Single-molecule interfacial electron transfer dynamics manipulated by an external electric current

Interfacial electron transfer (IET) dynamics in a 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) dye molecule/indium tin oxide (ITO) film system have been probed at the ensemble and single-molecule levels. By comparing the difference in the external electric current (EEC) dependence of the fluorescence intensities and lifetimes of the ensembles and single molecules, it is shown that the single-molecule probe can effectively demonstrate IET dynamics. The backward electron transfer and electron transfer from the ground state induce single-molecule fluorescence quenching when an EEC is applied to the DiD/ITO film system.     

Heterogeneous diffusion in thin polymer films as observed by high-temperature single-molecule fluorescence microscopy

Single-molecule fluorescence microscopy was used to investigate the dynamics of perylene diimide (PDI) molecules in thin supported polystyrene (PS) films at temperatures up to 135 °C. Such high temperatures, so far unreached in single-molecule spectroscopy studies, were achieved using a custom-built setup which allows for restricting the heated mass to a minimum. This enables temperature-dependent single-molecule fluorescence studies of structural dynamics in the temperature range most relevant to the processing and to applications of thermoplastic materials. In order to ensure that polymer chains were relaxed, a molecular weight of 3000 g/mol, clearly below the entanglement length of PS, was chosen. We found significant heterogeneities in the motion of single PDI probe molecules near T(g). An analysis of the track radius of the recorded single-probe molecule tracks allowed for a distinction between mobile and immobile molecules. Up to the glass transition temperature in bulk, T(g,bulk), probe molecules were immobile; at temperatures higher than T(g,bulk) + 40 K, all probe molecules were mobile. In the range between 0 and 40 K above T(g,bulk) the fraction of mobile probe molecules strongly depends on film thickness. In 30-nm thin films mobility is observed at lower temperatures than in thick films. The fractions of mobile probe molecules were compared and rationalized using Monte Carlo random walk simulations. Results of these simulations indicate that the observed heterogeneities can be explained by a model which assumes a T(g) profile and an increased probability of probe molecules remaining at the surface, both effects caused by a density profile with decreasing polymer density at the polymer-air interface.     

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.     

Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction

It is experimentally challenging to directly obtain structural information of the transition state (TS), the high-energy bottleneck en route from reactants to products, for solution-phase reactions. Here, we use single-molecule experiments as well as high-level quantum chemical calculations to probe the TS of disulfide bond reduction, a bimolecular nucleophilic substitution (S N2) reaction. We use an atomic force microscope in force-clamp mode to apply mechanical forces to a protein disulfide bond and obtain force-dependent rate constants of the disulfide bond reduction initiated by a variety of nucleophiles. We measure distances to the TS or bond elongation (Delta x), along a 1-D reaction coordinate imposed by mechanical force, of 0.31 +/- 0.05 and 0.44 +/- 0.03 A for thiol-initiated and phosphine-initiated disulfide bond reductions, respectively. These results are in agreement with quantum chemical calculations, which show that the disulfide bond at the TS is longer in phosphine-initiated reduction than in thiol-initiated reduction. We also investigate the effect of solvent environment on the TS geometry by incorporating glycerol into the aqueous solution. In this case, the Delta x value for the phosphine-initiated reduction is decreased to 0.28 +/- 0.04 A whereas it remains unchanged for thiol-initiated reduction, providing a direct test of theoretical calculations of the role of solvent molecules in the reduction TS of an S N2 reaction. These results demonstrate that single-molecule force spectroscopy represents a novel experimental tool to study mechanochemistry and directly probe the sub-?ngstr?m changes in TS structure of solution-phase reactions. Furthermore, this single-molecule method opens new doors to gain molecular level understanding of chemical reactivity when combined with quantum chemical calculations.     

Investigation of photobleaching and saturation of single molecules by fluorophore recrossing events

A method for investigation of photobleaching and saturation of single molecules by fluorophore recrossing events in a laser beam is described. The diffraction-limited probe volumes encountered in single-molecule detection (SMD) produce high excitation irradiance, which can decrease available signal. The single molecules of several dyes were detected and the data was used to extract interpeak times above a defined threshold value. The interpeak times revealed the number of fluorophore recrossing events. The number of molecules detected that were within 2 ms of each other represented a molecular recrossing for this work. Calcein, fluorescein and R-phycoerythrin were analyzed and the saturation irradiance and photobleaching effects were determined as a function of irradiance. This approach is simple and it serves as a method of optimizing experimental conditions for single-molecule detection.     

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb5 molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.     

单分子流式检测仪的研制

采用激光诱导荧光和流体动力学聚焦技术成功地研制出单分子流式检测仪, 实现了对水溶液中单个藻红蛋白及单个DNA分子片段的检测, 检测速率可达到每秒几十次. 与单分子荧光显微术相比, 流式分析将固定的标本台改为流动的单分子悬液, 大大提高了检测速率和统计精确性, 更加适合生物样品的快速、超高灵敏分析.     

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。     

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.     

Quantitative Single-Molecule Electrochemiluminescence Bioassay

A single-molecule electrochemiluminescence bioassay is developed here which allows imaging and direct quantification of single biomolecules. Imaging single biomolecules is realized by localizing the electrochemiluminescence events of the labeled molecules. Such an imaging system allows mapping the spatial distribution of biomolecules with electrochemiluminescence and contains quantitative single-molecule insights. We further quantify biomolecules by spatiotemporally merging the repeated reactions at one molecule site and then counting the clustered molecules. The proposed single-molecule electrochemiluminescence bioassay is used to detect carcinoembryonic antigen, showing a limit of detection of 67 attomole concentration which is 10 000 times better than conventional electrochemiluminescence bioassays. This spatial resolution and sensitivity enable single-molecule electrochemiluminescence bioassay a new toolbox for both specific bioimaging and ultrasensitive quantitative analysis.     

Interaction of an antituberculosis drug with nano-sized cationic micelle: Förster resonance energy transfer from dansyl to rifampicin in the microenvironment

In this contribution, we report studies on the interaction of an antituberculosis drug rifampicin (RF) in a macromolecular assembly of CTAB with an extrinsic fluorescent probe, dansyl chloride (DC). The absorption spectrum of the drug RF has been employed to study Förster resonance energy transfer (FRET) from DC, bound to the CTAB micelle using picosecond resolved fluorescence spectroscopy. We have applied a kinetic model developed by Tachiya to understand the kinetics of energy transfer and the distribution of acceptor (RF) molecules around the donor (DC) molecules in the micellar surface with increasing quencher concentration. The mean number of RF molecules associated with the micelle increases from 0.24 at 20 μm RF concentration to 1.5 at 190 μm RF concentration and consequently the quenching rate constant (k_q) due to the acceptor (RF) molecules increases from 0.23 to 0.75 ns^?1 at 20 and 190 μm RF concentration, respectively. However, the mean number of the quencher molecule and the quenching rate constant does not change significantly beyond a certain RF concentration (150 μm ), which is consistent with the results obtained from time resolved FRET analysis. Moreover, we have explored the diffusion controlled FRET between DC and RF, using microfluidics setup, which reveals that the reaction pathway follows one‐step process.     

Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 × 10⁻¹⁴-3.0 × 10⁻¹² mol L⁻¹ was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.     

Diffusion of lipid-like single-molecule fluorophores in the cell membrane

The dicyanomethylenedihydrofuran (DCDHF) class of single-molecule fluorophores contains an amine donor and a dicyanomethylenedihydrofuran acceptor linked by a conjugated unit (benzene, naphthalene, or styrene). Molecules in this class have a number of useful properties in addition to those usually required for single-molecule studies (such as high fluorescence quantum yield and photostability), including second-order optical nonlinearity, large ground-state dipole moment, and sensitivity to local environment. Moreover, most DCDHF molecules have amphiphilic structures, with a polar dicyanomethylenedihydrofuran headgroup and nonpolar hydrocarbon tails on the amine or furan ring, and can be used as fluorescent lipid analogues for live cell imaging. Here we demonstrate that individual molecules of several different DCDHF lipid analogues can be observed diffusing in the plasma membrane of Chinese hamster ovary cells. The photophysical and diffusive behaviors of the DCDHF lipid analogues in membranes are described and are found to be competitive with the well-known lipid probe N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine.     

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.     

Spin-coated polyethylene films probed by single molecules

We have studied ultrathin spin-coated high-density polyethylene films by means of single-molecule spectroscopy and microscopy at 1.8 K. The films have been doped with 2.3,8.9-dibenzanthanthrene (DBATT) molecules, which function as local reporters of their immediate environment. The orientation distributions of single DBATT probe molecules in 100-200 nm thin films of high-density polyethylene differ markedly from those in low-density films. We have found a preferential orientation of dopant molecules along two well-defined, mutually perpendicular directions. These directions are preserved over at least a 2 mm distance. The strong orientation preference of the probe molecules requires the presence of abundant lateral crystal faces and is therefore not consistent with a spherulitic morphology. Instead, a "shish-kebab" crystal structure is invoked to explain our results.     

Analytical assays based on detecting conformational changes of single molecules.

One common strategy for the detection of biomolecules is labeling either the target itself or an antibody that binds to it. Herein, a different approach, based on detecting the conformational change of a probe molecule induced by binding of the target is discussed. That is, what is being detected is not the presence of the target or the probe, but the conformational change of the probe. Recently, a single-molecule sensor has been developed that exploits this mechanism to detect hybridization of a single DNA oligomer to a DNA probe, as well as specific binding of a single protein to a DNA probe. Biomolecular recognition often involves large conformational changes of the molecules involved, and therefore this strategy may be applicable to other assays.     

Structure of single-molecule single crystals of isotactic polystyrene and their radiation resistance

The structure of single-molecule single crystals of isotactic polystyrene (i-PS) was investigated by electron diffraction (ED). The nanoscale single-molecule single crystals were found to be more resistant to electron irradiation when compared to the larger crystals of many molecules, as indicated by both observation of ED and high-resolution electron microscopy with increasing radiation dose. It is proposed that since the single-molecule single crystals are very small, the secondary electrons escape more frequently from the crystal so that the radiation damage is reduced. Lattice imaging was achieved at room temperature in the case of single-molecule single crystals because of their stability to electron irradiation. Published 1998 John Wiley & Sons, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

J Polym Sci B: Polym Phys 36 : 105–112, 1998     

Quantitative detection of antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling

We presented a sensitive method to quantify antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling. In this method, the biotinylated monoclonal anti-human IgG molecules were immobilized on the silanized glass substrate surface. By the strong biotin-streptavidin affinity, streptavidin-coated quantum dots 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. Finally, the number of fluorescent spots corresponding to single molecules in the subframe images was counted, 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.