Single-Cell Monitoring And Nucleic Acid Detection Technology For Early Cancer Diagnosis

As a protease,matrix metalloproteinase(MMP)can destroy the histological barrier of tumor cell invasion,and its activity is related to the ability of tumor metastasis and invasion.Due to the heterogeneity of tumors,the average activity of MMP obtained from population cells cannot accurately reflect the invasion ability of tumors.In this paper,a high-throughput single-cell real-time monitoring platform based on static droplets was developed,to study the difference in enzyme activities of MMP at the single-cell level.A thermosetting oil has been developed which has good biocompatibility and gas permeability.It can solidify and fix the droplet within 30 minutes after the droplet formed,and doesn’t need structures to capture droplets,so single cells are continuously monitored for a long time.Compared with microwell chip and droplet technology based on capturing structure,this method has high detection flux,simple design and flexible operation.In this paper,the physical and mechanical properties of the developed thermosetting oil were measured,and the experimental system for detecting enzyme activity of cells in droplets was optimized.The MMP enzyme activity of a single A549 cell was detected by fluorescence microscope timing shooting.It was found that the expression of MMP in A549 cells was highly heterogeneous at the single cell level,and a few A549 cells had higher MMP enzyme activity.Digital PCR(dPCR)technology is a high-sensitivity accurate nucleic acid quantification technology based on single-molecule amplification.It has huge application prospects in circulating tumor DNA detection,but the narrow dynamic detection range isn’t good for the practical application of dPCR.In order to increase the dynamic detection range of dPCR,a self-driven multivolume dPCR chip was developed in this paper.The chip contains four types of microwells with volumes of 0.50 nL,2.74 nL,14.34 nL,and 68.60 nL.The overall size is 2.8cm×3.1 cm.This chip,which microwells contains only 192 reaction units per types,has a detection limit of 2.0×10~5 copies,which is equivalent to a chip containing 20,000 same volume reaction units,which greatly reduces the processing difficulty of the chip.The chip utilizes the porosity of polydimethylsiloxane(PDMS)material,which can achieve the precise distribution of negative pressure-driven samples without the assistance of other instruments.The water channel layer embedded in the chip can effectively inhibit the evaporation of water during the PCR reaction and ensure the PCR amplification efficiency and quantitative accuracy.A series of diluted 18s ribosomal plasmids were used to verify the quantitative performance of the chip.The correlation coefficient reached 0.9943,and the quantitative accuracy was comparable to the commercial instrument LifeTech Quantstudio 3DdPCR system.In addition,this paper also proposes a concept of a high-throughput multivolume dPCR chip based on droplet microfluidic technology.Through COMSOL Multiphysics simulation and experimental verification,a one-step generation of multi-volume droplets is initially achieved.Combining microfluidic droplet technology with multivolume dPCR can further improve the dynamic range and detection throughput of dPCR,and has important reference significance for the development of a multiplex gene commercialization platform.The single-cell enzyme activity real-time detection platform developed in this thesis can realize the detection of MMP enzyme activity at the single-cell level flexibly and high-throughput,which is helpful for the study of heterogeneity of single-cell migration capacity.The developed negative pressure-driven multivolume dPCR chip broadens the dynamic range of nucleic acid detection.The combination with microfluidic droplet technology will further increase the detection flux and dynamic range of dPCR,and will play an important role in early diagnosis and monitoring of cancer.