Research On Microfluidic-based Immunofluorescence Analysis System And Application In Disease Detection

The early diagnosis,treatment and monitoring of therapeutic effects are of great significance to human health promotion.The effective treatment of many critical diseases depends on the timeliness and accuracy of diagnosis.However,traditional detection methods such as tissue biopsy still have limitations such as difficulty in sampling,inability to achieve early detection,patient suffering,and low sampling frequency.In Vitro Diagnostics has accompanied the development of biochemistry,immunology,molecular biology and other fields,as well as the rise of molecular diagnostics and precision medicine.It has been used in point-of-care-testing and other applications have achieved remarkable results.However,current IVD products still have limitations such as low test accuracy,single test items,and unstable performance,which limit the development of the domestic IVD industry.The microfluidic chip system has promoted the development of portable in vitro diagnostic technology with its miniaturization and integration features,which can achieve clinical needs such as miniaturization of test consumption samples and multi-parameter simultaneous detection and deepened its application in biochemistry and translational medicine,such as micro-biochemical analysis,high-throughput screening,real-time detection,and formation of novel biological materials.Compared with traditional immunoassays,it has the advantages of short analysis time,high sensitivity,reduced consumption of expensive reagents and samples,and easy integration.Most nowadays microfluidic immunodetection chips rely heavily on affinity capture of target proteins by probes immobilized on the channel surface.In this interface process,the mass transfer efficiency of the target to the surface and the balance and kinetics of the binding reaction are the basic factors controlling the sensing performance.However,due to the extremely low Reynolds number,the flow in the microchannel is generally laminar,and the mass transfer efficiency is low,making it difficult for the antibodies on the wall to bind to the target protein in the fluid,resulting in biosensors in the micro/nano fluid system inefficiency.In this study,we combined microfluidic chip technology with immunofluorescence analysis technology to develop a continuous,fast,and efficient microfluidic immunodetection chip.The microfluidic chip was filled with antibody beads to disturb the laminar flow state of the liquid sample improves the mass transfer efficiency,while increasing the contact area of the immunoaffinity reaction,greatly enhancing the binding of the antibody to the target protein,amplifying the fluorescent signal,and significantly enhancing the sensitivity and efficiency of detection.By optimizing the reaction conditions and detection parameters,a rapid quantitative detection of disease target protein levels in blood samples was achieved.The research work mainly includes the following aspects:1.Design and manufacture of microfluidic chip.The microfluidic chip was legally designed and fabricated using soft lithography technology and pressure-sensitive adhesive bonding method.The chip mainly includes three parts: a fluid transfer chamber,a reaction chamber,and a micro trap column.2.Preparation and characterization of antibody microbeads.The surface of the glass microbeads was silanized and covalently linked to the monoclonal antibody to complete the antibody modification on the surface of the microbeads.Characterize and analyze the morphology,surface elements,biological properties,and immune detection effects of antibody microbeads.3.Research and optimization of reaction conditions and fluorescence signal acquisition parameters,and performance evaluation of microfluidic chip detection system.Improving the efficiency and reliability of MCIAS by optimizing the sensitivity ISO,exposure time,bead size,immunological binding reaction time,and velocity of flow.And evaluate the reproducibility,stability and specificity of the method and device.4.Application and evaluation of MCIAS for protein detection in plasma.Using human breast cancer antigen CA15-3 protein as the detection object,the standard curve method was successfully used to quantitatively detect CA15-3 in the plasma of breast cancer patients,and the results were in good agreement with the results of commercial Enzyme-Linked ImmunoSorbent Assay kits.This shows that the method can fast and quantitatively detect the level of disease protein in blood samples.In summary,this study established a continuous,fast,and efficient method for detecting disease target proteins based on microfluidic in vitro detection technology,and successfully achieved the quantitative detection of CA15-3 protein in the plasma of breast cancer patients.The results show that the method can accurately detect the disease target protein in blood samples,and has good reproducibility,stability,specificity and accuracy,and provides a new method for the early diagnosis of major diseases such as cancer.The further development and implementation of integrated disease point-of-care-testing and devices have laid the foundation.