Simultaneous High Throughput Single Molecule Electrophoresis and Single Molecule Spectroscopy

Single molecule detection (SMD) has developed rapidly in recent years, especially high-throughput single molecule detection. Such research facilitated several fundamental studies at the single molecule level. In the fixture, SMD may be successfully applied to biological, clinical and medical research for DNA sequencing and single-molecule scans for disease detection. Presently, single-molecule identification of DNA and proteins is performed using fluorescence intensity, mobility or hybridization with a selective probe. In some cases, such methods are insufficient for confident single-molecule identification. Therefore, we invented a high-throughput combination single-molecule spectroscopy/imaging technique for monitoring the spectroscopic differences of several different individual molecules while they migrate in solution. The technique can offer three-dimensional data for each molecule:mobility, fluorescence intensity and spectroscopy information. Two sample systems were selected as test cases. In the first case, λ DNA is labeled with YOYO-Ⅰ,POPO-Ⅲ and a combination of the two dyes. Many individual λ DNA molecules are simultaneously imaged and identified by their spectroscopic differences. In the second case, a biotinylated 2.1 kb PCR product (also labeled with YOYO-Ⅰ) was reacted with avidin-conjugated R-phycoerythrin. The individual reactants and products are also simultaneously imaged and identified by their spectroscopic differences. This technique can be used for high-throughput DNA screening, molecular identification and monitoring intermolecular interactions with a speed of over 2,000,000 molecules per second. The existing method is the highest and most powerful single-molecule screening method available to date. Such technology is expected to have a great impact on single-molecule diagnosis and monitoring molecular interaction at the single molecule level and will be beneficial to early detection and diagnosis of disease (e.g. cancer, HIV). Furthermore, this technique allows one to directly observe and evaluate the data without any complicated calculations.     

单分子光谱检测法的新进展

自上世纪90年代以来，单分子检测已经取得了巨大的进步。它提供了大量非平衡体系中个体或平衡态脉动体系的信息，而这些信息是无法从传统的统计测量中获得的。本文主要介绍近10年来，单分子光谱检测技术从低温到室温、从非溶液介质到水溶液、从固体到活细胞、从有机染料分子到纳米粒子等方面的进展情况。     

单分子水平的酶催化与基因表达研究

近半个多世纪以来生命科学取得了非凡的进展, 从DNA双螺旋结构的提出, 到第一个酶晶体结构的被解析, 都得益于像X射线衍射、核磁共振、质谱这样的物理化学工具的发展. 如今, 在深入细致地定量研究生物活体系统中我们正面临新的挑战, 例如:了解酶及其他大分子复合物在体内是如何实时工作的, 它们在分子数很少时是怎样工作的, 在活细胞中大分子复合物是如何协调工作的, 以及不同的基因在活细胞中分子数很少的情况下是如何实现表达和不表达的等等. 近十多年来, 单分子成像, 超高分辨率显微镜和单分子操纵技术在世界范围内被广泛地运用于生物医学研究, 对生物化学和分子生物学的发展产生着深远的影响, 因为运用这些单分子、超高分辨技术, 使很多如上述的令人感兴趣的生物学问题实现了单分子层面上的研究和理解. 本文拟就近年来相关的物理化学方法特别是单分子方法和技术在生物医学中的应用做一个简要介绍.     

单根ZnO纳米线的室温气敏特性

利用改进的CVD法制备了(002)面取向生长的ZnO纳米线, 通过光刻/剥离的自下而上的组装技术制备了基于单根ZnO纳米线的气敏元件, 其中的ZnO纳米线直径为50-300 nm, 有效长度为2-10 μm. 测试表明在室温条件下, 元件对浓度为500 μL·L-1的氧气和乙醇的灵敏度可以分别达到1.3 和1.2, 而气敏响应时间分别为10 和5 s, 由于单根纳米线的一维小尺寸结构加剧了电流的自加热效应, 因此元件在外界环境为室温的条件下的灵敏度才略有提高.     

Dynamics of Nucleic Acids at Room Temperature Revealed by Pulsed EPR Spectroscopy

The investigation of the structure and conformational dynamics of biomolecules under physiological conditions is challenging for structural biology. Although pulsed electron paramagnetic resonance (like PELDOR) techniques provide long‐range distance and orientation information with high accuracy, such studies are usually performed at cryogenic temperatures. At room temperature (RT) PELDOR studies are seemingly impossible due to short electronic relaxation times and loss of dipolar interactions through rotational averaging. We incorporated the rigid nitroxide spin label Ç into a DNA duplex and immobilized the sample on a solid support to overcome this limitation. This enabled orientation‐selective PELDOR measurements at RT. A comparison with data recorded at 50 K revealed averaging of internal dynamics, which occur on the ns time range at RT. Thus, our approach adds a new method to study structural and dynamical processes at physiological temperature in the <10 μs time range with atomistic resolution.     

基于原子力显微镜的高分子单分子力学研究

原子力显微镜(AFM)从根本上改变了人们对单个原子和分子的作用和认识方式。单分子力谱是基于原子力显微镜力的测量方法。概速了近年来利用基于原子力显微镜的单分子力谱研究单个高分子分子内及分子闻作用力的进展。     

Observation of Quantum Interference at Room Temperature in a Single Perovskite Quantum Dot

Break junction technique allows researchers to probe charge transport properties in a single Perovskite quantum dot(QD)with anÅngstrom scale resolution,and observe signatures of quantum interference effects at room temperature.     

常温下C60晶体中分子旋转势垒的研究

采用Ｌ－Ｊ加和势对晶体中Ｃ６０分子的旋转进行了研究，求得了对六种不同旋转轴的旋转势垒，由于常温下Ｃ６０晶体中分子在高速旋转，计算过程中除所研究的旋转分子实际处理外，其它分子对旋转分子的相互作用按原子均匀分布在Ｃ６０球面上处理。计算过程中除了考虑晶体中最近邻分子之间的相互作用外，对次近邻分子之间的相互作用本文也进行了考虑。次近邻分子之间的相互作用对旋转势垒影响很小。     

球形全碳分子C60的谱学表征研究

对一种新型的全碳分子C_(60)的谱学表征进行了研究,其UV-VIS、IR及~(13)C-NMR的分析方法和给出的数据与文献报道基本一致。在质谱研究中首次采用DEI及FD方法,给出了非常清晰和可靠的结果。通过亚稳离子方法对DEI中各碎片峰的来源进行了准确的归属。     

Kinetic Spectroscopy of Erythrosin Phosphorescence and Delayed Fluorescence in Aqueous Solution at Room Temperature*

The photophysics and polarization of the phosphorescence and delayed fluorescence of erythrosin in conditions compatible with the current biological applications of the dye (aqueous buffers at pH 7.4 at ambient temperatures) and in ethanol have been studied as a function of dye concentration (10 ^?7-10^?5M) and temperature (245–333K). The emission decay is strictly single exponential and the detailed kinetic analysis of all the rate processes connected with the emitting T₁ state showed that (1) the lowering of the emission lifetime at the higher temperatures is due to a very efficient self-quenching process, (2) the back intersystem crossing rate T_x S₁ is temperature dependent (δE_TS7 kcal mol^?1) but the T₁S₀ is not (E_a0.1 kcal mol^?1) and (3) both intersystem crossing processes are very sensitive to solvent polarity, which accounts for the solvent dependence of the phosphorescence yield and lifetime. The high value of the phosphorescence anisotropy (r₀= 0.25 lt 0.006) is independent of the excitation and emission wavelengths, and its evolution in time accurately reflects the rotational restrictions in solid solutions. The relevance of these findings to studies with protein-dye conjugates is also outlined to facilitate the design and interpretation of phosphorescence depolarization experiments that probe the (μs-ms dynamics of biomolecules and supramolecular systems.     

荧光相关光谱单分子检测系统的研究

基于激光共焦构型,构建了一套高灵敏度和高稳定性的荧光相关光谱单分子检测系统。采用高数值孔径物镜使激光束高度聚焦,并用同一物镜收集样品的荧光,单光子计数器用于实时记录荧光信号。优化系统保持物镜焦点、针孔以及单光子计数器光敏区高度同轴。采用60×1.2(NA)的水浸物镜及直径为160μm的针孔得到的检测系统结构参数为ω0和z0分别为0.42μm和1.33μm,照射微区的体积为1.3fL。单个RhodamineGreen分子可获得13倍的信噪比。用该系统获得了小分子和生物大分子(DNA片段和蛋白质)的高质量荧光相关图谱,成功实现了单个分子的检测。     

Combination of Micro-fluidic Chip with Fluorescence Correlation Spectroscopy for Single Molecule Detection

A single molecule detection technique was developed by the combination of a single channel poly (dimethylsiloxane)/glass micro-fluidic chip and fluorescence correlation spectroscopy (FCS). This method was successfully used to determine the proportion of two model components in the mixture containing fluorescein and the rhodamine-green succinimidyl ester.     

硫酸钠诱导单链聚(N-异丙基丙烯酰胺)相变的单分子力谱研究

聚（N-异丙基丙烯酰胺） （PNIPAM）具有独特的相变行为,已成为人们研究蛋白质折叠等生命过程发生机理的模型体系. 我们利用基于原子力显微镜（AFM）的单分子力谱技术（SMFS）研究了单链PNIPAM在硫酸钠诱导下的相转变过程,并定量化了相变后所形成塌缩结构的稳定性. 通过对单链PNIPAM的单分子力谱实验得知：在相变前,得到单调上升的力曲线,对应着PNIPAM无规线团结构的形变过程;相变后,得到的锯齿型力曲线,对应着PNIPAM塌缩结构在外力诱导下的解折叠过程. 首次从单分子水平观察到在外加盐的作用下,单链PNIPAM低温相转变和高温相转变的差异：相比于低温相转变,高温相转变生成的塌缩结构更加稳定.     

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。     

基于原子力显微镜的单分子力谱在生物研究中的应用

原子力显微镜被广泛应用于生物研究领域,基于原子力显微镜的单分子力谱可以在单分子、单细胞水平上研究生物分子内和分子间的相互作用。 本文介绍了原子力显微镜单分子力谱在生物分子间相互作用、蛋白质去折叠、细胞表面生物分子、细胞力学性质和基于单分子力谱成像等研究中的最新进展。     

Spectral Versatility of Single Reef Coral Fluorescent Proteins Detected by Spectrally‐Resolved Single Molecule Spectroscopy

We use spectrally‐resolved room temperature single molecule spectroscopy to yield insights into the occurrence and dynamics of spectral forms of single tetramers of DsRed and its variants DsRed2, Fluorescent Timer, DsRed_N42H and AG4. The red‐emitting chromophore in DsRed and all studied variants readily converts into a high quantum efficiency super‐red emitting form. We propose the existence of two super‐red forms of different quantum efficiencies. The observed emission from the green‐emitting chromophore is consistent with bulk spectroscopy. We further observe distinct new spectral forms from each variant, which we attribute to a photoinduced chemical reaction leading to a truncated form of the red‐emitting chromophore analogous to the chromophore in the visible fluorescent protein zFP538. Our results have implications for the accurate interpretation of biological and biochemical processes illuminated by fluorescent proteins as well as for choosing appropriate experimental configurations.     

Giant Hysteretic Single‐Molecule Electric Polarisation Switching above Room Temperature

Continual progress has been achieved in information technology through unrelenting miniaturisation of the single memory bit in integrated ferromagnetic, ferroelectric, optical, and related circuits. However, as miniaturisation approaches its theoretical limit, new memory materials are being sought. Herein, we report a unique material exhibiting single‐molecule electric polarisation switching that can operate above room temperature. The phenomenon occurs in a Preyssler‐type polyoxometalate (POM) cluster we call a single‐molecule electret (SME). It exhibits all the characteristics of ferroelectricity but without long‐range dipole ordering. The SME affords bi‐stability as a result of the two potential positions of localisation of a Tb³⁺ ion trapped in the POM, resulting in extremely slow relaxation of the polarisation and electric hysteresis with high spontaneous polarisation and coercive electric fields. Our findings suggest that SMEs can potentially be applied to ultrahigh‐density memory 1 and other molecular‐level electronic devices operating above room temperature. 2