Construction Of FT-SPR Sensitization Sensor Based On Nanomaterials And Its Application In PRRSV Detection

Fourier Transform Surface Plasmon Resonance(FT-SPR)biosensor is a new type of high-sensitivity and selective detection of biosensor analysis technology,which changes the refractive index of incident light through the combination of the test object and the surface of the sensor chip,it is mainly used in various fields such as analysis and detection,biology,pharmacology and environmental research;medical nanomaterials are widely used in preclinical basic medical research due to their good biocompatibility and special biological effects;Au nanocluster(AuNCs)has the advantages of small size,good optical properties and controllable,good stability and biocompatibility,and is often used in analysis and testing and basic research in the field of biomedicine.Graphene Oxide(GO)is rich in functional groups such as carboxyl,hydroxyl,epoxy,etc.,which increases its modification ability and has good biocompatibility.Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease caused by porcine blue ear disease virus(PRRSV).At present,the detection of this virus mainly revolves around nucleic acid detection,but it has disadvantages such as high false negatives time-consuming and failure to realize direct detection of undamaged samples..Based on it,our project selected different functional peptides as templates to synthesize four functional Au nanoclusters,and the prepared Au nanoclusters and graphene oxide were applied to the sensitization analysis of SPR to realize the rapid detection of PRRSV.In addition,the experiment also preliminarily studied the antibacterial effect of synthetic clusters.The specific contents are as follows:In the first part,using peptides with different chain lengths as templates,changing the preparation conditions and synthesizing gold nanoclusters modified by different peptide ligands(GSH-AuNCs,CV-AuNCs,CY-AuNCs,BSA-AuNCs),the physical and chemical properties of the material were characterized by super-resolution transmission electron microscopy(HR-TEM),fluorescence spectroscopy,ultraviolet spectroscopy,and dynamic light scattering(DLS).Through the analysis of the catalytic activity of cluster materials to TMB,the enzyme-like catalytic activity of the materials was studied.The biosafety of the material was studied by hemolysis test and cell CCK-8 activity test.Finally,an SPR biosensor was constructed to analyze the sensitization effect of different materials.The results show that the prepared functional gold clusters were all smaller than 2 nm,and the distribution was relatively uniform.They emitted strong fluorescence under ultraviolet light,had good stability and peroxidase-like activity.Through hemolysis test and CCK-8 toxicity test analysis,it has good biological safety.In the analysis of the sensitization effect of the chain length effect on the SPR biosensor,Au nanoclusters have obvious size dependence and are closely related to the chain length of ligands.The experimental results showed that the sensitization effect of GSH-AuNCs was not obvious,the sensitization effect of CY-AuNCs,CV-AuNCs and BSA-AuNCs were 1.32,1.56 and 2times,respectively,and the reproducibility was good.The experimental results show that the sensitization effect becomes better and better with the growth of peptide chain,and indirectly proves the weak surface plasmon resonance effect of clusters.The experimental results provide a new idea and theory for studying the interaction between targeted cluster materials and target analytes by changing the ligands of gold cluster materials.In the second part,the SPR sensitization effect of graphene oxide was studied.The morphology and structure of graphene oxide were characterized by scanning electron microscope(SEM)and infrared spectroscopy(IR).In order to explore the influence of modification conditions in the process of constructing SPR biosensor,the effects of adding mode,concentration and time of graphene oxide were explored in this experiment.Under the best experimental conditions,the sensitizing effect of gold nanoclusters was compared with that of previously prepared gold nanoclusters.Finally,under the optimized experimental conditions,the real-time on-line detection of PRRSV was realized by graphene oxide SPR biosensor modified by targeted peptide,and the detection effect was compared with that of CY peptide targeted PRRSV.The results showed that the graphene oxide was flaky and and the surface was rich in functional groups such as oxygen-containing hydroxyl,carboxyl,and epoxy groups.It has good biocompatibility,and the best sensitization condition of the gold sheet was soaked in2.0 g/L graphene oxide for 5 h.And compared with the sensitization effect of Au nanoclusters,it is found that the sensitization effect of graphene oxide is higher than Au nanoclusters.The modification of graphene oxide can increase the signal of the SPR sensor by about 2.9 times,and there was a good linear relationship between the concentration of papain and the wavenumber of SPR biosensor in the range of 100-500 mg·L^-1.The linear equation was y=0.306x+91.8,and the linear correlation coefficient was R²=0.99438.Applying it to the detection of PRRSV,the results show that the sensitization effect is about 2.8 times,which is basically in line with the linear correlation coefficient.A new method for rapid and real-time detection of PRRSV using SPR sensors has been established.In the third part,CV-AuNCs with blue fluorescence were synthesized using functional peptides and applied to antibacterial experiments.The physicochemical properties of the materials were characterized by fluorescence,ultraviolet,and infrared spectroscopy,and the biological safety of the materials was preliminarily evaluated through hemolysis experiments.The influence of the concentration on the antibacterial effect was studied by changing the incubation concentration.The results showed that the maximum excitation of CV-AuNCs was at 320 nm,the maximum emission was at 400 nm,and it had good biological safety and stability.The gold clusters of 100?M,250?M and 500?M were applied to the antibacterial activity experiments of Staphylococcus aureus(S.aureus)and methicillin-resistant Staphylococcus aureus(MRSA),respectively.The results show that in vitro culture,CV-AuNCs had inhibitory effect on S.aureus and MRSA,and and with the concentration of Au cluster increases,the antibacterial effect is more obvious.And through the calculation of the antibacterial rate,when the material concentration reached 500?M,the antibacterial rate of the two bacteria was greater than 50%,It indicated that the experimental synthesis of CV-AuNCs(blue)had a good bacteriostatic effect on S.aureus and MRSA bacteria.The experiment provides a new method for studying new antibacterial materials.