Experimental Study On Specific Detection Of Herpes Simplex Virus By Magnetic Upconversion Fluorescent Nanospheres

Objectives A new method will be designed to rapidly and sensitively detect the herpes virus,which is based on the fluorescent and magnetic encoding nanospheres.Polystyrene seeds were synthesized by dispersion polymerization.Prepare the porous carboxylated polystyrene nanoospheres by Seed Polymerization.High-temperature swelling method was applied to prepare multifunctional magnetic upconversion fluorescent encoding microspheres simultaneously with robust fluorescent intensity and stable magnetic performance.Furthermore,multifunctional MNP-UCN-encoded nanospheres were used to detect herpes simplex virus 1(HSV-1),in order to make the detection convenient rapid and sensitive.The novel method will undoubtedly facilitate the clinical diagnosis and prosper the personalizedmedicine in periodontitis treatment.Methods Dispersion polymerization method was used to synthesize the polystyrene seeds.Prepare the porous carboxylated polystyrene nanoospheres by Seed Polymerization.High-temperature swelling method was applied to prepare multifunctional magnetic upconversion fluorescent encoding microsphereswith robust fluorescent intensity and stable magnetic performance.Herpes simplex virus 1 were cultured in HELA cells,then the TCID50 of the selected virus were determined by Reed-Muench method.The nanospheres carboxyl groups(–COOH)were activated to be conjugated with HSV-1 specific antibodies.After modification with antibodies,nanospheres were added to the virus suspension of HSV-1.Then,the rhodamine-labeled antibodies were added to the suspension.Detect the virus by double antibody sandwich method.Experiment designed to verify the specificity of magnetic upconversion nanospheres and to explore the minimum detectable virus concentration.Results The polystyrene microspheres were homogeneous in size and smaller than 5?m in diameter.The magnetic upconversion nanoparticles were obtained by encapsulating the magnetic particles and the upconversion particles in polystyrene seeds with the high-temperature swelling method.The nanoparticles did not polymerize,possessed both magnetic and upconversion fluorescence properties.The herpesviruses with biological activity were obtained by culturing in HELA cells,and the collected herpes virus solution was assayed by Reed-Muench method at a concentration of 10-4/ 0.1ml TCID50.The magnetic up-conversion nanospheres combined with the monoclonal antibody to HSV-1 is specific for HSV-1,which can specifically bind to HSV-1 and display the purple color under fluorescent microscopy with rhodamine-labeled secondary antibody reaction.By detecting HSV-1 at different gradient concentrations,HSV-1 was still detected when only 10TCID50 HSV-1 was present in the virus solution.Conclusion HSV-1 can be specifically detected by double-antibody sandwich method by connecting magnetic upconversion nanospheres and HSV-1 monoclonal antibody.Through the magnetism of nanoparticles,rapid separate the object,and its color reaction was found quickly and easily in fluorescence microscopy.Electron microscopy showed that the smooth surface of magnetic nanospheres,which were attached to antibody,became rough,and could connect with virus particles.Specific microspheres were reacted with HSV-1 and VZV respectively.The results showed that there was a specific color reaction only in HSV-1 group,indicating that monoclonal antibody-bound nanospheres has specificity.By detecting HSV-1 at different gradient concentrations,HSV-1 was still detected when only 10 TCID50HSV-1 was present in the virus solution.