基于裂结技术的单分子尺度化学反应研究进展

分子电子学是研究单分子器件的构筑、性质以及功能调控的一门新兴学科。其中,金属/分子/金属结的构筑和表征是现阶段分子电子学的主要研究内容。裂结技术是当前分子电子学研究的主要实验方法,主要包括机械可控裂结技术和扫描隧道显微镜裂结技术。本文对裂结技术进行了介绍,并对近年来利用这些技术,在单分子尺度化学反应的检测和动力学研究,以及将这些技术与溶液环境、静电场、电化学门控等方法相结合,调控单分子器件的电输运性质等方面所取得的进展进行了概述。     

Towards single-molecule optoelectronic devices

Benefiting from the development of molecular electronics and molecular plasmonics, the interplay of light and electronic transport in molecular junctions has attracted growing interest among researchers in both fields, leading to a new research direction of “single-molecule optoelectronics”. Here, we review the latest developments of photo-modulated charge transport, electroluminescence and Raman spectroscopy from single-molecule junctions, and suggest future directions for single-molecule optoelectronics.     

Single-molecule triplet-state photon antibunching at room temperature

We have probed single-molecule metal-to-ligand charge transfer (MLCT) dynamics of a ruthenium complex at room temperature. Using photon antibunching measurements under continuous wave (CW) laser excitation, nonclassical photon statistics, and excitation power dependent measurements, we were able to selectively measure the single-molecule MLCT state lifetime. This work demonstrated, as the first single-molecule photon antibunching measurement of the triplet excited state, a new application of single-molecule spectroscopy on excited-state dynamics and ground-state recovering dynamics of an important class of chemical species that have often been used and studied in energy conversion and electron transfer.     

Probing ground-state single-electron self-exchange across a molecule-metal interface

We have probed single-molecule redox reaction dynamics of hemin (chloride) adsorbed on Ag nanoparticle surfaces by single-molecule surface-enhanced Raman spectroscopy (SMSERS) combined with spectroelectrochemistry. Redox reaction at the molecule/Ag interface is identified and probed by the prominent fluctuations of the Raman frequency of a specific vibrational mode, ν(4), which is a typical marker of the redox state of the iron center in a hemin molecule. On the basis of the autocorrelation and cross-correlation analysis of the single-molecule Raman spectral trajectories and the control measurements of single-molecule spectroelectochemistry and electrochemical STM, we suggest that the single-molecule redox reaction dynamics at the hemin-Ag interface is primarily driven by thermal fluctuations. The spontaneous fluctuation dynamics of the single-molecule redox reaction is measured under no external electric potential across the molecule-metal interfaces, which provides a novel and unique approach to characterize the interfacial electron transfer at the molecule-metal interfaces. Our demonstrated approaches are powerful for obtaining molecular coupling and dynamics involved in interfacial electron transfer processes. The new information obtained is critical for a further understanding, design, and manipulation of the charge transfer processes at the molecule-metal interface or metal-molecule-metal junctions, which are fundamental elements in single-molecule electronics, catalysis, and solar energy conversion.     

A single-molecule study of the inhibition effect of Naringenin on transforming growth factor-β ligand-receptor binding

With single-molecule fluorescence imaging and single-molecule force measurement, we have found that the natural compound Naringenin exerts an inhibition effect on TGF-β ligand-receptor interaction, the initial step of TGF-β signaling.     

Single-molecule fluorescence spectroelectrochemistry of cresyl violet

Here we report a new path to study single molecule electron transfer dynamics by coupling scanning fluorescence microscopy with a potentiostat via a conventional electrochemical cell to enable single-molecule fluorescence spectroelectrochemistry of cresyl violet in aqueous solution, demonstrating that the single-molecule fluorescence intensity of cresyl violet is modulated synchronously with the cyclic voltammetric potential scanning.     

Dependence of single-molecule conductance on molecule junction symmetry

The symmetry of a molecule junction has been shown to play a significant role in determining the conductance of the molecule, but the details of how conductance changes with symmetry have heretofore been unknown. Herein, we investigate a naphthalenedithiol single-molecule system in which sulfur atoms from the molecule are anchored to two facing gold electrodes. In the studied system, the highest single-molecule conductance, for a molecule junction of 1,4-symmetry, is 110 times larger than the lowest single-molecule conductance, for a molecule junction of 2,7-symmetry. We demonstrate clearly that the measured dependence of molecule junction symmetry for single-molecule junctions agrees with theoretical predictions.     

A single-molecule probe based on intramolecular electron transfer

Detecting structure, dynamics, and chemical reactions at the single-molecule level represents the ultimate degree of sensitivity for sensing and imaging. There is a tremendous need to develop new molecular systems and methodology for single-molecule-based sensing. This work presents for the first time the single-molecule spectroscopy of a new molecular probe which uses an intramolecular electron transfer mechanism to detect binding, local structure, and interfacial processes. Moreover, we show how information about the interaction of these probes with their environment is obtained from an analysis of the intensity, duration and time-varying behavior of the single-molecule fluorescence.     

Facile method of constructing polyproteins for single-molecule force spectroscopy studies

Constructing polyproteins consisting of identical tandem repeats of proteins provides an unambiguous method of investigating the mechanical properties of proteins at the single-molecule level using force spectroscopy techniques. Here we report a maleimide-thiol coupling-based facile method of constructing polyproteins for single-molecule force spectroscopy studies on the mechanical properties of proteins. This method allows for the construction of polyproteins in an efficient fashion under room temperature. The resultant thioether bonds are resistant to reduction and make it possible to carry out single-molecule force spectroscopy studies under various redox conditions. This novel method complements existing polyprotein engineering methods and can be easily applied to a wide variety of proteins.     

聚合物单分子力谱的研究进展

在单分子水平研究聚合物体系的分子内及分子间相互作用, 对于揭示其结构-性能的关系, 进而实现对相应功能的调控极为重要. 基于原子力显微镜技术(AFM)的单分子力谱, 由于其操作简单且适用面广, 在单分子研究领域得到了广泛的应用. 本文概括了该技术在生物高分子及合成高分子体系的研究进展. 对于生物高分子体系, 主要介绍了核酸(DNA/RNA)、 蛋白质和多糖(淀粉)的单分子力谱研究及利用各自力学指纹谱对其它分子间的相互作用的研究. 对于合成高分子体系, 主要介绍了聚合物的一级结构与单链弹性的关系及溶剂和聚集态结构等对高分子单链力学性质的影响规律.     

Profiling single-molecule reaction kinetics under nanopore confinement

The study of a single-molecule reaction under nanoconfinement is beneficial for understanding the reactive intermediates and reaction pathways. However, the kinetics model of the single-molecule reaction under confinement remains elusive. Herein we engineered an aerolysin nanopore reactor to elaborate the single-molecule reaction kinetics under nanoconfinement. By identifying the bond-forming and non-bond-forming events directly, a four-state kinetics model is proposed for the first time. Our results demonstrated that the single-molecule reaction kinetics inside a nanopore depends on the frequency of individual reactants captured and the fraction of effective collision inside the nanopore confined space. This insight will guide the design of confined nanopore reactors for resolving the single-molecule chemistry, and shed light on the mechanistic understanding of dynamic covalent chemistry inside confined systems such as supramolecular cages, coordination cages, and micelles.

A four-state kinetics model is proposed to reveal the kinetics of a single-molecule reaction under nanopore confinement.     

On stochastic models of dynamic disorder

In this paper we investigate some general aspects of stochastic models of dynamic disorder. First, we reexamine the Zwanzig model for the kinetics of escape through a fluctuating hole. We show that this model is trivially connected to the canonical model of the broadening of the zero-phonon line (ZPL) in crystals. This provides a new perspective of the Wang-Wolynes expression for the rate of escape from a geometric bottleneck with non-Markovian Gaussian fluctuations. Motivated by recent single-molecule experiments, we examine more general examples of fluctuation processes from the perspective of cumulant expansions. Finally, we discuss recent single-molecule experiments probing enzyme turnover performed by Xie and co-workers.     

Quantum trajectory analysis of single-photon control from a single-molecule source

We investigate theory of single-photon control from a two-level single-molecule source irradiated by laser pulses of various shapes and pulse durations in terms of quantum trajectories which link stochastic dynamics of the radiating source with quantum measurement theory. Using Monte Carlo wave function simulation, we analyze the detailed dissipative dynamics of the single-molecule source and the photon statistics as revealed by repeated Gedanken photon measurement on the single radiating source. We show that much of the photon statistics from the two-level single-molecule single-photon sources, including few-photon emission probability, waiting time distribution, and two-time correlation function of the fluorescent light, can be understood qualitatively from the simple picture of Rabi nutation and pi pulse in terms of pulse areas.     

Molecular Spin Clusters: New Synthetic Approaches and Neutron Scattering Studies

We review our recent work in the field of molecular spin clusters and single-molecule magnets, showing how inelastic neutron scattering (INS) can be used to determine magnetic exchange interactions and anisotropy splittings. A general introduction to neutron scattering precedes selected examples, building upon the first determination of exchange coupling in a transition metal complex using INS, through anisotropic exchange in cobalt(II ) spin clusters to the determination of exchange interactions in a dodecanuclear nickel(II ) wheel. The strength of INS for the accurate determination of anisotropy splittings in single-molecule magnets is revealed. Not only can one determine the axial zero-field splitting parameter D, which plays a key role in single-molecule magnet behavior, but also higher-order terms important in understanding the quantum tunneling behavior. Finally, we review two of our synthetic approaches towards new single-molecule magnets based on nickel, manganese, and iron.     

Quantitative determination of the single-molecule detection regime in fluorescence fluctuation microscopy by means of photon counting histogram analysis

Fluorescence fluctuation experiments are performed in single-molecule detection regime if the fluorescence of at most one molecule is registered at a time. Although the significance of such experiments for investigations of complex nonergodic systems like those met in the biosciences has been stressed out by many scientists, the quantitative and accurate determination of the single-molecule detection regime received rather little attention. In this work we present a method based on the photon counting histogram (PCH) analysis, which enables the determination of the average number N of molecules within the observation volume, for which only the fluorescence of individual molecules is detected at a time. Thus, the accurate design of fluorescence fluctuation experiments performed in single-molecule detection regime is possible. Demonstrative fluorescence fluctuation experiments based on two-photon excitation are performed on diluted solutions of coumarin 153, in order to verify the potential of the PCH analysis in experiments on the single-molecule detection level. If the mean number N of molecules within the excitation volume is larger than 0.048, the probability to simultaneously detect the fluorescence of two or more molecules is no longer negligible, i.e., no single-molecule detection regime. If the mean number N of molecules is lower than 0.0057, the detection limit of the method is reached, i.e., the fluorescence signal cannot be distinguished from the background. Consequently, the concentration of coumarin 153 characteristic for the single-molecule detection regime lies in the range 13-110 pmol/l for the given experimental conditions. We also investigate the influence of the molecular brightness, i.e., detected photons per fluorophore molecule and sampling time, on the single-molecule detection regime.     

Elementary processes of DNA surface hybridization resolved by single-molecule kinetics: implication for macroscopic device performance

Direct monitoring of single-molecule reactions has recently become a promising means of mechanistic investigation. However, the resolution of reaction pathways from single-molecule experiments remains elusive, primarily because of interference from extraneous processes such as bulk diffusion. Herein, we report a single-molecule kinetic investigation of DNA hybridization on a metal surface, as an example of a bimolecular association reaction. The tip of the scanning tunneling microscope (STM) was functionalized with single-stranded DNA (ssDNA), and hybridization with its complementary strand on an Au(111) surface was detected by the increase in the electrical conductance associated with the electron transport through the resulting DNA duplex. Kinetic analyses of the conductance changes successfully resolved the elementary processes, which involve not only the ssDNA strands and their duplex but also partially hybridized intermediate strands, and we found an increase in the hybridization efficiency with increasing the concentration of DNA in contrast to the knowledge obtained previously by conventional ensemble measurements. The rate constants derived from our single-molecule studies provide a rational explanation of these findings, such as the suppression of DNA melting on surfaces with higher DNA coverage. The present methodology, which relies on intermolecular conductance measurements, can be extended to a range of single-molecule reactions and to the exploration of novel chemical syntheses.

Hybridization of a single DNA molecule on a surface was investigated by electrical conductance measurements. The hybridization efficiency increases with increasing the DNA concentration, in contrast to preceding studies with ensemble studies.     

Single-molecule emulsion PCR in microfluidic droplets

The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.     

生物纳米孔道技术在非基因测序方面的研究与应用

纳米孔道分析技术是一种低成本、快速、无需标记的单分子检测技术,仅有20多年的发展历史,在DNA单分子测序领域展示出较好的应用前景,现已有商业化的产品面世且趋于成熟.越来越多的研究表明,纳米孔可作为一个通用的单分子传感器.本文综述了生物纳米孔道分析技术对蛋白质、多肽和核酸等单个分子与孔道间相互作用、动力学和热力学过程的实时监测以及多种生物大分子和金属离子的定量检测等方面的研究进展.在纳米孔技术中,电化学检测系统也十分重要,本文还特别介绍了高带宽及超低电流分辨仪器和相关软件的相关进展.     

Protein unfolding and refolding under force: methodologies for nanomechanics.

An increasing number of inter- and intramolecular interactions can nowadays be probed using single-molecule manipulation techniques. Protein unfolding and refolding is the most representative--though complex--of these interactions. Herein, we review the main modes of performing a force unfolding experiment: the velocity clamp and the new force clamp mode. We also compare some of the physical aspects behind the two most frequently used single-molecule manipulation instrumentations: optical tweezers and atomic force microscopes.     

Understanding dynamic disorder fluctuations in single-molecule enzymatic reactions

Single-molecule studies of enzymatic reactions reveal fluctuations in the reaction rate, which cannot be explained by classic Markovian dynamics. This dynamic disorder is attributed to slow transitions in enzyme conformations that take place over timescales longer than reaction cycle times. In this review we summarize current theoretical models for reaction kinetics in fluctuating, single enzyme systems. Also examined are some of the implications of single-molecule fluctuations for reaction rates in systems such as cells or biosensors that contain a moderate number of molecular copies. We conclude that the dynamic disorder in single-molecule enzyme systems is well-described by available models. However, more work is required to study the effect of single-molecule fluctuations on finite systems over limited periods of time.