Construction Of Magnetic Mesoporous Silicon Nanoprobe And Its Research On Detection Of Newcastle Disease Virus

Newcastle disease(ND),also known as Asian fowl fever or pseudofowl fever,is an acute,febrile and highly infectious disease of poultry caused by NDV virus.It is mainly characterized by dyspnea and neurological symptoms,and serous and mucosal bleeding.In recent years,although the detection method of NDV has been continuously updated.Large-scale integrated farming models require more sensitive or faster detection techniques to prevent the presence of ND in poultry farming environments Conventional methods for NDV detection mainly include pathogen identification,PCR,RT-PCR,LAMP and ELISA.These detection techniques have their own advantages and disadvantages:etiological diagnosis is the gold standard for detecting NDV,but this method has long detection time and high cost.PCR is highly sensitive,but it requires nucleic acid extraction from samples,which requires high technical requirements for experimentalists and makes samples easy to be contaminated.LAMP,an isothermal amplification technology,is time-saving compared to trational PCR but the amplification efficiency is not so good and the sensitivity is much lower than PCR,therefore it has not been widely used.ELISA is sensitive and easy to handle for assay of large quantities of samples.However,due to its need for several incubations,therefore resulting in a relatively long detection time and non-specific adsorption that is difficult to completely avoid.Therefore,ELISA experiments often need to be operated several times to get better results.Magnetic mesoporous silica nanoparticle(M-MSN)has many outstanding merits,such as large pore volume(up to 2.5 cm3/g),high surface area(>1000 m2/g),customizable pore size,easy-to-be functionalized,biodegradable,highly active,good stability and good biocompatibility.And it has been used in biological detection,sensing as well as drug loading systems.In this study,using the loadability and modifiability of mesoporous silica,magnetic mesoporous silica nano-immune probe was prepared to replace the primary antibody incubation and enzyme-labeled secondary antibody incubation in ELISA technology.In the next step of the experiment,based on the enzyme-carrying magnetic mesoporous silica nano-immune probe was constructed.In brief:First,a kind of magnetic mesoporous silica nanoparticles with a particle size of?60 nm and aperture of?5 nm were choiced.PODs with a molecular weight of 44 kDa were mechanically diffused into the holes of mesoporous M-MSNs by mixing them in a solution system.Then,the surface of M-MSNs were coated with positive polymer Polyethylenimine(PEI)to make the POD molecules reside in pores of the M-MSNs.Then,the antibody was modified with negatively charged DNA to increase the electronegativity of the antibody.DNA modified antibody attaches PEI on the surface of M-MSNs by electrostatic adsorption to be the M-MSNs immunoprobe.DNA-functionalized antibodies were obtained by chemically clicking reaction between single strand of DNAs and NDV antibodies in a "one-to-one" form.SDS-PAGE electrophoresis,thermogravimetric analysis,DLS analysis and transmission electron microscopy were used to analyze and verify the successful preparation of the nanoprobes.The results showed that the mesopore of the enzyme-loaded mesoporous silica nano-immunoprobe was successfully loaded with?400 POD molecules,and the nanoprobe successfully used PEI to "encapsulate" the mesoporous structure to prevent POD leak.Through SDS-PAGE and the potential changes on the surface of the nanoprobe,it can be known that the DNA functionalized antibody is successfully modified on the surface of the nanoprobe,and there are about-200 antibody molecules on the surface of each nanoparticle.Subsequently,the probe was stored at 4? for two months,and the results of investigating the stability of the enzyme activity in the probe showed that the activity of the magnetic mesoporous silica nanoprobe POD remained stable,and the probe had no enzyme leakage.This shows that the enzyme-carrying mesoporous silica immunonanoprobe was successfully constructed.Next,we use the magnetic mesoporous silica nanoprobe constructed in this study to detect purified NDV samples.The results show that the probe is specific and the detection sensitivity reaches 102 PFU/100?L.At the same time,the traditional ELISA method is used for parallel detection for compare sensitivity and detection time.During the construction of the detection system,the PCR method was used to verify the accuracy and consistency of the detection results of the probe method,and the transmission electron microscope(TEM)characterization was used to verify the ability of the enzyme-carrying mesoporous silica nano-immune probe to capture the virus.result.Finally,the magnetic mesoporous silica nanoprobe method constructed in this paper was used to detect cloaca swab samples of birds infected with NDV.Simultaneously,several traditional ELISA methods were used to verify the results.The results found that the sensitivity of the ELISA method based on enzyme-carrying mesoporous silicon immunoprobe constructed in this study higher than traditional ELISA,and the detection time is shorter,which proves that this method can be used for clinical detection.In summary,technology on enzyme-loaded M-MSN is successfully constructed in this study,which is 10 times more sensitive than the traditional ELISA.Compared with indirect ELISA,the detection time is shortened.This technique is promising to replace the traditional ELISA method and be widely used in various biological laboratories and testing centers.