Separation And Concentration Of Pathogenic Microorganisms Based On Immunomagnetophoresis

In recent years,the outbreaks of serious animal diseases caused by high pathogenic avian influenza viruses and foodborne diseases caused by pathogenic bacteria have resulted in considerable economic losses and shown threaten to human's public health.The key to prevent and control these diseases is fast screening of pathogenic microorganisms,which usually includes sample collection,sample pretreatment and biological detection.For sample collection,the national standard methods are often employed and for the biological detection,such traditional or new methods as Polymerase Chain Reaction(PCR),Enzyme Linked Immuno-Sorbent Assay(ELISA),strips and biosensors are often used for fast detection of the microorganisms.However,animal and food samples are generally very complicated and require efficient pretreatment to achieve accurate and reliable results.The common methods,such as filtration and centrifugation,are based on the size and the mass of the targets respectively,which are not capable to specifically separate targets and have obvious limitations on the pretreatment of these kinds of samples.Although the immune magnetic separation method can specifically separate the targets with low enriching times,it still cannot meet the requirements for the pretreatment of the animal and food sample due to very low concentration of the target microorganisms.Based on immune magnetophoresis,lab on a chip and 3D printing,this study aimed to develop a novel method and device for specifically and efficiently separating a small amount of biological targets from a large volume of sample.High pathogenic avian influenza viruses H5N1 and E.coli 0157:H7 were used as research models for concept proof,system opmitization and performance evaluation.The main contents of this study included:(1)Based on the force and motion analysis on the magnetic particles in the magnetophoretic separation,a dynamic model of immune magnetophoresis was built through theoretical derivation,simulation calculation,experimental statistics and instrumental measurement.First,the spacial distribution of the gradient magnetic field were obtained using COMSOL Multiphysics.Then,Monte Carlo method was used to simulate the phenomenon of the magnetic particles' forming into chains at the presence of magnetic field,and fluorescence labelled magnetic particles were used to verify the concept.Finally,the dynamic model based on the force and motion analysis of the magnetic particles in a magnetophoretic system was built.(2)The method for immunomagnetophoretic separation of avian influenza viruses H5N1 was built based on 3D printing and trajectory simulation according to the dynamic model of magnetophoresis.First,the trajectories of the magnetic viruses were simulated and optimized for the development of the magnetophoretic chip.Then,the magnetophoretic chip was fabricated using 3D printing.Finally,the velocity and size of the magnetic particles were optimized,and inactive avian influenza viruses H5N1 were used as research model to evaluate the magnetophorectic system for separation and concentration of avian influenza viruses.The results showed this method could achieve a separation efficiency of more than 88%when the flow rate of the carrier was no more than 120?L/min.(3)The method for immunomagetophoretic separation of E.coli 0157:H7 was also built based on trajectory simulation,3D printing and active magnetic mixing.First,the trajectories of the magnetic bacteria were simulated and combined with an active magnetic mixer for the development of magnetophoretic chip.Then,the magnetophoretic chip was fabricated using 3D printing and the rotating magnetic field was generated using the DC motor to rotate the magnet and thus achieve the mixing.Finally,the flow rate of the carrier and the size of the magnetic particle were optimized and E.coli 0157:H7 was used as research model to evalate the magnetophoretic system for separation and concentration of foodborne pathogenic bacteria.The results showed that this method could achieve a separation of 87%with the mixing time of 20 min and the flow rate of 100?L/min.This study aimed at the practical requirements of the pretreatment of animal and food samples and proposed the dynamic model of magnetophoresis based on immunomagnetic separation,which was used for continuously separating a small number of biological targets from a large volume of sample.The avian influenza viruses H5N1 and E.coli 0157:H7 were used to verify the concenpt with good results,showing that this study could provide a specific separation and efficient concentration method for fast detection of pathogenic microorganisms.