Study On Phosphothreonine Lyase Electrochemical Immunosensor Based On Gold Nanoparticles

Novel electrochemical nano-gold particles biosensors for salmonella and shigellabased on electrochemical detection analysis which could achieve automation detection,free the effects of sample color and turbidity, and simple equipments were needed; theefficiency of enzymatic reaction; and immunoassay which pretreatments such asenrichment and purification were needed were prepared for food safety. Covalentbonding, electrostatic adsorption and self-assembled were used to immobilizebiomolecules onto the glassy carbon electrode combined the advantages of chitosanand gold nanoparticles.And we choose a anti-SpvC monoclonal antibody strain(Salmonella antigenpreparation) which has the stongest affinity with purified protein SpvC(phosphothreonine lyase) from five anti-SpvC monoclonal antibody strains by Biomolecularinteraction analyzer means (Octet biolayer interferometry), the equilibriumdissociation constant KD is3.85E-10M, the binding constant and dissociation rateconstant Kdis and Kon are respective3.73.E+03L/mol s-1and9.62E-07s-.A novel SpvC immunosensor based on double-layer gold nanoparticles has been developed,which the first layer of gold nanoparticles fixed on glassy carbon electrode by the chitosan, andthe monoclonal antibody of SpvC was adsorbed; the second layer of gold nanoparticals was fixedby droping electrode with thionine-chitosan/NG-HRP complex. The assembly procedures weremonitored by cyclic voltammetric and alternating current impedance. The chronoamperometrywas used to measure salmonella and shigella.Based on the amplification system of hydrogen peroxide biosensor based on horseradishperoxidase/GNPs-thionine/chitosan, the electrochemical immunosensor for anti-SpvCmonoclonal antibody will be fabricated using double-layer gold nanoparticles and chitosan. The sensing element of the immunosensor was comprised of gold nanoparticles fixed with the SpvCmonoclonal antibodies. To examine the quality of the synthesized gold nanoparticles, the size,shape and dispersion of the gold nanoparticles will be characterized by transmission electronmicroscopy (TEM), atomic force microscope (AFM) and UV–visible spectral scanning. Theinteractional affinities between the monoclonal antibodies and SpvC will be determined andoptimized by BioLayer Interferometry (ForteBio).The chronoamperometry was used to measure salmonella and shigella. The SpvCimmunosensor linear range of detection is form1×101to1×104cfu/mL with acorrelation coefficient of0.9913, while the detection limit up to10cfu/mL accordingto three times of the blank value standard deviation. And the preliminary practicalapplication was studied what could lay the foundation for dairy quality control.