Research On Integrated Immunoassay Based On Digital Microfluidics

In recent years,microfluidic chip has been widely used in the rapid separation and analysis of life science because of its advantages of portability,low energy consumption,easy preparation and easy operation.Digital microfluidics（DMF）does not rely on 3D fluid channels and external auxiliary equipment such as micro pump,micro valve,etc.,and it is easy to develop integrated devices combined with the measuring parts.So,it is considered as a powerful tool to realize the real-time detection.This paper presents an integrated immune detection method based on digital microfluidic.The theoretical model of driving droplets,the optimized design parameters of chip,the reliability of driving biological reagents,the strategy of biological detection and the factors affecting the detection results are studied.（1）The lumped parameter method is used to establish an equivalent circuit model for the DMF chip and the droplet driving force formula have been derived.Simulation calculations revealed the frequency dependence of the driving force.The influence of the electrical properties of the droplet,the physical properties of the dielectric layer material,and the distance between the upper and lower substrates on the driving force have been studied.The results show that in the low frequency region,the thickness of the dielectric layer has a negative correlation with the driving force,while the dielectric constant of the dielectric layer has a positive correlation with the driving force.In the high frequency region,the driving force is positively correlated with the dielectric constant of the droplet and negatively correlated with the thickness of the dielectric layer.The dynamic equation of the droplet movement is established,and the energy dissipation ratio of the resistance of the droplet is obtained.（2）Optimized the design and preparation of the DMF chip for immunoassay.The electrowetting properties of PZT,Ba Ti O₃/Teflon,SU8,Parylene C and other materials as the dielectric layer have been studied.The contact angle curve with the loading voltage during the electrowetting process have been obtained.The experimental results verify the simulation results that affect the driving force.The performance of the superhydrophobic microstructured dielectric layer of the DMF chip have been studied.The voltages at which the contact angle mutation occurred on micro post and microgroove structured dielectric layer are 300 V and 330 V,respectively.Through comparative analysis of the advantages and disadvantages of various materials,Parylene C is selected as the dielectric layer material of the DMF chip for biological immunoassay.Finally,the DMF chip is prepared by the MEMS microfabrication process,and the DMF on-chip droplet automatic control system have been completed using Lab VIEW software and relay switches.（3）An integrated immunoassay strategy based on DMF have been constructed.Luminol-horseradish peroxidase（HRP）-hydrogen peroxide（H₂O₂）chemiluminescence system have been used to establish human chorionic gonadotropin（HCG）antigen detection methods.A detection system for fluorescence signals have been designed and prepared.In order to verify the reliability of the system,fluorescent microspheres and fluorescently labeled E.coli have been tested.Finally,a linear curve between the sample concentration and the intensity of the luminescence signal is obtained.（4）Optimized of the cleaning method of magnetic microspheres have been designed.The magnetic field force on the magnetic microspheres have been calculated by COMSOL,and its influencing factors have been analyzed.The optimal magnetic field loading position and the concentration of magnetic microspheres have been obtained.Based on the simulation results,the droplets and magnetic microspheres have been successfully separated and resuspended.The cleaning effect of magnetic microspheres have been studied experimentally.Compared with the traditional method,the cleaning efficiency of the method used in this article has been significantly improved.The improvement effect of the surfactant Pluronic L64 on the biofouling have been studied.The influence of surfactant concentration on contact angle and droplet driving life was investigated.The results show that it has the best performance when the surfactant concentration is 0.1%.（5）The automated immunoassay for HCG was carried out using the built-up immunoassay platform based on DMF.The movement of the droplets can be automatically controlled by program.The experimental results show that both the control group and the experimental group have good linearity.It takes less than 20 minutes to complete all immunoassay.Finally,the optimal immunoassay time was determined,and other factors affecting the experimental results were analyzed.Using digital microfluidic technology to realize integrated on-chip immunoassay not only saves reagent cost and time cost,but also avoids the error caused by human operation,which is of great significance in biological detection and medical diagnosis.