磁性上转换荧光编码微球特异性检测单纯疱疹病毒的实验研究
发布时间：2019-04-19 12:02
【摘要】：目的本实验拟探讨一种基于磁性上转换纳米粒子能够快速特异性检测疱疹病毒的新方法,使用分散聚合法制备聚苯乙烯种子微球,利用种子聚合法制备多孔羧基化聚苯乙烯微球,采用高温熔胀法将上转换纳米粒子(upconversion nanoparticles,UCNs)NaYF4:Yb,Tm和磁纳米粒子(magnetic nanoparticles,MNPs)包埋至多孔聚苯乙烯微球中,从而制得一种磁响应性能和荧光强度最佳的磁性上转换荧光编码微球,并且能运用于单纯疱疹病毒1型(herpes simplex virus1,HSV-1)检测的磁性上转换荧光纳米材料,实现操作简单、快速,准确度高的目的。旨在为临床椅旁检验、精准个性化医疗提供实验基础。方法实验使用分散聚合法制备聚苯乙烯种子微球,利用种子聚合法制备多孔羧基化聚苯乙烯微球,再采用高温熔胀法将上转换(UCNs)NaYF4:Yb,Tm纳米粒子和磁纳米粒子(MNPs)包埋至多孔聚苯乙烯微球中,获得磁响应性能和荧光强度最佳的磁性上转换荧光编码微球。利用HELA细胞培养单纯疱疹病毒1型并利用Reed-Muench两氏法确定所培养的HSV-1的半数细胞培养物感染量TCID50。活化微球表面羧基基团,HSV-1的单克隆抗体与磁性上转换荧光编码微球偶联,加入病毒待检液,再用标记罗丹明染料的二抗与之反应,双抗夹心法检测病毒。验证磁性上转换微球的特异性,探究其能检出的最低病毒浓度。结果实验制得的聚苯乙烯微球大小均一,粒径小于5μm,高温溶胀法将磁粒子和上转换粒子包入其中后制得磁性上转换纳米粒子,纳米粒子无聚合现象。所制得的磁性上转换纳米微球同时具有磁性和上转换荧光粒子特性。通过HELA细胞培养病毒的方法获得了具有生物活性的疱疹病毒,收集疱疹病毒溶液通过Reed-Muench两氏法测定,浓度为10-4/0.1mlTCID50。磁性上转换纳米子结合HSV-1的单克隆抗体后具有特异性,能特异性结合HSV-1后与罗丹明标记的二抗反应荧光显微镜下显示紫色。通过检测不同梯度浓度的HSV-1发现当病毒液中仅有10TCID50HSV-1时仍可检出其中的HSV-1。结论通过连接磁性上转换纳米粒子和HSV-1单克隆抗体后利用双抗夹心法能特异性检出HSV-1,并通过纳米粒子的磁性,快速分离待检物,通过其上转换特性在荧光显微镜下快速简便的发现其显色反应。电镜下显示连接抗体的磁性上转换纳米微球表面由光滑变得粗糙,且能够连接病毒颗粒。特异性纳米微球分别与含有HSV-1、带状疱疹病毒(varicella-zoster virus,VZV)和牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)的三组待检溶液反应,结果显示仅在HSV-1组有特异性显色反应,表明连接了单克隆抗体的微球具有特异性。通过检测不同浓度的HSV-1病毒液,发现其能检出10TCID50HSV-1病毒液中的HSV-1,表明此方法在病毒液中仅含10TCID50HSV-1时仍能检测出HSV-1的存在。
[Abstract]:Objective to explore a new method based on magnetic upconversion nanoparticles for rapid and specific detection of herpesvirus, and to prepare polystyrene seed microspheres by dispersion polymerization. Porous carboxylated polystyrene microspheres were prepared by seed polymerization. The upconversion nanoparticles (upconversion nanoparticles,UCNs) NaYF4:Yb,Tm and magnetic nanoparticles (magnetic nanoparticles,MNPs) were embedded in porous polystyrene microspheres by high temperature melt expansion method. Thus, a magnetic up-conversion fluorescent nanoparticle with the best magnetic response and fluorescence intensity was prepared, and it can be used in the detection of herpes simplex virus type 1 (herpes simplex virus1,HSV-1) by magnetic up-conversion fluorescent nanomaterials, the operation is simple, and the performance of the magnetic up-conversion fluorescent nanospheres is simple. Fast, high accuracy of the purpose. The purpose of this paper is to provide experimental basis for clinical chair-side examination and accurate personalized medical treatment. Methods the seed microspheres of poly (styrene) were prepared by dispersion polymerization method, and the porous carboxy polystyrene microspheres were prepared by seed polymerization method. Then the upconversion of (UCNs) NaYF4:Yb, was carried out by high temperature melt expansion method. Tm nanoparticles and magnetic nanoparticles (MNPs) were embedded in porous polystyrene microspheres, and the magnetic up-conversion fluorescent coded microspheres with the best magnetic response and fluorescence intensity were obtained. Herpes simplex virus type 1 (HSV-1) was cultured with HELA cells and TCID50. of 50% cell culture medium of cultured HSV-1 was determined by Reed-Muench 's two-way method. The surface carboxyl groups of activated microspheres, the monoclonal antibody against HSV-1 were coupled with magnetic upconversion fluorescent coded microspheres, the virus was added into the solution, then the second antibody labeled with Rhodamine dye was used to react with it, and the virus was detected by double antibody sandwich method. To verify the specificity of magnetic up-conversion microspheres, to explore the lowest virus concentration it can detect. Results the size of the polystyrene microspheres was uniform and the particle size was less than 5 渭 m. Magnetic particles and upconversion particles were encapsulated in the microspheres by high temperature swelling method, and the magnetic up-conversion nanoparticles were prepared. The nano-particles did not polymerize. The magnetic up-conversion nanospheres have both magnetic and upconversion fluorescence particle properties. The herpesvirus with biological activity was obtained by HELA cell culture method. The herpesvirus solution was determined by Reed-Muench two-way method with the concentration of 10 ~ 4 脳 0.1 ml TCID _ (50). The magnetic up-conversion nanoson-binding monoclonal antibody to HSV-1 is specific. It can specifically bind HSV-1 to Rhodamine-labeled second antibody and show purple under fluorescence microscope. It was found that only 10 TCID _ (50) HSV-1 could be detected in the HSV-1 of different gradient concentrations of the virus. The HSV-1. could still be detected in the virus solution. Conclusion by connecting magnetic up-conversion nanoparticles and monoclonal antibodies to HSV-1, HSV-1, can be detected specifically by double-antibody sandwich method, and the samples to be detected can be separated quickly by magnetic properties of nanoparticles. Through its up-conversion property, the color reaction was found quickly and simply under the fluorescence microscope. Electron microscopy showed that the surface of the antibody-linked magnetic up-conversion nanospheres changed from smooth to coarse and could connect virus particles. The specific nanospheres reacted with three groups of solutions containing HSV-1, zoster virus (varicella-zoster virus,VZV) and Porphyromonas gingivalis (Porphyromonas gingivalis,Pg) respectively. The results showed that there was only a specific color reaction in HSV-1 group. The results showed that the microsphere with monoclonal antibody was specific. Through the detection of different concentrations of HSV-1 virological fluid, it was found that it could detect the HSV-1, in 10TCID50HSV-1 viral fluid. It indicated that this method could still detect the presence of HSV-1 in the viral liquid containing only 10 TCID _ (50) HSV ~ (- 1).
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4
本文编号：2460927
[Abstract]:Objective to explore a new method based on magnetic upconversion nanoparticles for rapid and specific detection of herpesvirus, and to prepare polystyrene seed microspheres by dispersion polymerization. Porous carboxylated polystyrene microspheres were prepared by seed polymerization. The upconversion nanoparticles (upconversion nanoparticles,UCNs) NaYF4:Yb,Tm and magnetic nanoparticles (magnetic nanoparticles,MNPs) were embedded in porous polystyrene microspheres by high temperature melt expansion method. Thus, a magnetic up-conversion fluorescent nanoparticle with the best magnetic response and fluorescence intensity was prepared, and it can be used in the detection of herpes simplex virus type 1 (herpes simplex virus1,HSV-1) by magnetic up-conversion fluorescent nanomaterials, the operation is simple, and the performance of the magnetic up-conversion fluorescent nanospheres is simple. Fast, high accuracy of the purpose. The purpose of this paper is to provide experimental basis for clinical chair-side examination and accurate personalized medical treatment. Methods the seed microspheres of poly (styrene) were prepared by dispersion polymerization method, and the porous carboxy polystyrene microspheres were prepared by seed polymerization method. Then the upconversion of (UCNs) NaYF4:Yb, was carried out by high temperature melt expansion method. Tm nanoparticles and magnetic nanoparticles (MNPs) were embedded in porous polystyrene microspheres, and the magnetic up-conversion fluorescent coded microspheres with the best magnetic response and fluorescence intensity were obtained. Herpes simplex virus type 1 (HSV-1) was cultured with HELA cells and TCID50. of 50% cell culture medium of cultured HSV-1 was determined by Reed-Muench 's two-way method. The surface carboxyl groups of activated microspheres, the monoclonal antibody against HSV-1 were coupled with magnetic upconversion fluorescent coded microspheres, the virus was added into the solution, then the second antibody labeled with Rhodamine dye was used to react with it, and the virus was detected by double antibody sandwich method. To verify the specificity of magnetic up-conversion microspheres, to explore the lowest virus concentration it can detect. Results the size of the polystyrene microspheres was uniform and the particle size was less than 5 渭 m. Magnetic particles and upconversion particles were encapsulated in the microspheres by high temperature swelling method, and the magnetic up-conversion nanoparticles were prepared. The nano-particles did not polymerize. The magnetic up-conversion nanospheres have both magnetic and upconversion fluorescence particle properties. The herpesvirus with biological activity was obtained by HELA cell culture method. The herpesvirus solution was determined by Reed-Muench two-way method with the concentration of 10 ~ 4 脳 0.1 ml TCID _ (50). The magnetic up-conversion nanoson-binding monoclonal antibody to HSV-1 is specific. It can specifically bind HSV-1 to Rhodamine-labeled second antibody and show purple under fluorescence microscope. It was found that only 10 TCID _ (50) HSV-1 could be detected in the HSV-1 of different gradient concentrations of the virus. The HSV-1. could still be detected in the virus solution. Conclusion by connecting magnetic up-conversion nanoparticles and monoclonal antibodies to HSV-1, HSV-1, can be detected specifically by double-antibody sandwich method, and the samples to be detected can be separated quickly by magnetic properties of nanoparticles. Through its up-conversion property, the color reaction was found quickly and simply under the fluorescence microscope. Electron microscopy showed that the surface of the antibody-linked magnetic up-conversion nanospheres changed from smooth to coarse and could connect virus particles. The specific nanospheres reacted with three groups of solutions containing HSV-1, zoster virus (varicella-zoster virus,VZV) and Porphyromonas gingivalis (Porphyromonas gingivalis,Pg) respectively. The results showed that there was only a specific color reaction in HSV-1 group. The results showed that the microsphere with monoclonal antibody was specific. Through the detection of different concentrations of HSV-1 virological fluid, it was found that it could detect the HSV-1, in 10TCID50HSV-1 viral fluid. It indicated that this method could still detect the presence of HSV-1 in the viral liquid containing only 10 TCID _ (50) HSV ~ (- 1).
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4
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相关期刊论文 前2条
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2 丁芳;孟焕新;李启强;赵亦兵;冯向辉;张立;;牙周机械治疗对慢性牙周炎患者龈沟液中疱疹病毒的影响[J];中华口腔医学杂志;2010年07期
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