The Application Of Nano Carbon Materials In Electrochemical Immunoassay Of Avian Leukosis Viruses

The mortality rate and attrition rate of poultry are increasing year by year due to the avian leukosis viruses subgroup J(ALVs-J),which led to economic losses and threat to food safety.The traditional detection methods of ALVs-J usually take several days to complete the whole progress and have high costs.Therefore,we focus our study on immunoassay which has advantages such as low consumption,high sensitivity,rapidness,low cost,easy operation and etc.Several different immunosensors were developed based on nano carbon materials like N-doped Porous Carbon(PCN),graphitic carbon nitride(g-C3N4),nanocrystalline cellulose(NCC)to detect ALVs-J with ultra-sensitivity.This paper is mainly divided into four parts as following:(1)A new sandwich-type electrochemical immunosensor was fabricated to detect the subgroup J of avian leukosis viruses(ALVs-J)by employing the Au nanoparticles(AuNPs)and concanavalin A(Con A)as the platform and Pt nanoparticles functioned N-doped porous carbon material(Pt/PCN)as the label.According to the previous report of the researchers from our group,the prepared Pt/PCN was found to have much higher catalytic activity than the individual Pt nanoparticles or the PCN.The utilization of the Pt/PCN greatly improved the sensitivity of the immunosensor.The immunosensor was constructed based on the excellent peroxide enzyme mimicking property of the Pt/PCN,which can catalyze the reaction of hydroquinone.Under the optimum conditions,the reduction peak currents of the obtained benzoquinone were proportional to the ALVs-J concentration over the range from 102.02 to 104.30 TCID50/mL with a detection limit of 107 TCID50/mL(S/N=3).The electrochemical immunosensor also showed good selectivity,reproducibility and stability.(2)A novel sandwich-type electrochemical immunosensor was fabricated for ultrasensitive detection of ALVs-J based on the composite of Au nanoparticles and graphitic carbon nitride(Au NPs/g-C3N4).The detection sensitivity was highly improved by the triple amplification strategy.The employment of Au NPs/g-C3N4 increased the surface area of electrode to capture more primary antibodies(Ab1);Au NPs can catalyze glucose from the detecting solution into hydrogen peroxide and gluconic acid for the oxidase mimicking property;the conjugated horseradish peroxidase(HRP)exhibited extraordinary electrochemical biocatalysis towards toluidine blue(Tb).The analytical properties of various immunosensor platforms and labels were compared in detail to prove the signal amplification property of this protocol.Under the optimum conditions,the oxidation peak currents of Tb were proportional to the ALVs-J concentration over the range from 102.22 to 104.30 TCID50/mL with a detection limit of 122 TCID50/mL(S/N=3).The electrochemical immunosensor also showed good selectivity,reproducibility and stability.(3)A novel electrochemical immunosensor was fabricated for ultrasensitive detection of ALVs-J by employing functional graphene as the platform and the complex of Au nanoparticles and nanocrystalline cellulose as the label.Graphene has the ability to capture more toluidine blue as well as the primary antibodies(Ab1);In the presence of hydrogen peroxidase,the conjugated horseradish peroxidase(HRP)exhibited extraordinary electrochemical biocatalysis towards Tb.The analytical properties of various immunosensor labels were compared in detail to prove the signal amplification property of this protocol.Under the optimum conditions,the reduction peak currents of Tb were proportional to the ALVs-J concentration over the range from 102.08 to 104.00 TCID50/mL with a detection limit of 116 TCID50/mL(S/N=3).The electrochemical immunosensor also showed good selectivity,reproducibility and stability.(4)An alternative imunosensor for ultrasensitive detection of ALVs-J was designed utilizing the self-enhanced electrochemiluminescence(ECL)of a novel Ru(Ⅱ)complex functionalized nanocrystalline cellulose as signal label.The desirable self-enhanced ECL luminophore was achieved by the combination of the coreactant polyamidoamine(PAMAM)and the luminophor bis(2,2’-bipyridine)-4’-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate).Compared with the common intermolecular ECL reactions,the intramolecular reaction has shown improved luminous efficiency due to the shorter electron-transfer path and less energy loss.What’s more,the employment of Au-PAMAM can greatly increase the loading amount of Ab1 and Ru.Under the optimum conditions,the ECL intensity was proportional to the ALVs-J concentration over the range from 102.22 to 104.30 TCID50/mL with a detection limit of 101 TCID50/mL(S/N=3).The electrochemiluminescence immunosensor also showed good selectivity,reproducibility and stability.