磁性上轉(zhuǎn)換熒光編碼微球特異性檢測單純皰疹病毒的實驗研究
發(fā)布時間：2019-04-19 12:02
【摘要】：目的本實驗擬探討一種基于磁性上轉(zhuǎn)換納米粒子能夠快速特異性檢測皰疹病毒的新方法,使用分散聚合法制備聚苯乙烯種子微球,利用種子聚合法制備多孔羧基化聚苯乙烯微球,采用高溫熔脹法將上轉(zhuǎn)換納米粒子(upconversion nanoparticles,UCNs)NaYF4:Yb,Tm和磁納米粒子(magnetic nanoparticles,MNPs)包埋至多孔聚苯乙烯微球中,從而制得一種磁響應(yīng)性能和熒光強度最佳的磁性上轉(zhuǎn)換熒光編碼微球,并且能運用于單純皰疹病毒1型(herpes simplex virus1,HSV-1)檢測的磁性上轉(zhuǎn)換熒光納米材料,實現(xiàn)操作簡單、快速,準確度高的目的。旨在為臨床椅旁檢驗、精準個性化醫(yī)療提供實驗基礎(chǔ)。方法實驗使用分散聚合法制備聚苯乙烯種子微球,利用種子聚合法制備多孔羧基化聚苯乙烯微球,再采用高溫熔脹法將上轉(zhuǎn)換(UCNs)NaYF4:Yb,Tm納米粒子和磁納米粒子(MNPs)包埋至多孔聚苯乙烯微球中,獲得磁響應(yīng)性能和熒光強度最佳的磁性上轉(zhuǎn)換熒光編碼微球。利用HELA細胞培養(yǎng)單純皰疹病毒1型并利用Reed-Muench兩氏法確定所培養(yǎng)的HSV-1的半數(shù)細胞培養(yǎng)物感染量TCID50�；罨⑶虮砻骠然鶊F,HSV-1的單克隆抗體與磁性上轉(zhuǎn)換熒光編碼微球偶聯(lián),加入病毒待檢液,再用標記羅丹明染料的二抗與之反應(yīng),雙抗夾心法檢測病毒。驗證磁性上轉(zhuǎn)換微球的特異性,探究其能檢出的最低病毒濃度。結(jié)果實驗制得的聚苯乙烯微球大小均一,粒徑小于5μm,高溫溶脹法將磁粒子和上轉(zhuǎn)換粒子包入其中后制得磁性上轉(zhuǎn)換納米粒子,納米粒子無聚合現(xiàn)象。所制得的磁性上轉(zhuǎn)換納米微球同時具有磁性和上轉(zhuǎn)換熒光粒子特性。通過HELA細胞培養(yǎng)病毒的方法獲得了具有生物活性的皰疹病毒,收集皰疹病毒溶液通過Reed-Muench兩氏法測定,濃度為10-4/0.1mlTCID50。磁性上轉(zhuǎn)換納米子結(jié)合HSV-1的單克隆抗體后具有特異性,能特異性結(jié)合HSV-1后與羅丹明標記的二抗反應(yīng)熒光顯微鏡下顯示紫色。通過檢測不同梯度濃度的HSV-1發(fā)現(xiàn)當病毒液中僅有10TCID50HSV-1時仍可檢出其中的HSV-1。結(jié)論通過連接磁性上轉(zhuǎn)換納米粒子和HSV-1單克隆抗體后利用雙抗夾心法能特異性檢出HSV-1,并通過納米粒子的磁性,快速分離待檢物,通過其上轉(zhuǎn)換特性在熒光顯微鏡下快速簡便的發(fā)現(xiàn)其顯色反應(yīng)。電鏡下顯示連接抗體的磁性上轉(zhuǎn)換納米微球表面由光滑變得粗糙,且能夠連接病毒顆粒。特異性納米微球分別與含有HSV-1、帶狀皰疹病毒(varicella-zoster virus,VZV)和牙齦卟啉單胞菌(Porphyromonas gingivalis,Pg)的三組待檢溶液反應(yīng),結(jié)果顯示僅在HSV-1組有特異性顯色反應(yīng),表明連接了單克隆抗體的微球具有特異性。通過檢測不同濃度的HSV-1病毒液,發(fā)現(xiàn)其能檢出10TCID50HSV-1病毒液中的HSV-1,表明此方法在病毒液中僅含10TCID50HSV-1時仍能檢測出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).
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781.4
本文編號：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).
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781.4
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相關(guān)期刊論文 前2條
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2 丁芳;孟煥新;李啟強;趙亦兵;馮向輝;張立;;牙周機械治療對慢性牙周炎患者齦溝液中皰疹病毒的影響[J];中華口腔醫(yī)學雜志;2010年07期
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