Study On Detection Of Two Avian Viruses And Transgenic Protein Cry1Ab By SPR Biosensors

Subgroup J avian leukosis virus(ALV-J) and avian influenza virus H9N2(H9N2 AIV) are two viruses which infect poultry seriously and are responsible for enormous economic losses to the poultry industry in the world including our country. At present, most conventional detection methods of the two avian viruses require long processing period and have high costs, and sometimes the detection easy to produce false positive result. In additon, the gradually increasing cultivation of transgenic crops has raised concerns about the ecosystem, and whether the genetically modified food is harm to the human health has caused a considerable controversy. However, the current detection technologies of the transgenic protein have various disadvantages such as complex procedures, long operation time, high costs, poor repeatability and accuracy. Therefore we have adopted surface plasmon resonance(SPR) biosensors which outstanding features are label-free sample, real-time online monitoring, and sensitive and rapid detection to detect the two avian viruses and transgenic protein Cry1 Ab. Meanwhile, in order to increase the sensitivity of SPR biosensors and make the detection results more accurate, Au/Ag composite nanoparticles and triangular silver nanoplates as the sensitization materials have been introduced to the biosensors. In this article, three experiments were performed for the three kinds of samples.(1) Two SPR biosensors were fabricated for ALV-J detection. The biosensors were based on the functionalized gold substrate and the immobilized Au/Ag composite nanoparticles, respectively. For the first biosensor, the gold substrate was functionalized with a dense self-assembled monolayer(SAM) of 3-mercaptopropionic acid(MPA) through Au-S bonds firstly. Then protein A was modified onto the sensor chip surface via the covalent bonds formed between the amino groups and the activated carboxyl groups of MPA. After being inactivated by ethanolamine, the sensor chip immobilized ALV-J antibody through the combination with protein A. Finally the biosensor realized ALV-J antigen detection. For the second biosensor, 1,6-hexanedithiol(HDT) formed a SAM on the sensor chip surface through Au-S bonds firstly. Then Au/Ag composite nanoparticles were immobilized onto the sensor chip via Au-S and Ag-S bonds. Next the method of biosensor modification of various reagents and detection of ALV-J antigen was as same as the first biosensor. As a result, both of the two biosensors could detect the ALV-J antigen successfully and the lowest analyte concentrations were 659 TCID50·mL–1 and 1054 TCID50·mL–1, respectively. The result suggested that introducing Au/Ag composite nanoparticles to FT-SPR biosensor is beneficial to effectively improve the sensitivity of biosensor. Moreover, the contrast experiment presented that the biosensors showed high specificity for ALV-J detection.(2) A novel SPR biosensor was fabricated for detection of H9N2 AIV. The biosensor was modified orderly by HDT, triangular silver nanoplates, MPA, protein A and H9N2 monoclonal antibody. As a result, the biosensor could detect the whole H9N2 AIV and the lowest analyte concentration was 103 ELD50·mL–1. In addition, there was a linear relationship between the SPR signal and the concentration of lytic H9N2 AIV in the range from 50 to 103 ELD50·mL–1 and the detection limit was 28 ELD50·mL–1(S/N=3). Moreover, the biosensor showed high specificity for H9N2 AIV detection.(3) Two SPR biosensors were fabricated for detection of transgenic protein Cry1 Ab. Sensor 2 was modified by HDT, Au/Ag composite nanoparticles, MPA, and protein A(or not [sensor 1]), with Cry1 Ab monoclonal antibody. As a result, both of the biosensors exhibited satisfactory linear responses in the Cry1 Ab protein concentration ranges of 10 to 500 ng·mL–1 and 8 to 1000 ng·mL–1, and the detection limits were 5.0 and 4.8 ng·mL–1(S/N=3), respectively. In addition, the biosensors possessed good specificity and acceptable reproducibility.