【作者】 曹元成；

【导师】 刘曼西；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2007， 博士

【摘要】 多元化与微型化是目前分析技术的发展趋势,他们能够在极短的时间内实现对大量候选物的分析,筛选出特定的目标分子如抗体、抗原、短肽等,而且所需要的样品体积很少,因此在药物开发和疾病诊断等领域具有重要应用前景。在各种新型的分析方法中,悬浮芯片无疑是这些技术中的代表。本文的主要工作是基于量子点(QDs)编码聚苯乙烯微球构建新型的悬浮芯片,完成的工作主要包括以下几个方面:通过磺化接枝制备表面羧基修饰的尺寸在100μm左右的微球,其含量约为2.1 mmol/g;而且此微球表面多孔性大大提高。该羧基化聚苯乙烯微球用不同比例的、发射峰分别在576 nm和628 nm处的两种量子点进行了准确编码;然后,该编码微球通过硅包被,在微球的表面形成了一层硅颗粒外壳,以“封”住掺入多孔中的量子点,从而克服泄漏的问题使之形成稳定的编码。系统地研究了硅包被对编码微球抗漂白性能的影响,结果表明硅包被处理4小时后微球抗光漂白性能为未经硅包被处理的微球的7倍。利用Sulfo-NHS和EDC共价偶联不同序列的DNA探针到不同的编码微球上,其探针密度可以达到2.0 mmol/g。利用此载有探针的编码微球对复杂DNA体系中的靶DNA序列进行杂交检测,结果表明单个微球的荧光光谱可以显著地区分出QDs编码信号和靶信号,并能对复杂体系中的靶DNA进行有效的检测,其检测的最低浓度可以达到0.2μg/mL。DNA探针实验证实基于该种微球的多元化分析是可行的。采用Layer-by-Layer技术,研究了包被完整、结构致密、稳定性好的纳米金包被μm级聚苯乙烯微球(Au@PS)的制备方法。实验中首先合成了柠檬酸稳定的电负性的20 nm左右金颗粒,又通过接枝使作为内核的微球带上与纳米金颗粒相反的电性,通过控制包被时间和金胶体的量实现对微球包被程度的准确控制。研究了一种基于金纳米颗粒包被聚苯乙烯微球的免疫分析方法。人IgG被固定于金包被微球上,其固定量可以达到每克微球160μg;荧光成像证实,基于Au@PS微球的免疫反应具有很好的特异性;进一步实验表明,通过单微球水平的荧光光谱分析可以检测出目标分子最低浓度达到0.01μg/mL,其线性范围为0.05μg/mL～15μg/mL。通过与酶联免疫分析方法比较证实,该分析方法具有良好的可靠性。研究了基于金包被聚苯乙烯微球的QDs编码性能。实验证明金包被微球多孔性表面对QDs的吸附固定量可达到1.0182×10-4 mmol/g,是金包被前的7倍多;能够显著降低QDs的泄漏程度;金包被编码微球的抗漂白性也得到一定程度的提高;通过比较游离QDs和固定QDs的光谱发现,掺入的QDs保持原有的光学性质。多色QDs编码实验证实,金包被微球的编码光谱重叠少,荧光稳定,不同编码信号能够被很好的识别。采用量子点编码微球制备基于微通道的探针阵列,并研究该阵列在免疫学分析中的应用。试验证明该方法所需样品体积小,具有简便、快捷等特点;微球上固定的探针能够很好的保持其生物活性;其编码信号能够被准确地识别。该方法可以通过增加编码微球种类实现同时对微量样品的多元化检测。综上所述,该论文主要研究了基于QDs编码微球分析系统的构建及其在生物分析中的若干应用,其内容主要包括微球表面的改性、探针偶联、QDs荧光编码、分析系统构建等。该论文的研究成果拓宽了基于微球分析系统在化学和生物学领域作为更广泛的检测平台的适用范围,为未来微型化分析技术的发展奠定了基础。更多还原

【Abstract】 Multiplexing and miniaturization are becoming pervasive themes in bioanalysis. Drug discovery, drug screening and diagnostics commonly involve carrying out assays on large numbers of molecules and screening for particular target molecules, such as antigens, antibodies, nucleotides and peptides, in test samples. Promising tools for this purpose are the‘multiplex technologies’, which allow multiple discrete assays to be carried out simultaneously in the same micro-volume sample. The push to measure ever-increasing numbers of species from smaller and smaller sample volumes has led to innovative devices for sample manipulation and ingenious approaches to simultaneous measurement capabilities, and in these approaches, one of the outstanding is suspendant chips. This thesis mainly discusses the construction of quantum dots （QDs） based encoding suspendant microarrays. The main results are as follow:Carboxyl modified 100μm polystyrene （PS） beads were prepared by sulfonation grafting. The surface area and pore volume are greatly improved, and the corresponding carboxyl content is about 2.1 mmol/g. Carboxyl grafted polystyrene beads were precisely encoded by the various ratios of two types QDs whose emission wavelength are 576 nm and 628 nm respectively. The leakage of QDs was greatly decreased and the barcode stability improved greatly by the deposited silica particles on the surface of PS. The anti-photo bleaching of silica-coated beads was systemically studied, which is 7 times longer than the uncoated ones.Then the different encoded beads were covalently immobilized with different human IgG probes and the probe density could reach to 2.0 mmol/g. These probe-linked encoded beads were used to detect the target DNA sequences in complex DNA solution by hybridization. The results show that the QDs and target signals can be obviously identified from single-bead-level spectrum. This strategy can be used to detect DNA targets effectively with a detection limit of 0.2μg/mL in complex solution. DNA probe hybridization experiments indicate that it is feasible to use this kind of beads for multiplex analysis.By Layer-by-Layer assembly technology, well-covered, high-density and stability Au nanoparticles coated PS （Au@PS） was prepared. First, citrate-stabilized and negative charged 20 nm Au nanoparticles were synthesized, then different charged PS were prepared by grafting, and the deposition and assembly of Au nanoparticles on the charged PS core were studied. The coverage degree and shell thickness can be controlled by the assembly time and Au colloids quantity.A novel immunoassay method based on Au@PS was studied. Human IgG was immobilized at the concentration of 16μg/g. The fluorescence imagining experiments indicated the FITC signal could be detected even when the target antibody concentration was as low as 0.01μg/mL on single-bead level spectra and the linear range was 0.05～15μg/mL. The assay results were compared with the ELISA and showed a relatively good reliability.The optical encoding property of QDs based Au@PS beads was also studied. The experiment results showed that the QDs loaded into the porous Au@PS beads reached 1.0182×10^-4 mmol/g, which is 7 times more than before the Au coating; the leakage of the doped QDs was remarkably reduced and the anti-photo bleaching ability of the QDs encoded Au@PS beads was also improved; comparing the spectra of free QDs and the doped QDs, it indicated that the doped QDs kept the initial optical property. Mult-color QDs encoding experiments showed that the coded spectra of Au@PS beads had little over-lap; the encoding signals were easy distinguishable on single bead level.The micro-channel based probe-array was fabricated using the QDs-encoded Au@PS beads and its application in immunoassay was studied. The experiments indicated that the assay approach has theμ-volume sample, simple and convenient properties; the encoding could be well identified.Accordingly, several fabrications and applications of QDs encoding PS based bioassay technology were studied in this dissertation. It mainly includes the surface grafting, probe immobilization, QDs fluorescent encoding and fabrication of bioassay systems. The results expand the application fields of the bead-based assay system in chemistry and biology, and establish the bedrock of micro-analysis technologies for future.更多还原