荧光相关光谱及其在单分子检测中的应用进展

单分子检测在生命科学、化学、物理学等领域具有重要的意义。荧光相关光谱是单分子检测的新技术,在生命科学领域有巨大的应用潜力。综述了荧光相关光谱单分子检测的原理、实验技术以及在生物分子相互作用、活细胞、核酸、疾病诊断、高通量筛选以及与毛细管电泳联用等领域的研究,并展望了其发展前景。     

《单细胞分析》

单细胞分析是分析化学、生物学和医学多学科相互渗透发展形成的跨学科前沿领域。 分析化学新方法新技术丛书——《单细胞分析》，由武汉大学程介克教授等著。全书共分15章，全面系统地介绍了单细胞分析的各种方法，包括毛细管电泳、微流控芯片、多种光学显微镜（荧光显微镜、聚焦荧光显微镜、全内反射荧光显微镜、多光子荧光显微镜、荧光相关显微镜、近场扫描光学显微镜等）、扫描电化学显微镜、质谱成像、原子力显微镜、扫描隧道显微镜图像分析、阿达玛变换显微光谱及成像、肿瘤电化学及免疫分析、动力学分析、荧光及发光探针、纳米技术以及实时动态检测等新技术和新方法。     

《单细胞分析》

单细胞分析是分析化学、生物学和医学多学科相互渗透发展形成的跨学科前沿领域。分析化学新方法新技术丛书———《单细胞分析》,由武汉大学程介克教授等著。全书共分15章,全面系统地介绍了单细胞分析的各种方法,包括毛细管电泳、微流控芯片、多种光学显微镜(荧光显微镜、聚焦荧光显微镜、全内反射荧光显微镜、多光子荧光显微镜、荧光相关显微镜、近场扫描光学显微镜等)、扫描电化学显微镜、质谱成像、原子力显微镜、扫描隧道显微镜图像分析、阿达玛变换显微光谱及成像、肿瘤电化学及免疫分析、动力学分析、荧光及发光探针、纳米技术以及实…     

小分子荧光探针在绿色农药开发中的应用

小分子荧光探针以其灵敏度高、特异性强、稳定性好、操作便捷和成本低等特点在生命科学、医药化学和环境科学等领域得到了广泛的应用。在农药化学领域,小分子荧光探针常被用作农药残留及重金属污染的检测手段。近年来随着全球开发绿色农药战略需求的不断增强,作为靶向型药物设计和高通量筛选的重要分子工具,荧光探针在绿色农药新产品研发领域的应用不断普及和深化。本文从探针分子的化学设计、靶点识别及药物筛选的角度出发,围绕不同类型的绿色农药重要生物靶点,综述了小分子荧光探针在绿色农药开发领域的研究现状,并对其未来的发展趋势和应用前景进行了展望。     

新型化学敏感器的合成及性能研究

分子识别 (Molecular Recognition)是超分子科学研究中的一个基本问题.在生命科学中酶的特殊催化功能不仅表现为其催化能力的高效性,更重要的是在于它能特征性的对某些化合物分子发生专一性的反应.这种能力的产生是建立在酶对于底物分子识别基础上的,因此设计和合成有光谱响应的荧光化学敏感器和显色试剂在生物化学、临床医学、环境科学等与人类生命科学密切相关的领域中都有着很强的应用前景.本论文较系统地总结了近年来分子识别的发展状况,特别注意到近年来发展起来的通过改变体系色调而进行检测的方法,并将此于本论文工作中加以实践.     

空间分辨荧光分析技术

空间分辨荧光分析技术突破了传统荧光分析的局限,为获得空间定位信息提供了技术保障。系统地综述了构成该技术的共焦荧光法、全内反射荧光法、多光子荧光法以及近场荧光法等4种方法的原理、特点、发展及其应用,并且强调了其在单分子测定中的作用。引用文献64篇。     

分子信标的构建及其应用研究进展

分子信标(molecular beacon,MB)是一种寡聚核苷酸荧光探针,其具有灵敏度高、特异性强、操作简单以及不必与未反应的探针分离即可实时检测等优点,在分子生物学和基因组学及分子医学等领域具有十分重要的应用价值。本文介绍了近年来出现的各种新型分子信标的结构及工作原理,概述了分子信标技术在生命科学领域中的应用,展望了分子信标技术的发展趋势。     

分子信标的构建及其应用研究进展

分子信标（molecular beacon,MB）是一种寡聚核苷酸荧光探针,其具有灵敏度高、特异性强、操作简单以及不必与未反应的探针分离即可实时检测等优点,在分子生物学和基因组学及分子医学等领域具有十分重要的应用价值。 本文介绍了近年来出现的各种新型分子信标的结构及工作原理,概述了分子信标技术在生命科学领域中的应用,展望了分子信标技术的发展趋势。     

应用全内反射单分子荧光成像研究蛋白复合物亚基组成

细胞的生化过程大都是由蛋白复合物完成的,研究蛋白复合物亚基的组成对于了解蛋白质的结构和生物学功能具有重要的意义,然而如何准确确定蛋白复合物中蛋白质亚基的数量（stoichiometry）仍然是一个挑战.近年来,活细胞体系单分子荧光成像技术的不断发展为原位实时动态地研究蛋白质的结构和性质提供了新的手段.本文主要介绍了应用活细胞全内反射单分子荧光成像技术表征细胞膜区蛋白复合物组成的3种方法,包括单分子漂白步数分析、荧光强度统计分布以及蛋白运动分析,并结合其基本原理介绍了这几种方法在活细胞体系膜蛋白研究中的应用.     

环境污染物的免疫传感检测方法进展

本文首先从免疫传感器的构建开始论述,对近5年有关免疫传感器用于环境污染物检测的文章进行了分类和归纳,对其中的三大热点进行了详细介绍,即全内反射荧光、光波导模式谱和表面等离子共振免疫传感器.其次对该领域的研究现状进行了分析,重点从信号放大技术、多组分检测、传感器的再生以及自动化和小型化等方面进行评述.最后,对免疫传感器用于环境污染物检测的发展趋势作了讨论.     

DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging

Single molecule visualization of protein-DNA complexes can reveal details of reaction mechanisms and macromolecular dynamics inaccessible to traditional biochemical assays. However, these techniques are often limited by the inherent difficulty of collecting statistically relevant information from experiments explicitly designed to look at single events. New approaches that increase throughput capacity of single molecule methods have the potential for making these techniques more readily applicable to a variety of biological questions involving different types of DNA transactions. Here we show that nanofabricated chromium barriers, which are located at strategic positions on a fused silica slide otherwise coated with a supported lipid bilayer, can be used to organize DNA molecules into molecular curtains. The DNA that makes up the curtains is visualized by total internal reflection fluorescence microscopy (TIRFM) allowing simultaneous imaging of hundreds or thousands of aligned molecules. These DNA curtains present a robust experimental platform portending massively parallel data acquisition of individual protein-DNA interactions in real time.     

单分子检测进展

单分子检测做为针对有限可数的化学微观个体性质和行为的测量分析方法,能够提供用传统的宏观测量方法得不到的分子微环境中微观个体信息,受到广泛关注。本文综述了近年来单分子检测领域的进展和单分子检测的基本技术,重点介绍了用电化学方法进行单分子检测的发展状况,并展望了单分子检测的发展前景。     

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.     

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

The ability to detect low concentrations of analytes and in particular low‐abundance biomarkers is of fundamental importance, e.g., for early‐stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single‐molecule bioaffinity assays. While many review articles have highlighted the potentials of single‐molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real‐world applications as one should expect. This Review provides a theoretical background on single‐molecule—or better digital—assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single‐molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.     

单分子毛细管电泳

对室温下液流中的单分子光学检测技术的基本原理、主要方法以及它在生物学中的应用做了综述。重点强调了毛细管电泳与单分子检测相结合-单分子毛细管电泳在分析科学中战略上的重要性，并展望了单分子光学检测技术为特征的单分子毛细管电泳在分析化学中的应用前景。     

Multiplexed detection of nucleic acids in a combinatorial screening chip

Multiplexed diagnostic testing has the potential to dramatically improve the quality of healthcare. Simultaneous measurement of health indicators and/or disease markers reduces turnaround time and analysis cost and speeds up the decision making process for diagnosis and treatment. At present, however, most diagnostic tests only provide information on a single indicator or marker. Development of efficient diagnostic tests capable of parallel screening of infectious disease markers could significantly advance clinical and diagnostic testing in both developed and developing parts of the world. Here, we report the multiplexed detection of nucleic acids as disease markers within discrete wells of a microfluidic chip using molecular beacons and total internal reflection fluorescence microscopy (TIRFM). Using a 4 × 4 array of 200 pL wells, we screened for the presence of four target single stranded oligonucleotides encoding for conserved regions of the genomes of four common viruses: human immunodeficiency virus-1 (HIV-1), human papillomavirus (HPV), Hepatitis A (Hep A) and Hepatitis B (Hep B). Target oligonucleotides are accurately detected and discriminated against alternative oligonucleotides with different sequences. This combinatorial chip represents a versatile platform for the development of clinical diagnostic tests for simultaneous screening, detection and monitoring of a wide range of biological markers of disease and health using minimal sample size.     

A method for visualization of biomolecules labeled by a single quantum dot in living cells by a combination of total internal reflection fluorescence microscopy and intracellular fluorescence microscopy

We developed a simple fluorescence microscopy for acquisition of high-resolution images of single quantum dots (QDs) labeled to biomolecules on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. The method was a combination of total internal reflection fluorescence microscopy (TIRFM) at apical cell surface and intracellular microscopy coupled with focusing objective. Insulin conjugated to single QD (insulin-QD) was chosen as the model system. In order to bind insulin-QDs to insulin receptors on the plasma membrane through the interaction between insulin and its receptor, as well as internalize them, the cells attached on a coverslip were incubated with biotinylated insulin and QD-streptavidin conjugate at 37 °C. Next, fluorescent molecules in the cells were photobleached by illuminating the cells using a 100-W mercury lamp with the wavelengths from 460 to 490 nm. Then, the incident angle of a laser beam was adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the insulin-QDs in the whole cell were excited, and the fluorescent molecules outside the cell were not illuminated. Finally, the images of single insulin-QDs on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell were taken by focusing the objective to different positions, respectively. The resolution and contrast of the fluorescent spots in the images were much higher than those obtained by using epi-fluorescence microscopy and comparable to those obtained by using the conventional TIRFM. The method improved the image acquisition speed for the images on the apical and basal plasma membrane using the conventional TIRFM, and could acquire the high-resolution images in the cell interior quickly.     

基于氧化还原循环信号放大技术的单分子电化学研究进展

现代分析科学的整体发展对分析方法的灵敏度、选择性以及快速响应等有了更高的要求。在单分子水平上实现对目标分子的检测及控制是化学家们长期以来梦寐以求的一项富有挑战性的前沿领域,也是近年来分析科学很重要的前沿发展方向。用电化学方法直接检测单分子面临的一项挑战是单个分子在氧化还原过程中得失电子产生的电流变化太小,现代仪器无法对如此小的电流进行识别。使电极表面氧化还原过程中的电子交换实现多次循环可以放大产生的电流,从而实现单分子水平的直接电化学分析。本文对近期通过循环电子交换过程放大电流信号的技术和装置进行了综述,将各类方法进行对比,并对单分子电化学未来的发展方向进行了展望。     

Statistics of single molecule SERS signals: is there a Poisson distribution of intensities?

This paper is aimed at clarifying the statistics of single molecule (SM) surface enhanced Raman scattering (SERS) signals. The argument of the possible existence of a Poisson distribution in the statistics of intensities in SM-SERS has been used many times in the last decade as a proof of single molecule detection. We show theoretically and experimentally that the conditions under which a Poisson distribution would be present are so unlikely to exist in a real system that there is no other option but to attribute the claims to poor statistical sampling. We believe the argument based on Poisson statistics should be dropped as a proof of single molecule detection in SERS.