高稳定性磁性荧光多功能化聚苯乙烯微球的制备及其初步应用
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摘要
磁性荧光聚合物微球由于其既具备了聚合物微球大的表面积、粒径可控、易于分离、表面可修饰多种官能团等优点,又具备了磁性材料独特的超顺磁性和量子点优异的光学特性而受到普遍关注,并广泛应用于生物医学领域,尤其在编码技术、细胞分离技术、共振显像、药物筛选和靶向、疾病的检测和诊断等方面有广阔的应用前景。
本文研究了磁性荧光多功能化聚苯乙烯微球的制备,探讨了不同的因素和条件对微球结构及性能参数的影响,并实现了在免疫检测方面的初步应用。首先,以分散聚合和种子溶胀聚合的方法制备了粒径在1-15μm的羧基化聚苯乙烯微球,并对分散聚合与种子聚合的机理,反应条件对种子微球和目标微球粒径、形貌及性能的影响进行了探讨;通过蛋白吸附实验、酶联免疫反应和免疫荧光反应评价了微球的表面吸附能力、表面反应能力以及羧基的活性。其次,以传统溶剂溶胀法配合高温溶剂溶胀法制备了高稳定性的磁性荧光多功能微球,并对微球的磁性能及荧光性能进行了表征,对反应机理进行了探讨,并且评价了其在有机溶剂、不同pH缓冲液和水环境下的稳定性。最后,以制备的磁性荧光微球为载体进行免疫诊断,并通过荧光显微镜和流式细胞仪对免疫反应后微球的荧光光谱进行分析。
由扫描电子显微镜分析可知,制得的微球形貌规则、粒径均匀;流式细胞术分析表明,不同粒径的微球分属不同的点群并能够完全分开,因此可通过微球粒径差异实现编码。红外谱图和核磁谱图证明,在种子聚合中通过共聚甲基丙烯酸的方法成功在微球表面引入羧基,由电导滴定测得微球表面羧基含量为0.4mmol/g;蛋白吸附试验和免疫实验进一步证实微球表面羧基具有良好的反应活性。激光共聚焦显微镜图片显示,纳米粒子通过多孔微球的孔道渗入了微球内部且均匀分布。震动磁强计和分离效果图显示通过常温-高温法制备的磁性荧光微球具有良好的磁响应性,而荧光图片及荧光光谱表明微球的荧光强度较高,可满足实际需求;通过稳定性研究发现,磁性荧光微球稳定性良好,免疫反应实验证明磁性荧光微球能够稳定地结合抗原(人IgG),并且能够特异识别相应的抗体。
本课题的研究成果为液相生物芯片研发、微球编码技术和样品分离技术提供了重要的理论支持和实验依据,在生物识别和检测领域具有广阔的应用前景。
Magnetic fluorescent multifunctional polymer beads have been attracted great interest in application of biomedical field not only due to their advantages of big surface area, size-controlled, separated facility and ease to be functionalized on the surface, but also performance unique superparamagnetization and excellent optical properties. In particular, these functional beads have broad application prospect in encoding, cell separation, resonance imaging, drug screening and targeting, disease detection and diagnosis, etc.
The preparation of monodisperse magnetic fluorescent multifunctional polystyrene beads and the effect of different factors and conditions on microsphere structure, performance and parameter were investigated in detail in this work. Bio-immunoassay and clinical diagnosis were operated on bead surface as a primary application. Firstly, two-step swelling seed polymerization was adopted to synthesize carboxylic beads in size of 1-15μm with polystyrene particles by dispersion polymerization in size of 1-3μm as seed latex. The schematics and reaction dynamics of dispersion polymerization and seed polymerization were described and the effects of conditions on size, morphology and properties of seed particles and target beads were discussed. Bead adsorption, reaction capacity and carboxylic activity were evaluated by BCA protein adsorption and ELISA assay. Secondly, an efficiently modified method combined conventional swelling method with high-temperature swelling method was applied to prepare high-stable multifunctional MNPs-QD-encoded polystyrene beads simultaneously with large encoding capacity and fast separation. Subsequently, fluorescent and magnetic performances were investaged in detail and the stability of multifunctional beads in organic solvent, buffers with different pH. The long-time stability in PBS buffer as an important characterization was used to perform that functional bead can store and apply for a long time, such as 6 months.
The regular morphology, uniform size can be observed from SEM images and beads with varied size can be completely separated from each other in flow cytomic data, so that encoding by bead size can be realized. The fact that carboxyl groups were successfully integrated onto bead surface can be obtained from NMR and FTIR spectra and the concentration is 0.4mmol/g by conductometric titration. QDs were packed into bead network and well-distributed seeing from laser scanning confocal microscope; meanwhile, magnetic performance was excellent getting from vibration magnetometer and separation photos. In this work, the results demonstrate that encapsulating QDs into pre-prepared MNPs-beads is the best sequence, which keeps high brightness and fast separation. The MNPs-QD-encoded microcarriers designed by our proposed method exhibit excellent performance for magnetic manipulation and optical encoding; what is more, they also have strong physical and chemical stabilities. Compared with beads prepared via conventional swelling method, the leakage of nanoparticles from the designed MNPs-QD-encoded PSEMBs induced by organic solvents (cyclohexane) is less than 6 % and fluorescence intensity of MNPs- QDs-encoded PSEMBs fluctuates more slightly in a wide range of pH 4-12 buffers; meanwhile, fluorescence intensity kept the low level less than 40% for 6 months in PBS buffer. In addition, immunoassay performance for human IgG detections indicates that carboxyl groups on fluorescence microsphere surface facilitate efficient attachment of biomacromolecule and therefore they can be further applied to fast separation and multiplexed biomolecular assays.
The results of research can provide important theoretical and experimental basis for the bead-encoding development of liquid biochip, and has broad application prospects in the field of testing biometrics.
本文研究了磁性荧光多功能化聚苯乙烯微球的制备,探讨了不同的因素和条件对微球结构及性能参数的影响,并实现了在免疫检测方面的初步应用。首先,以分散聚合和种子溶胀聚合的方法制备了粒径在1-15μm的羧基化聚苯乙烯微球,并对分散聚合与种子聚合的机理,反应条件对种子微球和目标微球粒径、形貌及性能的影响进行了探讨;通过蛋白吸附实验、酶联免疫反应和免疫荧光反应评价了微球的表面吸附能力、表面反应能力以及羧基的活性。其次,以传统溶剂溶胀法配合高温溶剂溶胀法制备了高稳定性的磁性荧光多功能微球,并对微球的磁性能及荧光性能进行了表征,对反应机理进行了探讨,并且评价了其在有机溶剂、不同pH缓冲液和水环境下的稳定性。最后,以制备的磁性荧光微球为载体进行免疫诊断,并通过荧光显微镜和流式细胞仪对免疫反应后微球的荧光光谱进行分析。
由扫描电子显微镜分析可知,制得的微球形貌规则、粒径均匀;流式细胞术分析表明,不同粒径的微球分属不同的点群并能够完全分开,因此可通过微球粒径差异实现编码。红外谱图和核磁谱图证明,在种子聚合中通过共聚甲基丙烯酸的方法成功在微球表面引入羧基,由电导滴定测得微球表面羧基含量为0.4mmol/g;蛋白吸附试验和免疫实验进一步证实微球表面羧基具有良好的反应活性。激光共聚焦显微镜图片显示,纳米粒子通过多孔微球的孔道渗入了微球内部且均匀分布。震动磁强计和分离效果图显示通过常温-高温法制备的磁性荧光微球具有良好的磁响应性,而荧光图片及荧光光谱表明微球的荧光强度较高,可满足实际需求;通过稳定性研究发现,磁性荧光微球稳定性良好,免疫反应实验证明磁性荧光微球能够稳定地结合抗原(人IgG),并且能够特异识别相应的抗体。
本课题的研究成果为液相生物芯片研发、微球编码技术和样品分离技术提供了重要的理论支持和实验依据,在生物识别和检测领域具有广阔的应用前景。
Magnetic fluorescent multifunctional polymer beads have been attracted great interest in application of biomedical field not only due to their advantages of big surface area, size-controlled, separated facility and ease to be functionalized on the surface, but also performance unique superparamagnetization and excellent optical properties. In particular, these functional beads have broad application prospect in encoding, cell separation, resonance imaging, drug screening and targeting, disease detection and diagnosis, etc.
The preparation of monodisperse magnetic fluorescent multifunctional polystyrene beads and the effect of different factors and conditions on microsphere structure, performance and parameter were investigated in detail in this work. Bio-immunoassay and clinical diagnosis were operated on bead surface as a primary application. Firstly, two-step swelling seed polymerization was adopted to synthesize carboxylic beads in size of 1-15μm with polystyrene particles by dispersion polymerization in size of 1-3μm as seed latex. The schematics and reaction dynamics of dispersion polymerization and seed polymerization were described and the effects of conditions on size, morphology and properties of seed particles and target beads were discussed. Bead adsorption, reaction capacity and carboxylic activity were evaluated by BCA protein adsorption and ELISA assay. Secondly, an efficiently modified method combined conventional swelling method with high-temperature swelling method was applied to prepare high-stable multifunctional MNPs-QD-encoded polystyrene beads simultaneously with large encoding capacity and fast separation. Subsequently, fluorescent and magnetic performances were investaged in detail and the stability of multifunctional beads in organic solvent, buffers with different pH. The long-time stability in PBS buffer as an important characterization was used to perform that functional bead can store and apply for a long time, such as 6 months.
The regular morphology, uniform size can be observed from SEM images and beads with varied size can be completely separated from each other in flow cytomic data, so that encoding by bead size can be realized. The fact that carboxyl groups were successfully integrated onto bead surface can be obtained from NMR and FTIR spectra and the concentration is 0.4mmol/g by conductometric titration. QDs were packed into bead network and well-distributed seeing from laser scanning confocal microscope; meanwhile, magnetic performance was excellent getting from vibration magnetometer and separation photos. In this work, the results demonstrate that encapsulating QDs into pre-prepared MNPs-beads is the best sequence, which keeps high brightness and fast separation. The MNPs-QD-encoded microcarriers designed by our proposed method exhibit excellent performance for magnetic manipulation and optical encoding; what is more, they also have strong physical and chemical stabilities. Compared with beads prepared via conventional swelling method, the leakage of nanoparticles from the designed MNPs-QD-encoded PSEMBs induced by organic solvents (cyclohexane) is less than 6 % and fluorescence intensity of MNPs- QDs-encoded PSEMBs fluctuates more slightly in a wide range of pH 4-12 buffers; meanwhile, fluorescence intensity kept the low level less than 40% for 6 months in PBS buffer. In addition, immunoassay performance for human IgG detections indicates that carboxyl groups on fluorescence microsphere surface facilitate efficient attachment of biomacromolecule and therefore they can be further applied to fast separation and multiplexed biomolecular assays.
The results of research can provide important theoretical and experimental basis for the bead-encoding development of liquid biochip, and has broad application prospects in the field of testing biometrics.
引文
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