Multiple Immunoassay Based On DEP Microfluidic Chip

Life and health has always been the focus of human attention,and the early diagnosis of disease is very important for human life and health,especially for cancer patients.Research shows that cancer exists in the human body in a certain incubation period,the earlier the diagnosis of cancer,means that it can greatly improve the cure rate and prolong the survival time of patients.At present,the diagnosis of many diseases usually requires the combination of a variety of biomarkers(such as interleukin,etc.),so it is of great significance to detect these biomarkers quickly,efficiently and at low cost.The traditional in vitro diagnostic method—enzyme-linked immunosorbent assay(ELISA)has been the mainstream,but there is some shortcomings,such as: accuracy is not high enough,the detection of single substance and so on.With the deepening of scientific research,multiple immunoassay technology developed on the basis of ELISA,mainly using solid phase plane array method and liquid suspension array method as two implementation schemes,have made many breakthroughs with the help of micro devices or detection equipment.In particular,the use of fluorescence-coded microbeads improves the multiplicity and accuracy of detection.Fluorescence-coded microbeads usually use surface biomolecular modification technology,micro-channel device and optical detection equipment to play their detection role,but there is still a large room for improvement in the development of micro-devices and detection methods.Therefore,it is of great significance to conduct stable and reliable microsphere surface reaction experiments,develop matching microdevices and testing equipment,and efficient data analysis and processing to improve the detection effect.In this thesis,we combine the microfluidic chip technology with the immunoassay technology based on fluorescence-coded microbeads,and integrate the automation technology,image processing and data processing technology,so as to realize a high sensitivity,high reusability and fast multiple detection immunoassay scheme.Firstly,through the deeply analysis of the theory of dielectric electrophoresis(DEP)and the simulation of the corresponding parameters in the chip with COMSOL Multiphysics simulation software to verified the feasibility of the scheme from theory.Then,a DEP microfluidic chip was fabricated by lithography and sputtering technology.An alternating current is applied between the indium tin oxide(ITO)conductive glass at the top and the gold-plated surface at the bottom of the microhole to provide a nonuniform electric field,which generates a DEP force to manipulate the microbeads,improving the proportion falling into microholes and the reusability of the chip.This thesis customized an optical detection platform according to the design requirements,using two kinds of excitation light,dichroic mirror,three kinds of filter and total internal reflection transmission principle.The excitation light source and image acquisition device are set below the chip,and the space saved above the chip is used to set up the automatic pipetting device,which expands the function of the detection platform.The platform uses two wavelengths of excitation light(532 nm and 638 nm)to photograph the target area in the chip with a charge-coupled element(CCD)camera,and combines three kinds of filters to obtain images under multiple channels.In the experiment,fluorescent coded microbeads containing two different dyes in a specific proportion were used in this thesis.A unified microbead surface modification experiment procedure was determined in repeated tests.Different microbead surfaces were combined with different capturing antibodies,so as to capture specific target substances.In addition,a gradient concentration experiment containing various concentrations is designed to obtain the relationship between image fluorescence brightness information and concentration.In the process of image processing and data processing,two kinds of pixel extraction algorithms are designed based on edge detection algorithm,and one of the more efficient algorithms is selected to realize the rapid extraction of fluorescence brightness information from images.According to the luminance distribution characteristics of fluorescence-coded microbeads,the threshold value was set to identify the microbeads.According to the different concentration of the target substance,adopts the data analysis method of segmented processing,and the standard curve between the fluorescence brightness information and the concentration of the target substance was obtained by using four-parameter model fitting.This thesis verifies the detection effect of the detection device and the detection scheme through a variety of comparative experiments.The image results with and without alternating current under white light verified the control effect of DEP on microbeads in the chip.The proportion falling into microholes in the chip increased from 24% to 44.3%,and the chip still had good usability after repeated use.In the simultaneous detection of four cytokines(IL-2,IL-6,IL-10 and TNF-?)in trace samples,the recognition effect of microbeads and the standard curve drawn indicate the effectiveness of the image processing and data processing methods,and the limit of detection(LOD)of detection results all reach the pg/m L level.Flow cytometry was used to compare the detection results,and the accuracy was calculated by comparing the measured concentration and the expected concentration,and the average accuracy was 86.9%,which verified the consistency and accuracy of the device and scheme in this thesis and the flow cytometry.Due to the advantages of the device and the scheme in the multiplicity,rapidity and sensitivity of the detection,the detection scheme can meet the high throughput requirement for rapid simultaneous detection of multiple small molecules(such as cancer or viral infection),and has a broad application prospect.