs in a single cell and used to classify the different tumor cells.We have developed a single-cell highthroughput package based on droplets,and the secondary isothermal signal amplification of mi RNA in droplets and use of the laboratory self-built laser-induced fluorescence detection device for detection of fluorescent signals within the droplet.Using this analysis scheme,high-throughput quantitative detection of miRNAs in single cells was achieved.By measuring the content of miRNA between different types of cells and mapping their distribution,different types of cancer cells are classified based on the difference in the expression level of miRNA.It provides a new method for the high-through analysis of miRNA in single cells and the classification of cell types.