The Research Of Detecting Avian Leukosis Viruses Based On Manganese Nanomaterials Signal Amplification Immunosensor

In recent years,the global outbreak of avian leukosis viruses subgroup J?ALV-J?has led to massive poultry deaths,causing huge economic losses to the poultry industry.However,there is no vaccine or drug for the treatment of ALV-J.It is important to find simple,efficient and sensitive ways to detect ALV-J.Immunosensor analysis is a detection technology combining immunoassay and sensors,which has the advantages of high sensitivity,fast response speed,simple operation and so on.Immunosensor provides an effective method for detection of ALV-J.In the construction of immunosensor,the use of excellent nanomaterials with good biocompatibility and large specific surface area to improve the load of signal molecules is the key to realize signal amplification and improve signal detection sensitivity.Manganese is a kind of element which widely exists in nature.Since its discovery,manganese and its compounds have been widely used in the production and life of human beings.In this paper,based on manganese-based nanomaterials,the properties of nanomaterials such as hollow manganese dioxide,three-dimensional flower clustered manganese dioxide,and spherical manganese carbonate were studied.Three kinds of sandwich type immunosensor were prepared by using these materials as signal probes or carriers.The effects on the sensor detection signal amplification was studied,and the sensitive detection of avian leukemia virus was realized.This paper is mainly divided into the following three parts:?1?A novel sandwich electrochemical immunosensor based on hollow manganese dioxide nanocomposite material?H-MnO₂?was fabricated for detecting avian leukosis virus subgroup J?ALV-J?.The sandwich electrochemical immunosensor was prepared by using the graphene oxide reduction with tannin?GR-TA?as the substrate to connect the primary antibody?Ab₁?,and the signal molecule was introduced into the sensor through hollow manganese dioxide coated copper nanoparticles as the carry of Ab₂.The nano-copper was released by changing the pH of the solution,and the stripping voltammetric current of copper was used as the detection signal to achieve the highly sensitive detection of avian leukemia virus.The using of GR-TA as electrode modification material can increase the amount of Ab₁,while enhancing the conductivity.Hollow structure of the MnO₂ increased the loading of the signal molecule,thereby amplifying the detection signal and enhancing the detection sensitivity.Under the optimal experimental conditions,high sensitive detection of ALV-J was achieved by the DPV.The linear range was 10^2.0210^4.5 TCID₅₀/mL,with the detection limit of 10^1.92 TCID₅₀/mL?S/N=3?.In addition,the sensor has the good selectivity,reproducibility and stability,which can be used for quantitative detection of ALV-J.?2?In order to further improve the detection sensitivity of the sensor,a novel sandwich-type electroluminescent immunosensor was constructed using the nano-composite materials of manganese dioxide and palladium nanoparticles for the highly sensitive detection of ALV-J.The GCE electrode was modified with nano gold?Au NPs?to fixed the primary antibody.And MnO₂-PEI-Pd complex was prepared as the carrier of luminescent Ru?bpy?₃²⁺and Ab₂ to form the signal probe.The detection of ALV-J was carried out in the solution containing potassium persulfate through the specific reaction between antigen and antibody.Three-dimensional flower-cluster manganese dioxide?3D-MnO₂?has a large specific surface area,which can increase the load of signal molecule Ru.In order to further amplify the signal and improve the detection sensitivity,the palladium nanoparticles?Pd NPs?were combined with 3D-MnO₂ to effectively catalyze the ECL reaction while increasing the amount of antibody binding.The introduction of palladium nanoparticles?Pd NPs?increased the binding of Ab₂ and effectively catalyzed the ECL reaction,leading to the signal amplification.Under the best experimental conditions,the immunosensor enab led a good linear response against ALV-J concentration from 10^1.98-10^4.30 TCID₅₀/mL with the low detection limit of 10^1.90TCID₅₀/mL?S/N=3?and the correlation coefficient of 0.994.In the same time,the immunosensor showed the characteristics of high stability,good selectivity and reproducibility for detection of ALV-J,providing an effective tool for clinical detection of ALV-J.?3?A sandwich-type electroluminescence immunosensor was constructed using manganese carbonate?MnCO₃?as a new type of electroluminescence material,which simplified the process of sensor construction and achieved sensitive detection of ALV-J.Firstly,the dendritic polymer-nanometer gold?PAMAM-Au?complexes was prepared as electrode modified materials.Then MnCO₃ was making as the luminescent material.After modified with PEI,the MnCO₃ was combining Ab₂ to form the signal probe.The ECL test was carried out in the solution containing glucose.To achieving the signal amplification,PAMAM-Au was used to increase the catch of Ab₁.And the addition of glucose can catalyze the activation of K₂S₂O₈ to amplify the reaction signal.The composite materials were characterized by SEM,XRD,and the electrode modification process was characterized by CV.The feasibility of the experiment,the optimization of experimental conditions and the quantitative detection of ALV-J were completed by ECL technology.The results showed that the immunosensor exhibited a good linear response with the wide range concentration from10^1.98-10^4.30 TCID₅₀/mL,and the detection limit was 10¹.88.88 TCID50/mL?S/N=3?.In addition,the sensor also has excellent selectivity,reproducibility and stability,indicating far-reaching significance for clinical application.