多酸基荧光检测微球的制备及布鲁氏菌检测方法的研究
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摘要
近年来，全球范围内新发公共传染病不断发生，其病原体75%源自动物或动物源性食品。新发公共传染病已越来越多的呈现出“人禽共患”或“人畜共患”的特点。其中由布鲁氏菌引起的传染病在我国尤其是在牧区发病率较高，且传染性较强。布鲁氏菌病的高发和流行不仅造成巨大的经济损失，而且严重威胁人群的健康。对布鲁氏菌病的预防、监督检查现已引起世界各国的广泛重视，加强对布鲁氏菌的预防和监控现已纳入世界卫生组织和我国卫生部传染病检测的主要工作内容之一。因此，开发快速、高效、特异的布鲁氏菌病检测技术现已成为公共卫生检测领域研究的重点课题之一。本课题研究主要是设计合成了多酸基荧光免疫探针，利用双抗体夹心荧光免疫原理，借助液相芯片检测技术平台，建立一种安全、准确、特异的布鲁氏菌检测方法。本课题研究分为四个部分：
第一部分布鲁氏菌多克隆抗体与单克隆抗体的制备及鉴定。①多克隆抗体的制备及鉴定：利用羊种布鲁氏菌M5疫苗皮下多点注射免疫新西兰白兔，每次免疫前耳缘静脉取血，测定其血清效价，达到所需标准，兔心脏采血并分离血清，用硫酸铵沉淀法结合Protein G亲和层析法进行抗体纯化，Bradford法测定抗体蛋白含量，间接ELISA、SDS-PAGE凝胶电泳试验测定效价和纯度，并利用生物素进行标记。结果显示纯化后布鲁氏菌多抗纯度较高，蛋白含量为8.22mg·L~(-1)，效价可达到1:64000，每摩尔多克隆抗体蛋白可标记生物素摩尔数为2.61。②单克隆抗体的制备及鉴定：采用杂交瘤细胞技术，以羊种布鲁氏菌M5为抗原免疫BALB/c小鼠，在细胞融合前4d加强免疫。取免疫小鼠脾细胞和SP2/0细胞进行融合。用已建立的间接ELISA进行杂交瘤细胞阳性筛选，采用有限稀释法进行亚克隆，筛选出能稳定分泌M5抗体的阳性杂交瘤细胞。阳性杂交瘤细胞经扩大培养后，小鼠体内诱生法制备单抗腹水，并对腹水中的单抗用硫酸铵沉淀法结合Protein G亲和层析法进行纯化， Bradford法测定蛋白含量，间接ELISA、SDS-PAGE凝胶电泳试验测定效价和纯度。结果显示经过亚克隆筛选出1株能稳定分泌M5单克隆抗体的杂交瘤细胞株，抗体亚型为IgG_3型，纯化浓缩后的抗体蛋白含量为4.39mg·L~(-1)，效价可达到1:6400，纯度较高，特异性较好。
第二部分多酸基荧光发光材料的合成与表征。①采用有机无机杂化法，设计合成新型多酸荧光N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2化合物。利用X-射线单晶衍射、氢核磁、碳核磁、红外光谱、紫外光谱和荧光光谱对其结构和性质进行表征。结果显示该化合物呈负电性，红外光谱特征峰为2958、2929、2870、1482、1378、811、795、718cm~(-1)，紫外特征吸收峰为244、270、283、389、345nm，激发峰为262、275、329、345nm，在345nm激发下的发射波谱范围较宽，最高发射峰在400nm处。②采用常规的多酸化学合成方法，合成Na_9EuW_(10)O_(36)，利用红外光谱、紫外光谱和荧光光谱对所合成的化合物结构和性质进行表征。结果显示，该化合物红外光谱特征峰为543、705、785、843、945nm，紫外特征吸收峰为208和258nm，在278nm激发下的发射峰位于579、592、619、651、692、700nm。
第三部分功能化多酸基荧光检测微球的制备与表征。①功能化多酸荧光染料[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]标记聚苯乙烯微球的制备与表征：采用分散聚合法合成聚苯乙烯微球，微球粒径约为2.8μm，选用聚电解质PDADMAC、PAA、PAH对微球进行活化，采用层接层静电自组装法将所制备的多酸荧光染料[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]包覆在微球表面，通过聚电解质进一步修饰，使微球表面带有羧基官能团。利用Zeta电位、荧光光谱和激光共聚焦显微镜对所制备的微球进行表征，结果显示多酸荧光染料和聚苯乙烯微球可以较好的偶联，且具有良好的荧光发光特性。②功能化多酸荧光染料[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]标记二氧化硅微球的制备与表征：采用种子聚合法合成二氧化硅微球，微球粒径约为1μm，选用聚电解质PEI和PAA对微球进行活化，采用层接层静电自组装法将所制备的多酸荧光染料[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]包覆在微球表面，通过聚电解质进一步修饰，使微球表面带有羧基官能团。利用Zeta电位、荧光光谱和流式细胞仪对所制备的微球进行表征。结果显示多酸荧光染料和二氧化硅微球可以较好的偶联，且具有良好的荧光发光特性。③功能化多酸荧光染料Na_9EuW_(10)O_(36)标记聚苯乙烯微球的制备与表征：采用细乳液聚合法将Na_9EuW_(10)O_(36)杂化引入聚苯乙烯微球中，通过聚电解质进一步修饰，使微球表面带有羧基官能团。利用紫外光谱、荧光光谱、Zeta电位和透射电镜对所制备的微球进行表征。结果显示Na_9EuW_(10)O_(36)与聚苯乙烯微球可以较好的杂化，且具有良好的荧光发光特性。
第四部分布鲁氏菌液相芯片检测方法的建立及评价。①布鲁氏菌液相芯片检测方法的建立：利用双抗体夹心荧光免疫分析原理，采用液相芯片检测技术平台，通过对反应条件的优化，建立一种布鲁氏菌液相芯片检测方法，并对该方法的检测效果进行验证。结果显示，本课题所建立的检测方法最佳反应条件为：单抗最佳偶联浓度8.3125μg·mL~(-1)，生物素标记的多克隆抗体工作浓度为432μg·mL~(-1)，SA-PE的工作浓度为8μg·mL~(-1)。该方法检测的特异性和重复性较好，利用该方法检测羊布鲁氏菌M5最低检出限为1.0×10~6CFU·mL~(-1)。②布鲁氏菌液相芯片检测方法的评价：制备40份实验室模拟样本，经过72h增菌培养后，分别按照所建立的液相芯片方法和分离培养鉴定法进行检测，并对两种检测方法的结果进行比较。结果显示，所建立的液相芯片法与分离培养鉴定法相比，准确性和检测限相近，但液相芯片法可大大缩短检测时间，且检测工作无需带菌操作，安全可靠。
In recent years, newly emerging public communicable diseases occurredfrequently, and75%of the pathogens are derived from animals or animal derived food.The pathogens for the newly emerging communicable diseases manifest distinctivecharacteristics of infecting birds or livestocks and humans. Among the communicablediseases, Brucellosis by Brucella is proved to be highly contagious and with highprevalence in China, especially in the pastoral areas. The high prevalence andincidence of Brucellosis cause a huge economic loss and pose a great threat topeople’s health. Thus the prevention and surveillance of Brucellosis has attracted wideattention in the world, and the improvement on the prevention and monitoring on thispathogen has become one of the major tasks of WHO and the Ministry of Health ofChina. Therefore, quick, efficient and specific technology for detection of Brucellahas become one of important issues in public health. The objective of the presentstudy is to synthesize POMs-based fluorescent probes to detect Brucella based on thedouble antibodies sandwich fluorescent immunological principles.
PartⅠ P reparation and identification ofpolyclonal antibody and monoclonalantibody of Brucella
1. Preparation and identification of polyclonal antibody: New Zealand white rabbitswere immunized with B. melitensis M5vaccine at multisites by subcutaneousinjection.Blood was collected through ear vein and serum antibody titer of anti-M5was determined. When the serum titer of anti-M5antibody reached the required levels,the rabbits were narcotized and blood was collected from heart by syringe. Theanti-M5antibodies were sedimented by ammonia sulfate and purified by Protein Gaffinity chromotography. The content, titer and purity of anti-M5antibody wasdetermined by Bradford, indirect ELISA and SDS-PAGE respectively. The purifiedantibody was labelled with biotin. The results showed that the content of the antibody was8.22mg·L~(-1), the titer was1:64000and the labelling efficiency was2.62of biotinto antibody.
2. Preparation and identificaion of monoclonal antibody: The B. melitensis M5vaccine was injected into BALB/c mice. The mice were given an intensivevaccination before the4thday of cell fusion. The hybridoma cell producing anti-M5monoclonal antibody was prepared by applying hybridoma technology, expandedculture and inoculated into mice introperitoneal to produce ascites. The anti-M5monoclonal antibodies in ascites were sedimented by ammonia sulfate and purified byProtein G affinity chromotography. The content, titer and purity of anti-M5monoclonal antibody were determined by Bradford, indirect ELISA and SDS-PAGErespectively. The results demonstated that the subtype of anti-M5monoclonalantibody, derived from hybridoma cell, was IgG3.The concentration of the purifiedand concentrated antibody was4.39mg·L~(-1)and the titer was1:6400.
Part Ⅱ Synthesis and characteristics of POMs-based fluorescent compounds
1. The compound N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2was designed andsynthesized by organic-inorganic hybridization. Constituents and characterisitics ofthis compound were determined through X-ray diffraction, H-NMR, C-NMR, IR, UVspectrophotometer and fluorospectro photometer. The results demonstrated that thecompound had negative charges. The characteristic peaks of IR were2958,2929,2870,1482,1378,811,795and718cm-1, UV absorption peaks were244,270,283,389and345nm. Excitation peaks were262,275,329and345nm，The range ofemission spectrum was wide under the excitation of345nm and the maximumemission peak was400nm.
2. Na_9EuW_(10)O_(36)was synthetized by conventional POMs synthesis method. Structuresand characterisitics were determined through IR, UV spectrophotometer andfluorospectro photometer.The IR characteristic peaks of this compound were543,705,785,843and945nm, The UV characteristic absorption peaks were208nm and258nm, The emission peaks under the excitation of278nm were579,592,619,651,692and700nm.
Part Ⅲ Preparation and characteristics of functional POMs-based fluorescentdetecting polytyrene microspheres
1. Preparation and characteristics of styrene microspheres labelled by[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2] compound: polystyrene micro-spheres were synthesized by dispersion polymerization, with the diameter of the beads2.8μm. The beads were activated by polyelectrolytes including PDADMAC, PAAand PAH. The activated beads were labelled by POMs-based fluorescent compound[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]. The beads were further modified by–COOH and characterized by Zeta potential, fluorospectro photometer and flowcytometer. The results showed that POMs-based fluorescence dye could be wellcoupled with polystyrene microspheres and had satisfactory fluorescencecharacteristics.
2. Preparation and characteristics of functional POMs-based fluorescent [N(C4H9)4]2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2] labeled SiO2microspheres. The microsphereswere synthesized by seeding polymerization method with the diameter of the beads1μm. The beads were activated by polyelectrolytes including PEI, PAA and PEI. Theactivated beads were labelled with POMs-based fluorescent compounds [N(C4H9)4]2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2] by layer-by-layer electrostatic self-assemblymethod. The beads were further modified by-COOH and characterized by Zetapotential, fluorospectro photometer and flow cytometer. The results showed thatPOMs-based fluorescence dye could be well coupled with SiO2microspheres andpossessed good fluorescent-emission characteristics.
3. Preparation and characteristics of functional POMs-based fluorescent dye labeledpolystyrene microspheres: Na_9EuW_(10)O_(36)were incorporated into styrene microspheresby fine emulsion polymerization. The beads were further modified by-COOH andcharacterized by UV spectrophotometer, fluorospectro photometer, Zeta potential andTEM. The results showed that Na_9EuW_(10)O_(36)could be well hybridized withpolystyrene microspheres, and the beads existed fine fluorescence characteristics.
Part ⅣEstablishment and evaluation of Liquid chip detection method for thedetection of Brucella
1. Establishment of Liquid chip detection method for the detection of Brucella: thedetection method was established based on the principles of double-antibodysandwich fluorescent immunology by optimized monoclonal antibody couplingconcentration, biotin labeled polyclonal antibody and SA-PE working concentration.And the detection effect of this method was confirmed.The results showed thatoptimal reaction conditions were as followed, optimal monoclonal antibody couplingconcentration8.3125μg·mL~(-1), biotin labeled polyclonal antibody workingconcentration432μg·mL~(-1), SA-PE working concentration8μg·mL~(-1). Specificity andrepeatability of the method was satisfying and the mimimum detection limit for M5Brucella was1.0×10~6CFU·mL~(-1).
2. Evaluation on Liquid chip method for the detection of Brucella: forty mimicSamples were multiplied and detected by Liquid chip detection method and separationculture assay method, respectively. The detection results were compared. The resultsindicated that both methods manifested similar accuracy and detection limit, butLiquid chip detection method could reduce the detection duration and made thedetection much safer because of no live bacteria available.
第一部分布鲁氏菌多克隆抗体与单克隆抗体的制备及鉴定。①多克隆抗体的制备及鉴定：利用羊种布鲁氏菌M5疫苗皮下多点注射免疫新西兰白兔，每次免疫前耳缘静脉取血，测定其血清效价，达到所需标准，兔心脏采血并分离血清，用硫酸铵沉淀法结合Protein G亲和层析法进行抗体纯化，Bradford法测定抗体蛋白含量，间接ELISA、SDS-PAGE凝胶电泳试验测定效价和纯度，并利用生物素进行标记。结果显示纯化后布鲁氏菌多抗纯度较高，蛋白含量为8.22mg·L~(-1)，效价可达到1:64000，每摩尔多克隆抗体蛋白可标记生物素摩尔数为2.61。②单克隆抗体的制备及鉴定：采用杂交瘤细胞技术，以羊种布鲁氏菌M5为抗原免疫BALB/c小鼠，在细胞融合前4d加强免疫。取免疫小鼠脾细胞和SP2/0细胞进行融合。用已建立的间接ELISA进行杂交瘤细胞阳性筛选，采用有限稀释法进行亚克隆，筛选出能稳定分泌M5抗体的阳性杂交瘤细胞。阳性杂交瘤细胞经扩大培养后，小鼠体内诱生法制备单抗腹水，并对腹水中的单抗用硫酸铵沉淀法结合Protein G亲和层析法进行纯化， Bradford法测定蛋白含量，间接ELISA、SDS-PAGE凝胶电泳试验测定效价和纯度。结果显示经过亚克隆筛选出1株能稳定分泌M5单克隆抗体的杂交瘤细胞株，抗体亚型为IgG_3型，纯化浓缩后的抗体蛋白含量为4.39mg·L~(-1)，效价可达到1:6400，纯度较高，特异性较好。
第二部分多酸基荧光发光材料的合成与表征。①采用有机无机杂化法，设计合成新型多酸荧光N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2化合物。利用X-射线单晶衍射、氢核磁、碳核磁、红外光谱、紫外光谱和荧光光谱对其结构和性质进行表征。结果显示该化合物呈负电性，红外光谱特征峰为2958、2929、2870、1482、1378、811、795、718cm~(-1)，紫外特征吸收峰为244、270、283、389、345nm，激发峰为262、275、329、345nm，在345nm激发下的发射波谱范围较宽，最高发射峰在400nm处。②采用常规的多酸化学合成方法，合成Na_9EuW_(10)O_(36)，利用红外光谱、紫外光谱和荧光光谱对所合成的化合物结构和性质进行表征。结果显示，该化合物红外光谱特征峰为543、705、785、843、945nm，紫外特征吸收峰为208和258nm，在278nm激发下的发射峰位于579、592、619、651、692、700nm。
第三部分功能化多酸基荧光检测微球的制备与表征。①功能化多酸荧光染料[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]标记聚苯乙烯微球的制备与表征：采用分散聚合法合成聚苯乙烯微球，微球粒径约为2.8μm，选用聚电解质PDADMAC、PAA、PAH对微球进行活化，采用层接层静电自组装法将所制备的多酸荧光染料[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]包覆在微球表面，通过聚电解质进一步修饰，使微球表面带有羧基官能团。利用Zeta电位、荧光光谱和激光共聚焦显微镜对所制备的微球进行表征，结果显示多酸荧光染料和聚苯乙烯微球可以较好的偶联，且具有良好的荧光发光特性。②功能化多酸荧光染料[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]标记二氧化硅微球的制备与表征：采用种子聚合法合成二氧化硅微球，微球粒径约为1μm，选用聚电解质PEI和PAA对微球进行活化，采用层接层静电自组装法将所制备的多酸荧光染料[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]包覆在微球表面，通过聚电解质进一步修饰，使微球表面带有羧基官能团。利用Zeta电位、荧光光谱和流式细胞仪对所制备的微球进行表征。结果显示多酸荧光染料和二氧化硅微球可以较好的偶联，且具有良好的荧光发光特性。③功能化多酸荧光染料Na_9EuW_(10)O_(36)标记聚苯乙烯微球的制备与表征：采用细乳液聚合法将Na_9EuW_(10)O_(36)杂化引入聚苯乙烯微球中，通过聚电解质进一步修饰，使微球表面带有羧基官能团。利用紫外光谱、荧光光谱、Zeta电位和透射电镜对所制备的微球进行表征。结果显示Na_9EuW_(10)O_(36)与聚苯乙烯微球可以较好的杂化，且具有良好的荧光发光特性。
第四部分布鲁氏菌液相芯片检测方法的建立及评价。①布鲁氏菌液相芯片检测方法的建立：利用双抗体夹心荧光免疫分析原理，采用液相芯片检测技术平台，通过对反应条件的优化，建立一种布鲁氏菌液相芯片检测方法，并对该方法的检测效果进行验证。结果显示，本课题所建立的检测方法最佳反应条件为：单抗最佳偶联浓度8.3125μg·mL~(-1)，生物素标记的多克隆抗体工作浓度为432μg·mL~(-1)，SA-PE的工作浓度为8μg·mL~(-1)。该方法检测的特异性和重复性较好，利用该方法检测羊布鲁氏菌M5最低检出限为1.0×10~6CFU·mL~(-1)。②布鲁氏菌液相芯片检测方法的评价：制备40份实验室模拟样本，经过72h增菌培养后，分别按照所建立的液相芯片方法和分离培养鉴定法进行检测，并对两种检测方法的结果进行比较。结果显示，所建立的液相芯片法与分离培养鉴定法相比，准确性和检测限相近，但液相芯片法可大大缩短检测时间，且检测工作无需带菌操作，安全可靠。
In recent years, newly emerging public communicable diseases occurredfrequently, and75%of the pathogens are derived from animals or animal derived food.The pathogens for the newly emerging communicable diseases manifest distinctivecharacteristics of infecting birds or livestocks and humans. Among the communicablediseases, Brucellosis by Brucella is proved to be highly contagious and with highprevalence in China, especially in the pastoral areas. The high prevalence andincidence of Brucellosis cause a huge economic loss and pose a great threat topeople’s health. Thus the prevention and surveillance of Brucellosis has attracted wideattention in the world, and the improvement on the prevention and monitoring on thispathogen has become one of the major tasks of WHO and the Ministry of Health ofChina. Therefore, quick, efficient and specific technology for detection of Brucellahas become one of important issues in public health. The objective of the presentstudy is to synthesize POMs-based fluorescent probes to detect Brucella based on thedouble antibodies sandwich fluorescent immunological principles.
PartⅠ P reparation and identification ofpolyclonal antibody and monoclonalantibody of Brucella
1. Preparation and identification of polyclonal antibody: New Zealand white rabbitswere immunized with B. melitensis M5vaccine at multisites by subcutaneousinjection.Blood was collected through ear vein and serum antibody titer of anti-M5was determined. When the serum titer of anti-M5antibody reached the required levels,the rabbits were narcotized and blood was collected from heart by syringe. Theanti-M5antibodies were sedimented by ammonia sulfate and purified by Protein Gaffinity chromotography. The content, titer and purity of anti-M5antibody wasdetermined by Bradford, indirect ELISA and SDS-PAGE respectively. The purifiedantibody was labelled with biotin. The results showed that the content of the antibody was8.22mg·L~(-1), the titer was1:64000and the labelling efficiency was2.62of biotinto antibody.
2. Preparation and identificaion of monoclonal antibody: The B. melitensis M5vaccine was injected into BALB/c mice. The mice were given an intensivevaccination before the4thday of cell fusion. The hybridoma cell producing anti-M5monoclonal antibody was prepared by applying hybridoma technology, expandedculture and inoculated into mice introperitoneal to produce ascites. The anti-M5monoclonal antibodies in ascites were sedimented by ammonia sulfate and purified byProtein G affinity chromotography. The content, titer and purity of anti-M5monoclonal antibody were determined by Bradford, indirect ELISA and SDS-PAGErespectively. The results demonstated that the subtype of anti-M5monoclonalantibody, derived from hybridoma cell, was IgG3.The concentration of the purifiedand concentrated antibody was4.39mg·L~(-1)and the titer was1:6400.
Part Ⅱ Synthesis and characteristics of POMs-based fluorescent compounds
1. The compound N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2was designed andsynthesized by organic-inorganic hybridization. Constituents and characterisitics ofthis compound were determined through X-ray diffraction, H-NMR, C-NMR, IR, UVspectrophotometer and fluorospectro photometer. The results demonstrated that thecompound had negative charges. The characteristic peaks of IR were2958,2929,2870,1482,1378,811,795and718cm-1, UV absorption peaks were244,270,283,389and345nm. Excitation peaks were262,275,329and345nm，The range ofemission spectrum was wide under the excitation of345nm and the maximumemission peak was400nm.
2. Na_9EuW_(10)O_(36)was synthetized by conventional POMs synthesis method. Structuresand characterisitics were determined through IR, UV spectrophotometer andfluorospectro photometer.The IR characteristic peaks of this compound were543,705,785,843and945nm, The UV characteristic absorption peaks were208nm and258nm, The emission peaks under the excitation of278nm were579,592,619,651,692and700nm.
Part Ⅲ Preparation and characteristics of functional POMs-based fluorescentdetecting polytyrene microspheres
1. Preparation and characteristics of styrene microspheres labelled by[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2] compound: polystyrene micro-spheres were synthesized by dispersion polymerization, with the diameter of the beads2.8μm. The beads were activated by polyelectrolytes including PDADMAC, PAAand PAH. The activated beads were labelled by POMs-based fluorescent compound[N(C_4H_9)_4]_2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2]. The beads were further modified by–COOH and characterized by Zeta potential, fluorospectro photometer and flowcytometer. The results showed that POMs-based fluorescence dye could be wellcoupled with polystyrene microspheres and had satisfactory fluorescencecharacteristics.
2. Preparation and characteristics of functional POMs-based fluorescent [N(C4H9)4]2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2] labeled SiO2microspheres. The microsphereswere synthesized by seeding polymerization method with the diameter of the beads1μm. The beads were activated by polyelectrolytes including PEI, PAA and PEI. Theactivated beads were labelled with POMs-based fluorescent compounds [N(C4H9)4]2[V_6O_(13){(OCH_2)_3CNH-CH_2-C_(16)H_9}_2] by layer-by-layer electrostatic self-assemblymethod. The beads were further modified by-COOH and characterized by Zetapotential, fluorospectro photometer and flow cytometer. The results showed thatPOMs-based fluorescence dye could be well coupled with SiO2microspheres andpossessed good fluorescent-emission characteristics.
3. Preparation and characteristics of functional POMs-based fluorescent dye labeledpolystyrene microspheres: Na_9EuW_(10)O_(36)were incorporated into styrene microspheresby fine emulsion polymerization. The beads were further modified by-COOH andcharacterized by UV spectrophotometer, fluorospectro photometer, Zeta potential andTEM. The results showed that Na_9EuW_(10)O_(36)could be well hybridized withpolystyrene microspheres, and the beads existed fine fluorescence characteristics.
Part ⅣEstablishment and evaluation of Liquid chip detection method for thedetection of Brucella
1. Establishment of Liquid chip detection method for the detection of Brucella: thedetection method was established based on the principles of double-antibodysandwich fluorescent immunology by optimized monoclonal antibody couplingconcentration, biotin labeled polyclonal antibody and SA-PE working concentration.And the detection effect of this method was confirmed.The results showed thatoptimal reaction conditions were as followed, optimal monoclonal antibody couplingconcentration8.3125μg·mL~(-1), biotin labeled polyclonal antibody workingconcentration432μg·mL~(-1), SA-PE working concentration8μg·mL~(-1). Specificity andrepeatability of the method was satisfying and the mimimum detection limit for M5Brucella was1.0×10~6CFU·mL~(-1).
2. Evaluation on Liquid chip method for the detection of Brucella: forty mimicSamples were multiplied and detected by Liquid chip detection method and separationculture assay method, respectively. The detection results were compared. The resultsindicated that both methods manifested similar accuracy and detection limit, butLiquid chip detection method could reduce the detection duration and made thedetection much safer because of no live bacteria available.
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