摘要
本文第一章对单分子检测(SMD)的意义进行了介绍。SMD在生命科学中的应用可以在单分子水平上揭示分子的动态行为、动力学过程和机制,并能够获取单个分子的信息和行为。本章对SMD中涉及的检测手段、荧光标记探针、检测环境等方面的内容进行了综述。对与之相对应的激光诱导荧光显微术(LIFM).量子点(QDs)、固相载体表面固定法等相关内容,以及它们在进行定量的SMD分析,即在单分子计数方面的应用进行了详细的综述。
第二章中我们提出了一种新的、灵敏的利用全内反射荧光显微术(TIRFM)定量基底表面固定了的抗体的分析方法。在我们的工作之前文献用TIRFM结合基底吸附平衡检测了Alexa Fluor标记的抗体溶液,检测限是5.4×10-11 mol L-1。我们用生物素化的单克隆抗体作为模型抗体,选择QDs作为荧光标记探针。首先将抗体固定在环氧基包被的硅烷化基底表面,然后用QDs对其进行荧光标记。采用TIRFM结合电子增强型电感耦合器(EMCCD)对靶分子进行荧光成像检测和信号收集。当溶液的浓度在8.0 X 10-14～5.0×10-12 mol L-1之间时,单分子计数得到的图像中的荧光点数目与抗体溶液浓度有良好的线性关系。线性范围的下限比文献报道的方法低了3个数量级。
第三章中我们通过单分子计数结合落射荧光显微术(EFM)对荧光染料(或染料-抗体结合物)分子在硅烷化基底表面的非特异性吸附进行了表征。非特异性吸附会引起假阳性信号并影响检测结果的准确性。功能性官能团包被的硅烷化基底被广泛用于生物大分子的固定,但由于其表面的疏水本性往往会引起一个强的非特异性吸附。我们制备了三种具有不同终端官能团的硅烷化基底表面,然后用QDs和QDs-抗体结合物(QDs-Ab)作为模型荧光染料和模型蛋白质对基底抑制非特异性吸附的能力进行表征。通过EFM结合EMCCD对非异性吸附的单个QDs或QDs-Ab分子进行识别和检测。采用单分子计数定量基底表面的非特异性吸附的分子数目,结果表明：相对于环氧基或氨基包被的疏水性的基底表面,羧基包被的亲水性的基底表面具有一个更好的抑制非特异性吸附的能力,也更适合SMD中靶分子的固定。
在第四章我们使用单分子计数结合EFM对亲水性玻片基底表面固定了的抗体进行定量。在我们的工作之前文献用亲水性的乙烯醇聚合物对二甲基硅醚聚合物(PDMS)基底进行修饰。修饰后的PDMS基底是亲水性的,其即可以有效地抑制疏水性相互作用引起的蛋白质非特异性吸附又可以用于靶蛋白质的超灵敏检测。我们制备了亲水性的羧基包被的硅烷化基底,并利用接触角测量对其亲水性进行了表征。在对基底表面抑制非特异性吸附的能力进行表征之后,利用抗体分子上的的氨基与基底上的羧基之间形成的酰胺键实现抗体在基底表面的固定。选用QDs作为荧光探针用于标记靶蛋白质分子。最后采用EFM结合EMCCD进行单个分子的识别和检测。利用单分子计数得到的方法的检测线性范围为5.0×10-14～3.0×10-12mol L-1。与文献相比,我们采用玻片代替PDMS作为基底可以降低基底噪声水平,简化基底处理过程；采用EFM代替原子力显微术(AFM)进行靶分子的识别和检测可以增加方法的适用性和仪器的性价比。
第五章构建了一种支持蛋白质多层(SPLs)基底来进行定向的、特异性的抗体分子固定。现有方法大多采用共价键合的方式将抗体固定在基底表面,这会造成抗体固定取向的随机性,降低抗体的生物活性和抗原结合能力。此外在基于固相载体固定的蛋白质检测中,采用BSA封闭未结合的活性位点可以有效地降低非特异性吸附,但这会阻碍结合位点,降低抗体的结合能力。根据这些背景我们进行了下列的研究工作：
1.通过在基底表面依次包被牛血清白蛋白(BSA)、抗-BSA (Anti-BSA)、G蛋白构建了SPLs玻片基底。SPLs基底上各个蛋白质层的功能如下：底层的BSA作为封闭剂用于抑制非特异性吸附；中间的Anti-BSA作为桥梁连接BSA与G蛋白；最上层的G蛋白通过生物亲和力特异性结合抗体分子的Fc端。与文献相比,这种SPLs基底即可以有效的抑制非特异性吸附,又可以实现靶抗体在固相载体表面的定向取向的生物亲和固定。
2.对构建的SPLs抑制非特异性吸附的能力进行了表征。我们用两种不同类型的染料-抗体结合物来表征SPLs基底抑制非特异性吸附的能力。采用单分子计数定量荧光结合物分子在基底表面上非特异性吸附的分子数量,结果表明：SPLs基底可以有效的抑制抗体的非特异性吸附,而且不受荧光标记染料类型的影响。
3.将SPLs基底用于抗体溶液的低浓度检测。通过定向取向的生物亲和力固定可以有效降低抗体分子之间的空间位阻,增加抗体在基底表面的结合数量。在我们的工作之前文献用单分子计数结合TIRFM检测了BSA封闭基底表面的蛋白质溶液,检测限是1.0×10-10mol L-1。我们用单分子计数结合EFM检测了BSA封闭的SPLs基底表面的抗体溶液,检测的线性范围为1.0×10-14到3.0×10-12mol L-1,线性范围的下限比文献报道的方法低了4个数量级。
In chapter one, significance of single-molecule detection (SMD) have been described. SMD, with its ability to detect single molecules, are powerful tools for investigation of dynamic and kinetics of single molecules. The related techniques and methods for SMD analysis have been described here. Corresponding to the mentioned techniques and methods, laser induced fluorescence microscopy (LIFM), quantum dots (QDs) and the strategies for solid-supported immobilization are reviewd. For a further step, a review of their applications for quantitative SMD analysis is provided here.
In chapter two, we developed a sensitive method for quantitative detection of antibody based on single-molecule counting by total internal reflection fluorescence microscopy (TIRFM) with QDs labeling. Alexa Fluor labeled antibody molecules have been detected using TIRFM with adsorption equibrium in literature. The limit of detection (LOD) was only 5.4×10-11 mol L-1. We chose biotinylated monoclonal anti-human IgG molecules as the model antibody. First, antibody molecules were immobilized on the silanized glass substrate surface. By the strong biotin-streptavidin affinity, streptavidin-coated QDs were labeled to the target molecules as fluorescent probe. Then, images of fluorescent spots in the evanescent wave field were obtained by a high-sensitivity electron multiplying charge coupled device (EMCCD). Finally, the number of fluorescent spots corresponding to single molecules in the subframe images was counted based on a single molecule counting approach, one by one. The linear range of 8.0×10-14 to 5.0×10"12 mol L-1 was obtained between the number of single molecules and the sample concentration. The lower limit of the linear range was 3 orders of magnitude lower than that reported in the literature.
In chapter three, we characterized nonspecific adsorption of fluorescent dyes (dye-labeled antibody) on silanized substrate surfaces using single-molecule counting with epi-fluorescence microscopy (EFM). Nonspecific adsorption causes false positive events, decreasing the accuracy and sensitivity of the assays. The silanization of substrate surfaces is a widely used method to attach functional groups such as amino, aldehyde, epoxy, or thiol groups for cross-linking antibody molecules onto the glass surface. Since the silanized surfaces are often hydrophobic as a result of their hydrophobic chains, these surfaces are liable to cause protein adsorption through the hydrophobic interaction between them. At first, three different silanized glass substrates with differently terminated-functional groups were obtained. QDs and QDs-Antibody conjugates were selected as the model target analytes for characterizing nonspecific adsorption. Then EFM coupled with EMCCD was used to indentify and detect single adsorped molecules. Finally, the nonspecific adsorption of adsorped molecules is quantified based on the direct counting of individual fluorescent spots. The results demonstrate that a hydrophilic silanized surface has the lowest nonspecific adsorption and is highly suitable for bioassays.
In chapter four, we described a hydrophilic substrate surface for antibody immobilization and presented a fluorescence single-molecule counting assays for antibody quantification using EFM. The covalent bonding of poly(vinyl alchol) (PVA) on a poly(dimethylsiloxane) (PDMS) surface provided a hydrophilic substrate surface in literature. Nonspecific adsorption of proteins was greatly reduced on the PVA-coated PDMS surface, and target molecules could be immolized with high loading. In our study, nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, QDs were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. EFM coupled with EMCCD was chosen as the tool for single-molecule fluorescence detection here. A linear range of 5.0×10-14 to 3.0×10-12 mol L-1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach. Compare with the literature, coverslip instead of PDMS as the substrate could reduce the background level; EFM instead of AFM as the detection tool could give a more widely application of the method mentioned here.
In chapter five, we presented a platform of supported protein layers (SPLs) surface for oriented and specific antibody immobilization. Current methods for antibody immobilization are mainly based on the covalent bonding of them to substrate surfaces, which often results in steric problems and an inevitable loss in binding affinity. A common strategy for preventing nonspecific adsorption is to block the surfaces with bovine serum albumin (BSA), however, this step often hinder access of molecules of interest to binding sites. Here, our work is described as follows:
1. Constructing the SPLs surface platform for antibody immobilization, which was achieved by attached BSA, anti-BSA, and protein G to carboxyl-terminated substrate surfaces by turns.
2. Nonspecific adsorption of single molecules on SPLs surfaces was investigated. Two different kinds of dye-antibody conjugates were chosen for the characterization of nonspecific adsorption. The results indicated that SPLs had the ability to resit the nonspecific adsorption of antibody, which did not effecet by the kinds of labeled dyes.
3. Quantifying the concentration of antibody binding to SPLs substrate. Protein immobilized on the BSA-blocked substrate surface has been detected using TIRFM with single-molecule counting in literature. The LOD was 1.0×10-10 mol L-1. We immobilized the target antibody molecules to the SPLs substrate surface. The oriented antibody immobilization with high affinity could reduce the steric problems and give a high binding capacity. QDs were labeled to the target molecules as fluorescent probe. Then, images of fluorescent spots were obtained using EFM coupled with EMCCD. The linear range of 1.0×10-14 to 3.0×10-12 mol L-1 was obtained via a single-molecule conting approach. The lower limit of the linear range was 4 orders of magnitude lower than that reported in the literature.
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[1]A. Agrawal, C. Zhang, T. Byassee, R.A. Tripp, and S. Nie, Counting single native biomolecules and intact viruses with color-coded nanoparticles. Anal Chem 78 (2006) 1061-70.
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