【作者】 张健；

【导师】 常津；

【作者基本信息】 天津大学 ， 材料学， 2017， 硕士

【摘要】 聚苯乙烯微球由于尺寸为微米级,表面易于功能化修饰,是液相生物芯片常用的载体材料,用于生物分子的高通量检测。分子信标是具有茎环结构的核酸探针,由于构象改变使其具有比线性核酸探针更好的特异性,可检测单碱基多态性(SNP)。本文将聚苯乙烯微球和分子信标结合起来,并利用金纳米粒子对多种荧光物质具有淬灭作用,用其取代分子信标传统的淬灭基团,开发一种基于聚苯乙烯-纳米金复合微球的核酸探针。具体研究内容如下:(1)采用分散聚合的方法制备单分散聚苯乙烯微球。实验结果显示,制得的微球单分散性好、表面光滑、粒径约为2μm且粒径分布窄。(2)通过多巴胺在碱性溶液中自聚使聚苯乙烯微球表面包覆一层聚多巴胺。通过该聚多巴胺原位还原氯金酸使金纳米粒子沉积在微球表面,粒子尺寸约为20nm。然后以该粒子为核,加入还原剂盐酸羟胺二次还原氯金酸,使金纳米粒子生长,最终得到表面富含金纳米粒子的复合微球。(3)将一端巯基功能化修饰的分子信标通过Au-S键连接在聚苯乙烯-纳米金微球的表面,制备核酸探针。探究了实验过程中,氯化钠浓度对微球表面分子信标载入量的影响,当氯化钠浓度为0.6M时,微球表面分子信标的载入量最大,达到40nmol/g。(4)系统地研究该探针的性能。该探针具有良好的特异性,可有效检测出单碱基多态性,而且检测时间短,10min就有90%的荧光信号产生。并通过多功能分子成像仪和流式细胞仪实现对目标核酸miRNA-21以及TK1 DNA的同时检测。本研究将为基于聚苯乙烯-纳米金复合微球的核酸探针应用于疾病诊断提供重要的实验数据。更多还原

【Abstract】 Polystyrene(PS)microspheres are often used as the carrier materials in liquid biochips for high-throughput detection of biomolecules because of the micrometer scale size and easy to functional modification.They are.Molecular beacon is the nucleic acid probe with stem-loop structure,which can detect single nucleotide polymorphism because of its better specificity than linear nucleic acid probe.In this paper,polystyrene microspheres were combined with molecular beacon and Au NPs were used to quench the fluorescent substances,replacing the traditional quenching groups of molecular beacon.As a result,a new nucleic acid probe based on polystyrene – gold nanoparticles composite microspheres was developed.The specific research contents are as follows:(1)Monodisperse polystyrene microspheres were prepared by dispersion polymerization.The results showed that the prepared monodisperse microspheres had good monodispersity and a narrow particle size distribution.The surface was smooth,and the diameter was about 2μm.(2)The surface of polystyrene microspheres was coated with a layer of poly dopamine by self-polymerization of dopamine in alkaline solution.The gold nanoparticles were deposited on the surface of the microspheres by the reduction of chloroauric acid in situ by the dopamine.The particle size was about 20 nm.Then,the particle was taken as the nucleus,the reducing agent hydrochloric acid hydroxylamine was used to reduce the chloroauric acid,and the gold nanoparticles were grown.Finally,the composite microspheres with gold nanoparticles were obtained.(3)A molecular beacon with one end thiolated was connected to the surface of polystyrene nanogold by Au-S bond to prepare a nucleic acid probe.The effect of NaCl concentration on the molecular beacon loading on the surface of the microspheres was investigated.The loading of molecular beacon on the surface of the microspheres was up to 40 nmol / g when the NaCl concentration was 0.6 M.(4)The performance of this probe was systematically investigated.The probe had a good specificity,can effectively detect the single nucleotide polymorphism,and detection time was short,about 90% of the fluorescent signal was generated within 10 min.And the target nucleic acid miRNA-21 and TK1 DNA were simultaneously detected by using multifunctional molecular imager and flow cytometer.This study will provide important experimental data for the application of nucleic acid probes based on polystyrene – gold nanoparticles composite microspheres for disease diagnosis.更多还原