【作者】 李璐；

【导师】 金文睿；

【作者基本信息】 山东大学 ， 分析化学， 2010， 博士

【摘要】 本文分为生物单分子定量检测新方法及电化学发光共振能量转移两部分,其中第一章至第六章为第一部分,第七章至第九章为第二部分。第一章主要对生物单分子激光诱导荧光检测涉及的各种检测方法及荧光探针进行了简要综述。在生物单分子激光诱导荧光检测中涉及的方法主要有共聚焦荧光显微术、全内反射荧光显微术、落射荧光显微术、双光子或多光子荧光显微术等。本章着重介绍了这些方法的原理及其在生物单分子定量研究中的应用,同时也对常用的荧光探针(包括有机荧光染料、纳米粒子、荧光蛋白及稀土离子等)的性质和应用做了简单介绍。第二章中我们建立了一种电化学吸附富集结合全内反射荧光显微术超灵敏的单分子成像定量新方法。我们选择罗丹明6G、Alexa-488标记的羊抗鼠IgG、以及罗丹明6G标记的DNA作为研究对象,利用电化学富集将这些分子吸附到镀有铟锡氧化物的光透导电玻片上,然后利用高灵敏的电子增强型电感耦合器(EMCCD)对玻片上的荧光分子进行全内反射成像。通过一个三维移动控制器移动玻片实现对玻片的顺次成像,对每一个样品获取100帧图片,并对其上对应单分子的荧光点计数。电化学富集的方法提高了已报道的单分子检测方法的灵敏度,结果显示浓度范围在5×10-15到5×10-12 mol/L的罗丹明6G,3×10-15到2×10-12 mol/L的Alexa-488标记的羊抗鼠IgG和3×10-15到2×10-12 mol/L罗丹明6G标记的DNA荧光点数和物质的浓度都具有良好的线性关系。第三章中我们建立了一种超灵敏的检测1.8 nL样品中蛋白质的单分子计数微阵列分析方法。有关微阵列芯片的单分子检测仅有少量文献报道,且这些工作都未能实现整个阵列点的成像。由于单分子检测是以检测到的单分子数作为定量基础,所以提高单分子计数微阵列分析法灵敏度的关键是将整个阵列点上的单分子完全成像。我们首先设计并研究出了微阵列单分子成像系统以实现整个阵列点的单分子成像,其次以量子点取代传统的有机荧光探针对生物分子进行标记,并针对量子点荧光闪烁问题提出了一种捕获微阵列上全部量子点图像的方法。再通过乙醇胺与牛血清白蛋白共同封闭基底降低背景使单分子计数微阵列分析法测定蛋白质的灵敏度比文献报道的方法提高100倍,达到1.8×10-21 mol(1080个分子)。该方法检测样品所需体积极小(1.8 nL),因此可以用来实现珍贵或者少量样品的多次取样测定。我们利用此方法测定了不同时间下人蜕膜基质细胞培养液中骨桥蛋白的含量,研究了活细胞中骨桥蛋白的表达动力学。第四章中我们利用单分子计数微阵列分析法实现了单细胞内无PCR扩增的多种基因表达的定量测定。首先用将不同的DNA1 s点样至玻片上形成形成10个亚阵列,每个亚阵列包括9个点,每个阵列点的直径约为300μm。3μL由单细胞中的mRNA反转录的cDNAs溶液滴至玻片上的亚阵列,不同的cDNAs通过杂交反应结合至亚阵列的各个点上。然后,通过与QDs标记的检测DNA2(QDs-DNA2)杂交反应,cDNAs被QDs标记。最后,使用我们在第三章中应用的单分子阵列成像设备对微阵列成像,对应单个cDNA的亮点被计数定量。这一方法可以检测至3μL样品体积中2×10-16 mol/L的DNA(360个分子),利用微阵列,10个细胞中9种基因表达可以被平行定量。实验的结果依赖于亮点的个数而不是荧光强度,减少了测定误差,保证了结果的可靠性。此方法实现了无PCR扩增的单细胞中多个基因表达的同时定量测定。第五章中我们利用亚微米粒径的磁珠作为标记物发展了一种新的简单易推广的可视单分子检测方法。以往单分子光学检测通常采用激光诱导荧光方法,常常需要高灵敏度的昂贵检测仪器,且荧光检测会受到荧光背景干扰和荧光标记物淬灭等影响。我们用粒径为300 nm的磁珠对DNA单分子标记,这些磁珠在普通光学显微镜下可以观察到。此可视单分子检测方法可以检测3μL样品中浓度低至4.0×10-16mol/L的DNA(720个分子),并实现了单细胞内多种基因的同时测定。这种直接可视的单分子检测方法有很多优点,首先普通光学显微镜即可对单分子信号成像,无需特殊的高灵敏仪器设备；其次由于单分子信号为磁珠的普通光学图像,背景不会干扰检测结果,且信号不会淬灭,因此保证了定量检测结果的可靠性。第六章中我们利用链酶亲和素修饰的磁珠结合AMCA,FAM及Cy5三种荧光染料标记的DNA,制备各种颜色的编码微珠。单个微珠上可以结合上千个染料分子,根据结合三种染料的比例不同,可以实现多种颜色微珠的制备。我们利用制备的各种微珠实现不同生物分子的单分子编码检测。第七章中我们对荧光共振能量转移(FRET)的原理及其在生物分析方面应用作了介绍。共振能量转移作为一种有力的生物分析工具在生物分子构型测定、免疫分析、核酸检测等方面广泛应用。半导体量子点(quantum dots, QD s)由于其优良的光学性能而被广泛应用于FRET体系中,将量子点作为能量供体或者受体引入生物发光共振能量转移(BRET)体系和化学发光共振能量转移(CRET)体系同样引起了人们的兴趣,本章也对QD在共振能量转移体系中的应用进行了综述。第八章中我们建立了一种新的共振能量转移模式—电化学发光共振能量转移(ECRET).以往报道的共振能量转移根据供体激发方式的不同分为荧光共振能量转移、生物发光共振能量转移、化学发光共振能量转移。以电化学发光作为光源激发供体只有能量被受体淬灭的研究,而受体得到能量发射荧光的现象未见报道。我们以鲁米诺作为能量供体,量子点作为能量受体实现了ECRET。本章对基于该体系的ECRET进行了理论计算,而且将ECRET应用至测定蛋白质之间相互作用和蛋白质构型的变化。第九章中我们研究了量子点作为能量供体,Cy5作为能量受体的电化学发光共振能量转移体系。对基于该体系的ECRET进行了理论计算,并将ECRET应用至测定蛋白质构型的变化。基于QDs-Cy5体系的ECRET不仅可以进行DNA的定性定量研究,也是蛋白质相互作用、蛋白质构型测定研究中一个有用的工具,它将为生命科学提供一种新的研究方法。更多还原

【Abstract】 In chapter one, fluorescence methods in single-molecule detection (SMD) for biomolecules and fluorescent probes were reviewed briefly. The main fluorescence methods in SMD are confocal fluorescence microscopy (CFM), total internal reflection fluorescence microscopy (TIRFM), epi-fluorescence microscopy (EFM) and two-photon or multi-photon laser scanning fluorescence microscopy (TPFM, MPFM). The principles of these techniques and methods and their applications for quantification of biomolecules were reviewed. The fluorescent probes including organic dye, nanoparticles, fluorescent protein, rare-earth-metal ions were also described.In chapter two, we developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat anti rat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6-CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope cover slip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40×40μm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying charge coupled device. One hundred images of the fluorescent molecules accumulated on the cover slip were taken consecutively, one by one, by moving the cover slip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5×10-15 to 5×10-12 mol/L for R6G,3×10-15 to 2×10-12 mol/L for IgG(H+L)-488, and 3×10-15 to 2×10-12 mol/L for DNA-R6G.In chapter three, a novel ultra-sensitive single-molecule-counting microarray assay (SMCMA) with a 1.8-nL sample volume for quantification of protein was developed using total internal reflection fluorescence microscopy coupled with fluorescent quantum dot (QD)-labeling. In life sciences, many protein microarray assays are required to detect proteins from small sample volumes and to describe low abundance levels in absolute terms (numbers or molar amounts). Therefore, in protein microarray assays, ultra-sensitive detection methods with ultra-small volumes have become increasingly important. SMD is rarely used in microarray assays, because the conventional scanners used in microarray assays cannot acquire images of single fluorescent molecules due to low sensitivity and resolution, and when SMD is used in microarray assays, the biggest difficulty is acquiring the whole microarray image. It is because the size of the images taken using SMD techniques is much less than that of a microspot on a microarray. In almost all reports concerning SMD-based microarray assays, only a part of each microspot was acquired. The readout system or method is crucial for the SMD-based microarray assays. In the SMCMA, a single-molecule microarray reader was used to acquire the whole images of microarrays at single-molecule level. Owing to the remarkably high photostability, QDs as labels are much better than fluorescent dyes and can withstand numerous illumination cycles without photoquenching during laser scanning the microarrays when acquiring single-molecule images. These perfect photochemical characteristics of QDs improved the signal and increased the sensitivity in the SMCMA. Using the present SMCMA, an amount as low as 1.8×10-21 mole (1080 molecules) for proteins in 1.8-nL samples could be detected. The SMCMA with 1.8-nL sample volume makes dynamic detection of protein expression for the same alive cells possible. Here, the SMCMA was applied to dynamically measure osteopontin (OPN) expression of decidual stromal cells (DSCs).In chapter four, A novel ultra-sensitive and high-selective single-molecule-counting microarray assay (SMCMA) of DNA for single-cell multi-gene expression was developed using a single-molecule microarray reader coupled with fluorescent quantum dot (QD)-labeling. In the SMCMA, a microarray fabricated on a silanized glass coverslip consists of 10 subarrays with 9 spots for each subarray. Each spot with a diameter of-300μm is modified with different capture DNAs (DNA1). The cDNAs corresponding to mRNAs in a single cell are captured to the complementary capture DNAls at different spots of a subarray. After the cDNAs are labeled with QDs using QD-labeled detection DNAs, the image of the microassay is acquired using a single-molecule microarray reader. The amounts of the cDNAs are quantified by counting the bright dots corresponding to single cDNA molecules on the microassy. Using the SMCMA,2×10-16 mol/L DNA in 3 μL sample or as low as 360 molecules of mRNAs in a single cell can be detected. For a microarray, nine different genes in ten different cells can be quantified in parallel. Since quantification relies on the number of bright dots corresponding to single DNA molecules rather than their signal size, the reproducibility of the detected signal intensity becomes irrelevant, thus guaranteeing reliability of the results and reducing detection error. To our knowledge, this is the first report on PCR amplification-independent quantification of multi-gene expression profiling in single cells.In chapter five, a novel visible SMD method for DNA analysis using conventional biological microscope was provided. In the method,300-nm-diameter magnet microbeads (MBs) as labels were bound to target DNA molecules immobilized on microarrays. Target DNA could be quantified based on counting the number of MBs corresponded to single target DNA molecules on the microarrays under a conventional microscope. Using the method, DNA as low as 1.2×10-21 mol (720 molecules) could be detected and multi-gene expression in single cell could be quantified. The method is simple without need of expensive instruments.In chapter six, multicolor optical coding for DNA single-molecule-detection has been achieved by binding dye-DNAs to magnetic microbeads at different ratios. The AMCA, FAM and Cy5 as dyes were used. About one thousand of dyes could be binded to a bead. The use of different ratios of dyes could make multicolor besds. Using these encoded besds, single-molecule-detection for DNA was achieved. This coding technology is expected to open new opportunities in gene expression studies, highthroughput screening, and medical diagnosticsIn chapter seven, the principal of fluorescence resonance energy transfer (FRET) and its biological application were reviewed briefly. The FRET as a powerful technique has been applied in measuring conformational change of biomolecules, immunnoassay and DNA analysis. Luminescent quantum dots (QDs) have been used in FRET, bioluminescence resonance energy transfer (BRET) and chemiluminescence resonance energy transfer (CRET) as acceptors or donors. The application of QDs in resonance energy transfer is also described.In chapter eight, we developed a novel resonance energy transfer, electrochemiluminescence resonance energy transfer (ECRET). In the ECRET technique, the emitters of N-(4-aminobutyl)-N-ethylisoluminol/H2O2 system generated at an electrode through electrochemical reactions act as electrochemiluminescent donors to emit light with a maximum emission of 460 nm and the red fluorescent luminescent semiconductor nanocrystals (quantum dots, QDs), having a maximum emission at 655 nm, serve as acceptors. When a potential is applied to the electrode, the electrochemiluminescent donors transfer energy to the proximal ground-state QD acceptors, producing efficient ECRET. As a result, the QD acceptors emit a light with a longer wavelength of 655 nm than that of the electrochemiluminescent donors (460 nm). From the ECRET spectra consisting of ECL spectra of the electrochemiluminescent donors and emission spectra of the QD acceptors via ECRET, many biological events can be evaluated. We report the ECRET between the luminol molecules and the QDs in luminol-DNA-DNA-QD, luminol-protein-protein-QD and lumino-protein-QD conjugates immobilized on the Au electrode. The ECRET technique could be applied to the investigation of interactions between nucleic acids or proteins and conformational changes of DNA and protein.In chapter nine, the ECRET between the QDs as donors and the Cy5 as acceptors was studied. The new ECRET system was applied to investigate the conformational changes of protein. The ECRET technique based on QDs-Cy5 system could provide a powerful tool to the study in chemistry and biology.更多还原