The Research Of Electrochemical Immunoassay By Organic-Inorganic Hybrid Nanomaterials Double Signal Amplification Detecting Avian Leukosis Viruses

In recent years,subgroup J of avian leukosis virus?ALV-J?is the main virus which has caused the death and elimination rate of poultry increased by years,causing economic losses and endangering food safety.Highly sensitive detection of leukemia virus?ALV-J?is important.Accordingly,highly sensitive methods need to be developed for the detection of ALV-J.Electrochemical immunosensors with sandwich-type structures are growing in popularity due to their numerous advantages which include low cost,high selectivity and sensitivity,fast analysis times and good reproducibility.Finding appropriate organic-inorganic hybrid nanomaterials and amplifying detection signal are the keys to improve the sensitivity of the immunosensor.In this work,we fabricated three kinds of immunosensors including electrochemicalimmunosensor,electrochemiluminescenceimmunosensorand photoelectrochemical immunosensor,using zeolitic imidazolate framework?ZIF-8?,nanocellulose?NC?,hollow MnO2?hMnO2?and black TiO2?bTiO2?to detect ALV-J.This paper is divided into four parts:?1?A novel sandwich-type electrochemical immunosensor for avian leukosis virus subgroup J?ALV-J?is described.The immunosensor was prepared by first modifying a glassy carbon electrode?GCE?with reduced graphene oxide that was functionalized with tannic acid and magnetite nanoparticles?rGO-TA-Fe3O4?.Primary antibodies?Ab1?were then deposited on the modified GCE.Hollow zeolitic imidazolate framework?eZIF?crystals functionalized with tannic acid and carrying secondary antibodies?Ab₂?and horseradish peroxidase?HRP?were used for signal amplification.The hollow eZIF crystals were found to be an excellent carrier for both Ab₂ and HRP,prompting the wider use of metal organic frameworks in electrochemical sensing.Under optimal conditions,the immunoassay afforded a detection range from 10^2.18 to10^4.0 TCID₅₀ mL^-1(where TCID₅₀ is the 50%tissue culture infective dose)and a low detection limit of 10^2.15 TCID₅₀ mL^-1?at S/N=3?.The immunoassay is highly selective for ALV-J,and it demonstrates excellent reproducibility and operational stability.?2?A sensitive sandwich-type electrochemical immunosensor was developed for the detection of ALV-J,which benefitted from multiple signal amplification involving graphene-perylene-3,4,9,10-tetracarboxylic acid nanocomposites?GR-PTCA?,nanocellulose-Au NP composites?NC-Au?and the alkaline phosphatase?ALP?catalytic reaction.GR-PTCA nanocomposites on glassy carbon electrodes served as the immunosensor platform.Due to their excellent electrical conductivity and abundant polycarboxylic sites,the GR-PTCA nanocomposites allowed fast electron transfer and good immobilization of primary antibodies,thereby affording a strong immunosensor signal in the presence of ALV-J.The detected signal could be further amplified by the introduction of NC-Au composites as a carrier of secondary antibodies?Ab₂?and by harnessing the catalytic properties of Au and ALP.Under optimized testing conditions,the electrochemical immunosensor displayed excellent analytical performance for the detection of ALV-J,showing a linear current response from 10^2.08 to 10^4.0TCID₅₀/mL with a low detection limit of 10^1.98 TCID₅₀/mL?S/N=3?.In addition to high sensitivity,the immunosensor showed very good selectivity,reproducibility and operational stability.?3?A novel pH-responsive sandwich-type electrochemiluminescence immunosensor using hollow MnO2?hMnO2?nanospheres encapsulating Ru?bpy?3Cl2 as carrier was successfully developed for the detection of avian leukosis virus subgroup J?ALV-J?.The immunosensor consisted of graphene oxide functionalized with tannic acid?rGO-TA?and primary antibodies?Ab₁?as the main ALV-J sensing platform.For signal amplification,pH-responsive hollow MnO₂?hMnO₂?nanospheres encapsulating Ru?bpy?₃Cl₂ and carrying secondary antibodies?Ab2?were employed.Under optimal conditions,the immunosensor exhibited a wide ALV-J detection range of 10^1.80 to 10^4.30 TCID₅₀/mL and a low detection limit of 10^1.71 TCID₅₀/mL?S/N=3?.Importantly,the immunosensor showed high sensitivity,good reproducibility and excellent operational stability.?4?A novel sandwich-type photoelectrochemical immunosensor using black TiO₂-THPP composites as platform was successfully fabricated for the detection of ALV-J.THPP can promote the photoelectric response of black titanium dioxide.The antibody is immobilized on the surface of the ITO electrode by MDI.The nano gold?Au?carrying secondary antibodies?Ab₂?and horseradish peroxidase?HRP?catalyzing the reaction of hydrogen peroxide?H₂O₂?and hydroquinone?QH₂?were used for signal amplification to detect ALV-J.Under optimal conditions,the immunosensor exhibited a wide ALV-J detection range of 10^1.7 to 10^4.5TCID₅₀/mL and a low detection limit of 10¹.62.62 TCID₅₀/mL?S/N=3?.Importantly,the immunosensor showed high sensitivity,good reproducibility and excellent operational stability.