【作者】 杜磊；

【导师】 骆清铭；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2004， 博士

【摘要】 在药物开发、新药筛选和快速诊断中,一般都要求对数量巨大的待测分子进行筛选检测。常规的高通量筛选技术是将所有的待测分子分别置于相互隔离的多孔板和微试管中,或者将其进行形如高密度DNA芯片的特殊定位,再进行多样品平行筛选。该法不能对不同样品进行同步混合筛选,且操作繁琐、技术复杂、设备昂贵。另一种技术是采用微载体如微球,将不同的待测分子定位于不同微球的表面进行筛选,并对微球进行编码。将载有不同待测分子的微球混合在一起进行筛选,只需识别筛选结果中挑出微球的编码,就可以确定与探针作用的待测分子。该技术具有显著的灵活性和优点,甚至可以实现多种不同的探针进行同步的微量混合筛选。目前常见的微球编码方法有化学编码和物理编码两大类,但在应用中仍存在诸如编码稳定性、反应兼容性和成本等方面的问题而受到限制。光谱自编码微球的原理是利用苯乙烯的同系物与苯乙烯进行共聚合成,得到与聚苯乙烯树脂在化学和物理性质上相似,并具有可识别的、稳定的光谱特征的编码微球;讨论了苯乙烯悬浮聚合反应中的反应体系、聚合温度、搅拌速率、引发剂和分散剂用量等诸因素对反应结果的影响;并对苯乙烯的乳液聚合、分散聚合和种子聚合等不同聚合方法进行了对比。将悬浮聚合与分散聚合法相结合,采用了双引发剂优化聚合法,反应总收率88.4%,其中过70目标准筛收率为22.8%。将苯乙烯及其6种烃基同系物按不同的排列组合配方进行共聚,合成出了20种具有不同红外特征光谱的编码微球,其稳定可识别的红外光谱特征峰主要出现在1400~1200cm-1和900~500cm-1两个波数区。光谱自编码微球表面的活性氯甲基浓度为2.96×10-4~3.27×10-4 mol/g。利用GAUSS软件,在计算机集群服务器上对光谱自编码微球的各种苯乙烯共聚单体进行了分子振动能级模拟计算,并换算成红外吸收峰。与实测的各单体红外光谱进行了对比。在PC机上利用Visual C++ 6.0编程,将20种编码微球的红外光谱编码转化成可视化条形码,并开发出了相应的编码识别软件。将组合策略中的定位扫描筛选策略与编码微球相结合,提出了两种新的编码组合策略:定位扫描筛选策略和定位编码解析策略。 <WP=4>比较了紫外吸收和荧光分光法对聚苯乙烯树脂的免疫吸附的测量精度,证实了荧光分光法在精度和可测量范围均优于前者。讨论了微球粒度、包被温度、包被液pH值和浓度等不同条件对聚苯乙烯树脂微球免疫吸附的影响。并以戊二醛表面处理法进行了聚苯乙烯树脂微球与免疫球蛋白共价交联。采用激光扫描荧光成像技术,对基于亚毫米级微球的免疫荧光筛选和荧光成像进行了探索。通过酶联免疫交叉矩阵对照实验证实,碱性磷酸酶标显色时,相同条件下光谱自编码微球的检测灵敏度50倍于96孔酶标比色板;而在辣根氧化物酶标显色时,相同条件下光谱自编码微球的检测灵敏度62.5倍于96孔酶标比色板。将20种光谱自编码微球与20种不同物种的免疫球蛋白和血清样品一一对映包被,然后将其全部混合在一起,进行多样品混合ELISA筛选。通过软件处理,挑出筛选结果中阳性信号最强微球,进行红外显微分析,识别出其红外特征光谱,从而解读出与探针相作用的阳性样品编号。将20种光谱自编码微球一一对映包被20种不同物种的血清样品,分别加入碱性磷酸酶标山羊抗兔免疫球蛋白、辣根氧化物酶标山羊抗人免疫球蛋白和异硫氰酸荧光素标记山羊抗鼠免疫球蛋白三种探针,对其进行同步混合筛选。一次性从中筛出了鼠血清、人血清和兔血清,再通过高效液相色谱分别对其进行了定量检测。成功开发出一种适用于高通量筛选的、基于编码微球的多探针——多样品的同步混合筛选技术。更多还原

【Abstract】 The discovery, screening and diagnostics of drug generally involve assay making on a great number of molecules. In conventional high throughput screening, each of the molecules is separated in the wells of a microtitre plate or separated by applying at a particular place in an array such as high-density DNA arrays or microchips at the price of expensive facilities and complicated practicing. In the second method, the reactions are carried out on individual microcarriers, for exemple, microbeads — each carrier having a particular molecule bound to its surface. The microbeads can be encoded for mixing the microbeads and subjecting them to assay simultaneously. When a microbead gives a positive reaction, reading the code can identify the substance on its surface. Obeviously this strategy is advantageously more flexibility because multiple discrete assays can be carried out simultaneously in mixing microvolume multi-samples. The present microbead encoding methods include chemical and physical encoding, but both of them are limited in application because of such problems as code stability, reaction compatibility and cost. The optical self-encoding microbeads are synthesized with styrene and its derivative co-monomers and they can be stably encoded by unique vibrational fingerprint of their spectra. Their chemical and physical properties are similar to the polystyrene resin. The reaction system, temperature, stirring speed, amounts of initiator and disperser are discussed in suspension polymerizing reaction of polystyrene. The latex polymerizing, disperse polymerizing and seeds polymerizing methods are compared with suspension polymerizing of polystyrene too. A novel dual initiators optimizing polymerizing way based on suspension and disperse polymerizing method is adopted. Twenty different self-encoding microbeads have been synthesized with styrene, and its six derivative co-monomers in combinatorial way. The identifiable and steady fingerprint of IR spectrum appears in 1400~1200cm-1 and 900~500cm-1. With GAUSS software in computer cluster server, the molecular vibration of styrene and its derivative co-monomers are simulated. The IR spectra of optical self-encoding <WP=6>microbeads are transformed to visible barcodes by identifying software. Based on optical self-encoding microbeads, two novel encoding combinatorial strategies named EPS(Encoding Position Scan) and PED(Position Encode Deconvolution) are put forward. The UV and fluorescence spectrophotometer analysis for measuring immunosorbent on polystyrene resins are compared. The influence of microbeads’ size, coating temperature, content of IgG and pH value of coating buffer solution upon immunosorbent on polystyrene resins are discussed. Applying Ar+-laser fluorescence scan-imaging technology, immunofluorescence screening and fluorescence imaging on sub-millimeter class microbeads are explored successfully.The sensitivity of AP enzyme conjugated antibody in ELISA matrix tests on optical self-encoding microbeads proves 50 times higher than on 96-wells ELISA plate. The sensitivity of HRP enzyme conjugated antibody in ELISA matrix tests on optical self-encoding microbeads proves 62.5 times higher than on 96-wells ELISA plate.Twenty samples are coated on 20 different optical self-encoding microbeads respectively. All beads were mixed after coating and washing for multi-samples ELISA mix-screening. The sample that has positive reaction with assay is defined by identifying IR spectrum of the bead with the strongest signal.Twenty different optical self-encoding microbeads are coated with 20 different kinds of animal serum samples respectively. Three assays are added after mixing all beads for multi-assays & samples ELISA mix-screening. All beads are divided into three groups on average corresponding to three assays. The beads with strongest signal in each group are picked out for analyzing IR spectra. The samples that have positive reaction with different assays are selected and inspected in HPLC for quantitative analysis. The technology of multi-assays & sam更多还原