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Sample records for single-molecule fret measurements

  1. In situ temperature monitoring in single-molecule FRET experiments

    Science.gov (United States)

    Hartmann, Andreas; Berndt, Frederic; Ollmann, Simon; Krainer, Georg; Schlierf, Michael

    2018-03-01

    Thermodynamic properties of single molecules including enthalpic and entropic contributions are often determined from experiments by a direct control and precise measurement of the local temperature. However, common temperature monitoring techniques using, for example, ultrafine temperature probes can lead to uncertainties as the probe cannot be placed in the vicinity of the molecule of interest. Here, we devised an approach to measure the local temperature in freely diffusing confocal single-molecule Förster Resonance Energy Transfer (smFRET) experiments in situ by directly adding the temperature-sensitive fluorescent dye Rhodamine B, whose fluorescence lifetime serves as a probe of the local temperature in the confocal volume. We demonstrate that the temperature and FRET efficiencies of static and dynamic molecules can be extracted within one measurement simultaneously, without the need of a reference chamber. We anticipate this technique to be particularly useful in the physicochemical analyses of temperature-dependent biomolecular processes from single-molecule measurements.

  2. Kinetic analysis of single molecule FRET transitions without trajectories

    Science.gov (United States)

    Schrangl, Lukas; Göhring, Janett; Schütz, Gerhard J.

    2018-03-01

    Single molecule Förster resonance energy transfer (smFRET) is a popular tool to study biological systems that undergo topological transitions on the nanometer scale. smFRET experiments typically require recording of long smFRET trajectories and subsequent statistical analysis to extract parameters such as the states' lifetimes. Alternatively, analysis of probability distributions exploits the shapes of smFRET distributions at well chosen exposure times and hence works without the acquisition of time traces. Here, we describe a variant that utilizes statistical tests to compare experimental datasets with Monte Carlo simulations. For a given model, parameters are varied to cover the full realistic parameter space. As output, the method yields p-values which quantify the likelihood for each parameter setting to be consistent with the experimental data. The method provides suitable results even if the actual lifetimes differ by an order of magnitude. We also demonstrated the robustness of the method to inaccurately determine input parameters. As proof of concept, the new method was applied to the determination of transition rate constants for Holliday junctions.

  3. Characterizing single-molecule FRET dynamics with probability distribution analysis.

    Science.gov (United States)

    Santoso, Yusdi; Torella, Joseph P; Kapanidis, Achillefs N

    2010-07-12

    Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single-molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two-state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis-testing models of conformational dynamics against experimental data.

  4. Excited-state annihilation reduces power dependence of single-molecule FRET experiments.

    Science.gov (United States)

    Nettels, Daniel; Haenni, Dominik; Maillot, Sacha; Gueye, Moussa; Barth, Anders; Hirschfeld, Verena; Hübner, Christian G; Léonard, Jérémie; Schuler, Benjamin

    2015-12-28

    Single-molecule Förster resonance energy transfer (FRET) experiments are an important method for probing biomolecular structure and dynamics. The results from such experiments appear to be surprisingly independent of the excitation power used, in contradiction to the simple photophysical mechanism usually invoked for FRET. Here we show that excited-state annihilation processes are an essential cause of this behavior. Singlet-singlet annihilation (SSA) is a mechanism of fluorescence quenching induced by Förster-type energy transfer between two fluorophores while they are both in their first excited singlet states (S1S1), which is usually neglected in the interpretation of FRET experiments. However, this approximation is only justified in the limit of low excitation rates. We demonstrate that SSA is evident in fluorescence correlation measurements for the commonly used FRET pair Alexa 488/Alexa 594, with a rate comparable to the rate of energy transfer between the donor excited state and the acceptor ground state (S1S0) that is exploited in FRET experiments. Transient absorption spectroscopy shows that SSA occurs exclusively via energy transfer from Alexa 488 to Alexa 594. Excitation-power dependent microsecond correlation experiments support the conclusion based on previously reported absorption spectra of triplet states that singlet-triplet annihilation (STA) analogously mediates energy transfer if the acceptor is in the triplet state. The results indicate that both SSA and STA have a pronounced effect on the overall FRET process and reduce the power dependence of the observed FRET efficiencies. The existence of annihilation processes thus seems to be essential for using FRET as a reliable spectroscopic ruler at the high excitation rates commonly employed in single-molecule spectroscopy.

  5. BOBA FRET: bootstrap-based analysis of single-molecule FRET data.

    Directory of Open Access Journals (Sweden)

    Sebastian L B König

    Full Text Available Time-binned single-molecule Förster resonance energy transfer (smFRET experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc. are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1/IBS1 is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET, as well as a graphical user interface, is available via http://www.aci.uzh.ch/rna/.

  6. 48-spot single-molecule FRET setup with periodic acceptor excitation

    Science.gov (United States)

    Ingargiola, Antonino; Segal, Maya; Gulinatti, Angelo; Rech, Ivan; Labanca, Ivan; Maccagnani, Piera; Ghioni, Massimo; Weiss, Shimon; Michalet, Xavier

    2018-03-01

    Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

  7. Single-molecule three-color FRET with both negligible spectral overlap and long observation time.

    Directory of Open Access Journals (Sweden)

    Sanghwa Lee

    Full Text Available Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF microscopy.

  8. Single molecule FRET data analysis procedures for FRET efficiency determination: probing the conformations of nucleic acid structures.

    Science.gov (United States)

    Krüger, Asger Christian; Birkedal, Victoria

    2013-11-01

    Single molecule FRET microscopy is an attractive technique for studying structural dynamics and conformational diversity of nucleic acid structures. Some of its strengths are that it can follow structural changes on a fast time scale and identify conformation distributions arising from dynamic or static population heterogeneity. Here, we give a description of the experiment and data analysis procedures of this method and detail what parameters are needed for FRET efficiency calculation. Using single molecule FRET data obtained on G-quadruplex DNA structures that exhibit large conformation diversity, we illustrate that the shape of the FRET distribution changes depending on what parameters are included in the data analysis procedure. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET

    Directory of Open Access Journals (Sweden)

    Mengyi Yang

    2018-01-01

    Full Text Available Summary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editing

  10. Fast-NPS-A Markov Chain Monte Carlo-based analysis tool to obtain structural information from single-molecule FRET measurements

    Science.gov (United States)

    Eilert, Tobias; Beckers, Maximilian; Drechsler, Florian; Michaelis, Jens

    2017-10-01

    The analysis tool and software package Fast-NPS can be used to analyse smFRET data to obtain quantitative structural information about macromolecules in their natural environment. In the algorithm a Bayesian model gives rise to a multivariate probability distribution describing the uncertainty of the structure determination. Since Fast-NPS aims to be an easy-to-use general-purpose analysis tool for a large variety of smFRET networks, we established an MCMC based sampling engine that approximates the target distribution and requires no parameter specification by the user at all. For an efficient local exploration we automatically adapt the multivariate proposal kernel according to the shape of the target distribution. In order to handle multimodality, the sampler is equipped with a parallel tempering scheme that is fully adaptive with respect to temperature spacing and number of chains. Since the molecular surrounding of a dye molecule affects its spatial mobility and thus the smFRET efficiency, we introduce dye models which can be selected for every dye molecule individually. These models allow the user to represent the smFRET network in great detail leading to an increased localisation precision. Finally, a tool to validate the chosen model combination is provided. Programme Files doi:http://dx.doi.org/10.17632/7ztzj63r68.1 Licencing provisions: Apache-2.0 Programming language: GUI in MATLAB (The MathWorks) and the core sampling engine in C++ Nature of problem: Sampling of highly diverse multivariate probability distributions in order to solve for macromolecular structures from smFRET data. Solution method: MCMC algorithm with fully adaptive proposal kernel and parallel tempering scheme.

  11. Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET

    NARCIS (Netherlands)

    Hohlbein, J.; Kapanidis, A.N.

    2016-01-01

    Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open

  12. Single-molecule FRET reveals structural heterogeneity of SDS-bound alpha-synuclein

    NARCIS (Netherlands)

    Veldhuis, Gertjan; Segers-Nolten, Ine; Ferlemann, Eva; Subramaniam, Vinod

    2009-01-01

    SDS-concentration-dependent alpha-synuclein structure: Upon interaction with SDS, alpha Syn folds into a structure with two antiparallel alpha-helices. We show from single-molecule FRET that alpha Synn adopts this conformation in an all-or-none fashion below the SDS critical micelle concentration.

  13. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    IAS Admin

    RESONANCE. February 2015. GENERAL ARTICLE. Single-Molecule Spectroscopy. Every Molecule is Different! Kankan Bhattacharyya. Keywords. Single-molecule ..... Resonance Energy. Transfer (FRET) is an elegant technique to measure the distance between a donor and an acceptor molecule. FRET refers to the.

  14. Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis.

    Science.gov (United States)

    Tomov, Toma E; Tsukanov, Roman; Masoud, Rula; Liber, Miran; Plavner, Noa; Nir, Eyal

    2012-03-07

    Among the advantages of the single-molecule approach when used to study biomolecular structural dynamics and interaction is its ability to distinguish between and independently observe minor subpopulations. In a single-molecule Förster resonance energy transfer (FRET) and alternating laser excitation diffusion experiment, the various populations are apparent in the resultant histograms. However, because histograms are calculated based on the per-burst mean FRET and stoichiometry ratio and not on the internal photon distribution, much of the acquired information is lost, thereby reducing the capabilities of the method. Here we suggest what to our knowledge is a novel statistical analysis tool that significantly enhances these capabilities, and we use it to identify and isolate static and dynamic subpopulations. Based on a kernel density estimator and a proper photon distribution analysis, for each individual burst, we calculate scores that reflect properties of interest. Specifically, we determine the FRET efficiency and brightness ratio distributions and use them to reveal 1), the underlying structure of a two-state DNA-hairpin and a DNA hairpin that is bound to DNA origami; 2), a minor doubly labeled dsDNA subpopulation concealed in a larger singly labeled dsDNA; and 3), functioning DNA origami motors concealed within a larger subpopulation of defective motors. Altogether, these findings demonstrate the usefulness of the proposed approach. The method was developed and tested using simulations, its rationality is described, and a computer algorithm is provided. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET.

    Science.gov (United States)

    Tutkus, Marijonas; Sasnauskas, Giedrius; Rutkauskas, Danielis

    2017-12-01

    Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules. © 2017 Wiley Periodicals, Inc.

  16. Photon-by-Photon Hidden Markov Model Analysis for Microsecond Single-Molecule FRET Kinetics.

    Science.gov (United States)

    Pirchi, Menahem; Tsukanov, Roman; Khamis, Rashid; Tomov, Toma E; Berger, Yaron; Khara, Dinesh C; Volkov, Hadas; Haran, Gilad; Nir, Eyal

    2016-12-29

    The function of biological macromolecules involves large-scale conformational dynamics spanning multiple time scales, from microseconds to seconds. Such conformational motions, which may involve whole domains or subunits of a protein, play a key role in allosteric regulation. There is an urgent need for experimental methods to probe the fastest of these motions. Single-molecule fluorescence experiments can in principle be used for observing such dynamics, but there is a lack of analysis methods that can extract the maximum amount of information from the data, down to the microsecond time scale. To address this issue, we introduce H 2 MM, a maximum likelihood estimation algorithm for photon-by-photon analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments. H 2 MM is based on analytical estimators for model parameters, derived using the Baum-Welch algorithm. An efficient and effective method for the calculation of these estimators is introduced. H 2 MM is shown to accurately retrieve the reaction times from ∼1 s to ∼10 μs and even faster when applied to simulations of freely diffusing molecules. We further apply this algorithm to single-molecule FRET data collected from Holliday junction molecules and show that at low magnesium concentrations their kinetics are as fast as ∼10 4 s -1 . The new algorithm is particularly suitable for experiments on freely diffusing individual molecules and is readily incorporated into existing analysis packages. It paves the way for the broad application of single-molecule fluorescence to study ultrafast functional dynamics of biomolecules.

  17. Single Molecule FRET Analysis of the 11 Discrete Steps of a DNA Actuator

    DEFF Research Database (Denmark)

    Hildebrandt, Lasse; Preus, Søren; Zhang, Zhao

    2014-01-01

    DNA hybridization allows the design and assembly of dynamic DNA-based molecular devices. Such structures usually accomplish their function by the addition of fuel strands that drive the structure from one conformation to a new one or by internal changes in DNA hybridization. We report here...... and nonautonomously. The 11 states of the actuator were investigated by single molecule Forster Resonance Energy Transfer (smFRET) microscopy to obtain information on the static and dynamic heterogeneities of the device. Our results show that the DNA actuator can be effectively locked in several conformations...... with the help of well-designed DNA lock strands. However, the device also shows pronounced static and dynamic heterogeneities both in the unlocked and locked modes, and we suggest possible structural models. Our study allows for the direct visualization of the conformational diversity and movement...

  18. Taking the ruler to the jungle: single-molecule FRET for understanding biomolecular structure and dynamics in live cells.

    Science.gov (United States)

    Sustarsic, Marko; Kapanidis, Achillefs N

    2015-10-01

    Single-molecule Förster resonance energy transfer (smFRET) serves as a molecular ruler that is ideally posed to study static and dynamic heterogeneity in living cells. Observing smFRET in cells requires appropriately integrated labeling, internalization and imaging strategies, and significant progress has been made towards that goal. Pioneering studies have demonstrated smFRET detection in both prokaryotic and eukaryotic systems, using both wide-field and confocal microscopies, and have started to answer exciting biological questions. We anticipate that future technical developments will open the door to smFRET for the study of structure, conformational changes and kinetics of biomolecules in living cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Organization of DNA partners and strand exchange mechanisms during Flp site-specific recombination analyzed by difference topology, single molecule FRET and single molecule TPM.

    Science.gov (United States)

    Ma, Chien-Hui; Liu, Yen-Ting; Savva, Christos G; Rowley, Paul A; Cannon, Brian; Fan, Hsiu-Fang; Russell, Rick; Holzenburg, Andreas; Jayaram, Makkuni

    2014-02-20

    Flp site-specific recombination between two target sites (FRTs) harboring non-homology within the strand exchange region does not yield stable recombinant products. In negatively supercoiled plasmids containing head-to-tail sites, the reaction produces a series of knots with odd-numbered crossings. When the sites are in head-to-head orientation, the knot products contain even-numbered crossings. Both types of knots retain parental DNA configuration. By carrying out Flp recombination after first assembling the topologically well defined Tn3 resolvase synapse, it is possible to determine whether these knots arise by a processive or a dissociative mechanism. The nearly exclusive products from head-to-head and head-to-tail oriented "non-homologous" FRT partners are a 4-noded knot and a 5-noded knot, respectively. The corresponding products from a pair of native (homologous) FRT sites are a 3-noded knot and a 4-noded catenane, respectively. These results are consistent with non-homology-induced two rounds of dissociative recombination by Flp, the first to generate reciprocal recombinants containing non-complementary base pairs and the second to produce parental molecules with restored base pairing. Single molecule fluorescence resonance energy transfer (smFRET) analysis of geometrically restricted FRTs, together with single molecule tethered particle motion (smTPM) assays of unconstrained FRTs, suggests that the sites are preferentially synapsed in an anti-parallel fashion. This selectivity in synapse geometry occurs prior to the chemical steps of recombination, signifying early commitment to a productive reaction path. The cumulative topological, smFRET and smTPM results have implications for the relative orientation of DNA partners and the directionality of strand exchange during recombination mediated by tyrosine site-specific recombinases. Copyright © 2013. Published by Elsevier Ltd.

  20. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

    Science.gov (United States)

    Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

    2018-01-09

    Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

    Science.gov (United States)

    Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Deckers-Hebestreit, Gabriele; Mayer, Günter; Henkel, Thomas; Heitkamp, Thomas; Börsch, Michael

    2016-02-01

    Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -- a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.

  2. Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α.

    Science.gov (United States)

    Kilic, Sinan; Felekyan, Suren; Doroshenko, Olga; Boichenko, Iuliia; Dimura, Mykola; Vardanyan, Hayk; Bryan, Louise C; Arya, Gaurav; Seidel, Claus A M; Fierz, Beat

    2018-01-16

    The dynamic architecture of chromatin fibers, a key determinant of genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in chromatin fibers. We show that nucleosomes engage in short-lived (micro- to milliseconds) stacking interactions with one of their neighbors. This results in discrete tetranucleosome units with distinct interaction registers that interconvert within hundreds of milliseconds. Additionally, we find that dynamic chromatin architecture is modulated by the multivalent architectural protein heterochromatin protein 1α (HP1α), which engages methylated histone tails and thereby transiently stabilizes stacked nucleosomes. This compacted state nevertheless remains dynamic, exhibiting fluctuations on the timescale of HP1α residence times. Overall, this study reveals that exposure of internal DNA sites and nucleosome surfaces in chromatin fibers is governed by an intrinsic dynamic hierarchy from micro- to milliseconds, allowing the gene regulation machinery to access compact chromatin.

  3. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

    KAUST Repository

    Rashid, Fahad

    2017-02-23

    Human flap endonuclease 1 (FEN1) and related structure-specific 5\\'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5\\'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually \\'locks\\' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

  4. Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

    Science.gov (United States)

    Hardin, John W.; Warnasooriya, Chandani; Kondo, Yasushi; Nagai, Kiyoshi; Rueda, David

    2015-01-01

    In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31. PMID:26503251

  5. Conformational dynamics of DNA hairpins at millisecond resolution obtained from analysis of single-molecule FRET histograms.

    Science.gov (United States)

    Tsukanov, Roman; Tomov, Toma E; Berger, Yaron; Liber, Miran; Nir, Eyal

    2013-12-19

    Here we provide high resolution study of DNA hairpin dynamics achieved by probability distribution analysis (PDA) of diffusion-based single-molecule Förster resonance energy transfer (sm-FRET) histograms. The opening and closing rates of three hairpins both free and attached to DNA origami were determined. The agreement with rates previously obtained using the total internal reflection (TIRF) technique and between free hairpins and hairpins attached to origami validated the PDA and demonstrated that the origami had no influence on the hairpin dynamics. From comparison of rates of four DNA hairpins, differing only in stem sequence, and from comparison with rates calculated using nearest-neighbor method and standard transition state theory, we conclude that the unfolding reaction resembles that of melting of DNA duplex with a corresponding sequence and that the folding reaction depends on counterion concentration and not on stem sequence. Our validation and demonstration of the PDA method will encourage its implementation in future high-resolution dynamic studies of freely diffusing biomolecules.

  6. A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

    Directory of Open Access Journals (Sweden)

    Alexander Gust

    2014-09-01

    Full Text Available Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

  7. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    IAS Admin

    GENERAL ARTICLE. Single-Molecule Spectroscopy. Every Molecule is Different! Kankan Bhattacharyya. Keywords. Single-molecule spectroscopy. (SMS), confocal microscopy,. FCS, sm-FRET, FLIM. 1 High-resolution spectrum re- fers to a spectrum consisting of very sharp lines. The sharp lines clearly display transitions to ...

  8. Single-molecule Förster resonance energy transfer (FRET) analysis discloses the dynamics of the DNA-topoisomerase II (Top2) interaction in the presence of TOP2-targeting agents.

    Science.gov (United States)

    Huang, Wan-Chen; Lee, Chun-Ying; Hsieh, Tao-Shih

    2017-07-28

    Topoisomerases play crucial roles in DNA replication, transcription, and recombination. For instance, topoisomerase II (Top2) is critically important for resolving DNA tangles during cell division, and as such, it is a broad anticancer drug target. Top2 regulates DNA topology by transiently breaking one double-stranded DNA molecule (cleavage), allowing a second double strand to pass through the opened DNA gate (opening), and then closing the gate by rejoining the broken ends. Drugs that modulate Top2 catalysis may therefore affect enzymatic activity at several different steps. Previous studies have focused on examining DNA cleavage and ligation; however, the dynamic opening and closing of the DNA gate has been less explored. Here, we used the single-molecule Förster resonance energy transfer (smFRET) method to observe the open and closed state of the DNA gate and to measure dwell times in each state. Our results show that Top2 binds and bends DNA to increase the energy transfer efficiency ( E FRET ), and ATP treatment further induces the fluctuation of E FRET , representing the gate opening and closing. Additionally, our results demonstrate that both types of Top2-targeting anticancer drugs, the catalytic inhibitor dexrazoxane (ICRF187) and mechanistic poison teniposide (VM26), can interfere with DNA gate dynamics and shorten the dwell time in the closed state. Moreover, Top2 bound to the nonhydrolyzable ATP analog 5'-adenylyl-β,γ-imidodiphosphate exhibits altered DNA gate dynamics, but the DNA gate appears to open and close even after N-gate closure. In summary, we have utilized single-molecule detection to unravel Top2 DNA gate dynamics and reveal previously unknown effects of Top2 drugs on these dynamics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

    Science.gov (United States)

    2011-01-01

    Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution. PMID:21338175

  10. Tunable PIE and synchronized gating detections by FastFLIM for quantitative microscopy measurements of fast dynamics of single molecules

    Science.gov (United States)

    Sun, Yuansheng; Coskun, Ulas; Ferreon, Allan Chris; Barbieri, Beniamino; Liao, Shih-Chu Jeff

    2016-03-01

    The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Förster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique -- FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.

  11. FRET structure with non-radiative acceptor provided by dye-linker-glass surface complex and single-molecule photodynamics by TIRFM-polarized imaging

    International Nuclear Information System (INIS)

    Tani, Toshiro; Mashimo, Kei; Suzuki, Tetsu; Horiuchi, Hiromi; Oda, Masaru

    2008-01-01

    We present our recent study of microscopic single-molecule imaging on the artificial complex of tetramethylrhodamine linked with a propyl chain onto silica glass surface, i.e. an asymmetric fluorescence resonance energy transfer (FRET) structure with non-radiative acceptor. In the synthesis of the complex, we used a mixture of two kinds of isomers to introduce rather small photodynamic difference among them. This isomeric structure change will provide more or less a distinctive photophysical change in e.g. non-radiative relaxation rate. Our recent observation at room temperatures, so far, shows that such contributions can be discriminated in the histograms of the fluorescent spot intensities; broad but distinctive multi-components appear. To identify the isomeric difference as a cause of structures, some configurational assumptions are necessary. One such basic prerequisite is that the transition dipoles of the chromophores should be oriented almost parallel to the glass surface. In order to make clear the modeling, we also provide preliminary experiments on the polarization dependence of the imaging under rotating polarization in epi-illumination

  12. Evaluation of the Electronic Structure of Single-Molecule Junctions Based on Current-Voltage and Thermopower Measurements: Application to C60 Single-Molecule Junction.

    Science.gov (United States)

    Komoto, Yuki; Isshiki, Yuji; Fujii, Shintaro; Nishino, Tomoaki; Kiguchi, Manabu

    2017-02-16

    The electronic structure of molecular junctions has a significant impact on their transport properties. Despite the decisive role of the electronic structure, a complete characterization of the electronic structure remains a challenge. This is because there is no straightforward way of measuring electron spectroscopy for an individual molecule trapped in a nanoscale gap between two metal electrodes. Herein, a comprehensive approach to obtain a detailed description of the electronic structure in single-molecule junctions based on the analysis of current-voltage (I-V) and thermoelectric characteristics is described. It is shown that the electronic structure of the prototypical C 60 single-molecule junction can be resolved by analyzing complementary results of the I-V and thermoelectric measurement. This combined approach confirmed that the C 60 single-molecule junction was highly conductive with molecular electronic conductances of 0.033 and 0.003 G 0 and a molecular Seebeck coefficient of -12 μV K -1 . In addition, we revealed that charge transport was mediated by a LUMO whose energy level was located 0.5≈0.6 eV above the Fermi level of the Au electrode. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Direct measurement and modulation of single-molecule coordinative bonding forces in a transition metal complex

    DEFF Research Database (Denmark)

    Hao, Xian; Zhu, Nan; Gschneidtner, Tina

    2013-01-01

    remain a daunting challenge. Here we demonstrate an interdisciplinary and systematic approach that enables measurement and modulation of the coordinative bonding forces in a transition metal complex. Terpyridine is derived with a thiol linker, facilitating covalent attachment of this ligand on both gold...... substrate surfaces and gold-coated atomic force microscopy tips. The coordination and bond breaking between terpyridine and osmium are followed in situ by electrochemically controlled atomic force microscopy at the single-molecule level. The redox state of the central metal atom is found to have......Coordination chemistry has been a consistently active branch of chemistry since Werner's seminal theory of coordination compounds inaugurated in 1893, with the central focus on transition metal complexes. However, control and measurement of metal-ligand interactions at the single-molecule level...

  14. On the Uncertainty in Single Molecule Fluorescent Lifetime and Energy Emission Measurements

    Science.gov (United States)

    Brown, Emery N.; Zhang, Zhenhua; McCollom, Alex D.

    1996-01-01

    Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least squares methods agree and are optimal when the number of detected photons is large, however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67 percent of those can be noise, and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous Poisson processes, we derive the exact joint arrival time probability density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. The ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background noise and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.

  15. Single-molecule conductance measurements of biomolecule translocation across biomimetic nuclear pores

    Science.gov (United States)

    Dekker, Cees

    2012-02-01

    After a brief overview of our recent work on solid-state nanopores, I will present single-molecule transport data across biomimetic nanopores that contain the key regulating parts of the nuclear pore complex (NPC). The mechanism for the remarkable selectivity of NPCs has remained unclear in a large part due to difficulties in designing experiments that can probe the transport at the relevant length and time scales. Building and measuring on biomimetic NPCs provides new opportunities to address this long-standing problem. covalently tether the natively unfolded Phe-Gly rich domains (FG-domains) of human nuclear binding proteins to a solid-state nanopore (a 10-100 nm sized hole in a SiN membrane). Ionic current measurements provide a probe to monitor single molecules that traverse the pore. Translocation events are observed for transport receptors (Impβ), whereas transport of passive molecules (BSA) is found to be blocked. Interestingly, a single type of nuclear pore proteins appears already sufficient to form a selective barrier for transport. A translocation time of about 2.5 ms is measured for Impβ. This time is found to be similar for transport across Nup153 and Nup98 coated pores, although the observed ionic conductance differs between these two types of pores. We compare two simple models for the pore conductance and find, for both Nups, that the data fits best to a model with an open central channel and a condensed layer along the outer circumference of the pore. reproducing the key features of the NPC, our biomimetic approach opens the way to study a wide variety of nucleo-cytoplasmic transport processes at the single-molecule level in vitro.

  16. Fast sensitive amplifier for two-probe conductance measurements in single molecule break junctions

    Science.gov (United States)

    Johnson, Tyler K.; Ivie, Jeffrey A.; Jaruvang, Jason; Monti, Oliver L. A.

    2017-03-01

    We demonstrate an amplifier based on the Wheatstone bridge designed specifically for use in single molecule break junctions. This amplifier exhibits superior performance due to its large bandwidth, flat frequency response, and high sensitivity. The amplifier is capable of measuring conductance values from 102 to 10-6G0 (G0 = 2e2/h), while maintaining a bandwidth in excess of 20 kHz, and shows remarkable resolution in the molecular conductance regime of 10-2 to 10-5 G0.

  17. A new multiplexing single molecule technique for measuring restriction enzyme activity

    Science.gov (United States)

    Harbottle, Allison; Cavanaugh, Jillian; Gordon, Wendy; Loparo, Joseph; Price, Allen

    2012-02-01

    We present a new multiplexing single molecule method for observing the cleavage of DNAs by restriction enzymes. DNAs are attached to a surface at one end using a biotin-streptavidin link and to a micro bead at the other end via a digoxigenin-antidigoxigenin link. The DNAs are stretched by applying a flow. After introduction of the restriction enzyme, the exact time of cleavage of individual DNAs is recorded with video microscopy. We can image hundreds to thousands of DNAs in a single experiment. We are using our technique to search for the signature of facilitated diffusion in the measured rate dependence on ionic strength.

  18. 3D-localization of the a-subunit in F 0F I-ATP synthase by time resolved single-molecule FRET

    Science.gov (United States)

    Düser, Monika G.; Zarrabi, Nawid; Bi, Yumin; Zimmermann, Boris; Dunn, Stanley D.; Börsch, Michael

    2006-02-01

    F °F I-ATP synthases catalyze the ATP formation from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. Internal rotation of subunits couples the chemical reaction at the F I part to the proton translocation through the F ° part. In these enzymes, the membrane-embedded a-subunit is part of the non-rotating 'stator' subunits and provides the proton channel of the F ° motor. At present, the relative position of the a-subunit is not known. We examined the rotary movements of the ɛ-subunit with respect to the non-rotating a-subunit by time resolved singlemolecule fluorescence resonance energy transfer (FRET) using a novel pulsed laser diode. Rotation of the ɛ-subunit during ATP hydrolysis was divided into three major steps. The stopping positions of ɛ resulted in three distinct FRET efficiency levels and FRET donor lifetimes. From these FRET efficiencies the position of the FRET donor at the asubunit was calculated. Different populations of the three resting positions of ɛ, which were observed previously, enabled us to scrutinize the models for the position of the a-subunit in the F ° part.

  19. Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

    Czech Academy of Sciences Publication Activity Database

    Fessl, Tomáš; Adamec, František; Polívka, Tomáš; Foldynová-Trantírková, Silvie; Vácha, František; Trantírek, L.

    2012-01-01

    Roč. 40, č. 16 (2012), s. 10 ISSN 0305-1048 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z60220518 Keywords : in-cell FRET * fluorescence * DNA * nucleic acid * ATTO * in vivo Subject RIV: BO - Biophysics Impact factor: 8.278, year: 2012

  20. Precision ESR Measurements of Transverse Anisotropy in the Single-molecule Magnet Ni4

    Science.gov (United States)

    Friedman, Jonathan; Collett, Charles; Allao Cassaro, Rafael

    We present a method to precisely determine the transverse anisotropy in a single-molecule magnet (SMM) through electron-spin resonance measurements of a tunnel splitting that arises from the anisotropy via first-order perturbation theory. We demonstrate the technique using the SMM Ni4 diluted via co-crystallization in a diamagnetic isostructural analogue. At 5% dilution, we find markedly narrower resonance peaks than are observed in undiluted samples. Ni4 has a zero-field tunnel splitting of 4 GHz, and we measure that transition at several nearby frequencies using custom loop-gap resonators, allowing a precise determination of the tunnel splitting. Because the transition under investigation arises due to a first-order perturbation from the transverse anisotropy, and lies at zero field, we can relate the splitting to the transverse anisotropy independent of any other Hamiltonian parameters. This method can be applied to other SMMs with zero-field tunnel splittings arising from first-order transverse anisotropy perturbations. NSF Grant No. DMR-1310135.

  1. Correlating single-molecule and ensemble-average measurements of peptide adsorption onto different inorganic materials.

    Science.gov (United States)

    Kim, Seong-Oh; Jackman, Joshua A; Mochizuki, Masahito; Yoon, Bo Kyeong; Hayashi, Tomohiro; Cho, Nam-Joon

    2016-06-07

    The coating of solid-binding peptides (SBPs) on inorganic material surfaces holds significant potential for improved surface functionalization at nano-bio interfaces. In most related studies, the goal has been to engineer peptides with selective and high binding affinity for a target material. The role of the material substrate itself in modulating the adsorption behavior of a peptide molecule remains less explored and there are few studies that compare the interaction of one peptide with different inorganic substrates. Herein, using a combination of two experimental techniques, we investigated the adsorption of a 16 amino acid-long random coil peptide to various inorganic substrates - gold, silicon oxide, titanium oxide and aluminum oxide. Quartz crystal microbalance-dissipation (QCM-D) experiments were performed in order to measure the peptide binding affinity for inorganic solid supports at the ensemble average level, and atomic force microscopy (AFM) experiments were conducted in order to determine the adhesion force of a single peptide molecule. A positive trend was observed between the total mass uptake of attached peptide and the single-molecule adhesion force on each substrate. Peptide affinity for gold was appreciably greater than for the oxide substrates. Collectively, the results obtained in this study offer insight into the ways in which inorganic materials can differentially influence and modulate the adhesion of SBPs.

  2. Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements.

    Directory of Open Access Journals (Sweden)

    Gil Rahamim

    Full Text Available Most active biopolymers are dynamic structures; thus, ensembles of such molecules should be characterized by distributions of intra- or intermolecular distances and their fast fluctuations. A method of choice to determine intramolecular distances is based on Förster resonance energy transfer (FRET measurements. Major advances in such measurements were achieved by single molecule FRET measurements. Here, we show that by global analysis of the decay of the emission of both the donor and the acceptor it is also possible to resolve two sub-populations in a mixture of two ensembles of biopolymers by time resolved FRET (trFRET measurements at the ensemble level. We show that two individual intramolecular distance distributions can be determined and characterized in terms of their individual means, full width at half maximum (FWHM, and two corresponding diffusion coefficients which reflect the rates of fast ns fluctuations within each sub-population. An important advantage of the ensemble level trFRET measurements is the ability to use low molecular weight small-sized probes and to determine nanosecond fluctuations of the distance between the probes. The limits of the possible resolution were first tested by simulation and then by preparation of mixtures of two model peptides. The first labeled polypeptide was a relatively rigid Pro7 and the second polypeptide was a flexible molecule consisting of (Gly-Ser7 repeats. The end to end distance distributions and the diffusion coefficients of each peptide were determined. Global analysis of trFRET measurements of a series of mixtures of polypeptides recovered two end-to-end distance distributions and associated intramolecular diffusion coefficients, which were very close to those determined from each of the pure samples. This study is a proof of concept study demonstrating the power of ensemble level trFRET based methods in resolution of subpopulations in ensembles of flexible macromolecules.

  3. Single Molecule Study of Photoconversion and Spectral Heterogeneities of Fluorophores

    DEFF Research Database (Denmark)

    Liao, Zhiyu

    of conformational changes and dynamics. The photophysical properties of organic dyes directly determine the quality of the experiments. So the better understanding of the photophysical properties of organic dyes, the better we are able to design the experiments and interpret the data, especially in single molecule...... 104 single molecule measurements. A simple and practical method is introduced to study the characteristics of the photoproducts at the ensemble level. Control experiments reveal that the reaction leading to photobleaching is oxygen related, but the composition of the photoproducts remains inconclusive...... stimulate new pathways in engineering and designing photoconvertible fluorophores, based on the reaction with oxygen or other chemicals. Besides, this results show that dyes that convert into other emissive species could give problems when interpreting single molecule FRET systems. The revealed mechanism...

  4. Single-Molecule Measurements of Synthesis by DNA Polymerase with Base-Pair Resolution

    Science.gov (United States)

    Christian, Thomas; Romano, Louis; Rueda, David

    2010-03-01

    The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule F"orster resonance energy transfer intensity drops in discrete steps to values consistent with single nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer-template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change following the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the pre-insertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.

  5. Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis

    Science.gov (United States)

    Seol, Yeonee; Strub, Marie-Paule; Neuman, Keir C.

    2016-01-01

    Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student’s t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism. PMID:27131595

  6. Quantum transport through single molecules

    NARCIS (Netherlands)

    Osorio Oliveros, E.A.

    2009-01-01

    This thesis describes three-terminal transport measurements through single molecules. The interest in this field stems from the dream that single molecules will form the building blocks for future nanoscale electronic devices. The advantages are their small size -nanometers-, and their synthetic

  7. Detailed single crystal EPR lineshape measurements for the single molecule magnets Fe8Br and Mn12-ac

    OpenAIRE

    Hill, S.; Maccagnano, S.; Park, K.; Achey, R. M.; North, J. M.; Dalal, N. S.

    2001-01-01

    It is shown that our multi-high-frequency (40-200 GHz) resonant cavity technique yields distortion-free high field EPR spectra for single crystal samples of the uniaxial and biaxial spin S = 10 single molecule magnets (SMMs) [Mn12O12(CH3COO)16(H2O)4].2CH3COOH.4H2O and [Fe8O2(OH)12(tacn)6]Br8.9H2O. The observed lineshapes exhibit a pronounced dependence on temperature, magnetic field, and the spin quantum numbers (Ms values) associated with the levels involved in the transitions. Measurements ...

  8. A distribution-based method to resolve single-molecule Förster resonance energy transfer observations.

    Science.gov (United States)

    Backović, Mihailo; Price, E Shane; Johnson, Carey K; Ralston, John P

    2011-04-14

    We introduce a new approach to analyze single-molecule Förster resonance energy transfer (FRET) data. The method recognizes that FRET efficiencies assumed by traditional ensemble methods are unobservable for single molecules. We propose instead a method to predict distributions of FRET parameters obtained directly from the data. Distributions of FRET rates, given the data, are precisely defined using Bayesian methods and increase the information derived from the data. Benchmark comparisons find that the response time of the new method outperforms traditional methods of averaging. Our approach makes no assumption about the number or distribution of underlying FRET states. The new method also yields information about joint parameter distributions going beyond the standard framework of FRET analysis. For example, the running distribution of FRET means contains more information than any conceivable single measure of FRET efficiency. The method is tested against simulated data and then applied to a pilot-study sample of calmodulin molecules immobilized in lipid vesicles, revealing evidence for multiple dynamical states.

  9. Intrinsic property measurement of surfactant-templated mesoporous silica films using time-resolved single-molecule imaging.

    Science.gov (United States)

    Kennard, Raymond; DeSisto, William J; Giririjan, Thanu Praba; Mason, Michael D

    2008-04-07

    Mesoporous silica membranes fabricated by the surfactant-templated sol-gel process have received attention because of the potential to prepare membranes with a narrow pore size distribution and ordering of the interconnected pores. Potential applications include ultrafiltration, biological separations and drug delivery, and separators in lithium-ion batteries. Despite advancements in synthesis and characterization of these membranes, a quantitative description of the membrane microstructure remains a challenge. Currently the membrane microstructure is characterized by the combination of results from several techniques, i.e., gas permeance testing, x-ray diffraction scanning electron microscopy, transmission electron microscopy, and permporometry. The results from these ensemble methods are then compiled and the data fitted to a particular flow model. Although these methods are very effective in determining membrane performance, general pore size distribution, and defect concentration, they are unable to monitor molecular paths through the membrane and quantitatively measure molecular interactions between the molecular specie and pore network. Single-molecule imaging techniques enable optical measurements that probe materials on nanometer length scales through observation of individual molecules without the influence of averaging. Using single-molecule imaging spectroscopy, we can quantitatively characterize the interaction between the probe molecule and the interior of the pore within mesoporous silica membranes. This approach is radically different from typical membrane characterization methods in that it has the potential to spatially sample the underlying pore structure distribution, the surface energy, and the transport properties. Our hope is that this new fundamental knowledge can be quantitatively linked to both the preparation and the performance of membranes, leading to the advancement of membrane science and technology. Fluorescent molecules, 1

  10. A Monopole Antenna at Optical Frequencies: Single-Molecule Near-Field Measurements

    NARCIS (Netherlands)

    Taminiau, Tim H.; Segerink, Franciscus B.; van Hulst, N.F.

    2007-01-01

    We present a monopole antenna for optical frequencies (~600 THz) and discuss near-field measurements with single fluorescent molecules as a technique to characterize such antennas. The similarities and differences between near-field antenna measurements at optical and radio frequencies are discussed

  11. Bridging the gap between single molecule and ensemble methods for measuring lateral dynamics in the plasma membrane

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Schwartzentruber, J.; Clausen, M. P.

    2013-01-01

    The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing...... the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label...... and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed...

  12. Thermophoretic forces on DNA measured with a single-molecule spring balance

    DEFF Research Database (Denmark)

    Pedersen, Jonas Nyvold; Lüscher, Christopher James; Marie, Rodolphe

    2014-01-01

    of the thermophoretic force in a static configuration finds forces up to 130 fN. This is eleven times stronger than the force experienced by the same molecule in the same thermal gradient in bulk, where the molecule shields itself. Our stronger forces stretch the middle of the molecule up to 80% of its contour length......We stretch a single DNA molecule with thermophoretic forces and measure these forces with a spring balance: the DNA molecule itself. It is an entropic spring which we calibrate, using as a benchmark its Brownian motion in the nanochannel that contains and prestretches it. This direct measurement...

  13. Role of ICAM-1 polymorphisms (G241R, K469E) in mediating its single-molecule binding ability: Atomic force microscopy measurements on living cells

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Rui [Chinese (301) General Hospital, 28 Fuxing Road, Haidian District, Beijing 100853 (China); Yi, Shaoqiong [Beijing Institute of Biotechnology, 20 Dongdajie, Fengtai, Beijing 100071 (China); Zhang, Xuejie [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry Chinese Academy of Sciences, 2 Zhongguancun North 1st Street, Beijing 100190 (China); Liu, Huiliang, E-mail: lhl518@vip.sina.com [Department of Cardiology, The General Hospital of Chinese People’s Armed Police Forces, Beijing 100039 (China); Fang, Xiaohong, E-mail: xfang@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry Chinese Academy of Sciences, 2 Zhongguancun North 1st Street, Beijing 100190 (China)

    2014-06-13

    Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP induced changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.

  14. Fabrication of birefringent nanocylinders for single-molecule force and torque measurement

    International Nuclear Information System (INIS)

    Li, Ping-Chun; T Yu, Edward; Chang, Jen-Chien; La Porta, Arthur

    2014-01-01

    Optically anisotropic subwavelength scale dielectric particles have been shown to enable studies of the mechanical properties of bio-molecules via optical trapping and manipulation. However, techniques emphasized to date for fabrication of such particles generally suffer from limited uniformity and control over particle dimensions, or low throughput and high cost. Here, an approach for rapid, low-cost, fabrication of large quantities of birefringent quartz nanocylinders with dimensions optimized for optical torque wrench experiments is described. For a typical process, 10 8 or more quartz cylinders with diameters of 500 nm and heights of 800 nm, with uniformity of ±5% in each dimension, can be fabricated over ∼10 cm 2 areas, for binding to a single bio-molecule, and harvested for use in optical trapping experiments. Use of these structures to measure extensional and torsional dynamics of single DNA molecules is demonstrated with measured forces and torques shown to be in very good agreement with previously reported results. (papers)

  15. Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange

    NARCIS (Netherlands)

    Loparo, Joseph J.; Kulczyk, Arkadiusz W.; Richardson, Charles C.; Oijen, Antoine M. van

    2011-01-01

    A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the

  16. Graphical models for inferring single molecule dynamics

    Directory of Open Access Journals (Sweden)

    Gonzalez Ruben L

    2010-10-01

    Full Text Available Abstract Background The recent explosion of experimental techniques in single molecule biophysics has generated a variety of novel time series data requiring equally novel computational tools for analysis and inference. This article describes in general terms how graphical modeling may be used to learn from biophysical time series data using the variational Bayesian expectation maximization algorithm (VBEM. The discussion is illustrated by the example of single-molecule fluorescence resonance energy transfer (smFRET versus time data, where the smFRET time series is modeled as a hidden Markov model (HMM with Gaussian observables. A detailed description of smFRET is provided as well. Results The VBEM algorithm returns the model’s evidence and an approximating posterior parameter distribution given the data. The former provides a metric for model selection via maximum evidence (ME, and the latter a description of the model’s parameters learned from the data. ME/VBEM provide several advantages over the more commonly used approach of maximum likelihood (ML optimized by the expectation maximization (EM algorithm, the most important being a natural form of model selection and a well-posed (non-divergent optimization problem. Conclusions The results demonstrate the utility of graphical modeling for inference of dynamic processes in single molecule biophysics.

  17. Single molecules and nanotechnology

    CERN Document Server

    Vogel, Horst

    2007-01-01

    This book focuses on recent advances in the rapidly evolving field of single molecule research. These advances are of importance for the investigation of biopolymers and cellular biochemical reactions, and are essential to the development of quantitative biology. Written by leading experts in the field, the articles cover a broad range of topics, including: quantum photonics of organic dyes and inorganic nanoparticles their use in detecting properties of single molecules the monitoring of single molecule (enzymatic) reactions single protein (un)folding in nanometer-sized confined volumes the dynamics of molecular interactions in biological cells The book is written for advanced students and scientists who wish to survey the concepts, techniques and results of single molecule research and assess them for their own scientific activities.

  18. Data-Driven Techniques for Detecting Dynamical State Changes in Noisily Measured 3D Single-Molecule Trajectories

    Directory of Open Access Journals (Sweden)

    Christopher P. Calderon

    2014-11-01

    Full Text Available Optical microscopes and nanoscale probes (AFM, optical tweezers, etc. afford researchers tools capable of quantitatively exploring how molecules interact with one another in live cells. The analysis of in vivo single-molecule experimental data faces numerous challenges due to the complex, crowded, and time changing environments associated with live cells. Fluctuations and spatially varying systematic forces experienced by molecules change over time; these changes are obscured by “measurement noise” introduced by the experimental probe monitoring the system. In this article, we demonstrate how the Hierarchical Dirichlet Process Switching Linear Dynamical System (HDP-SLDS of Fox et al. [IEEE Transactions on Signal Processing 59] can be used to detect both subtle and abrupt state changes in time series containing “thermal” and “measurement” noise. The approach accounts for temporal dependencies induced by random and “systematic overdamped” forces. The technique does not require one to subjectively select the number of “hidden states” underlying a trajectory in an a priori fashion. The number of hidden states is simultaneously inferred along with change points and parameters characterizing molecular motion in a data-driven fashion. We use large scale simulations to study and compare the new approach to state-of-the-art Hidden Markov Modeling techniques. Simulations mimicking single particle tracking (SPT experiments are the focus of this study.

  19. A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers

    NARCIS (Netherlands)

    Moayed, F.; Mashaghi, A.; Tans, S.J.

    2013-01-01

    Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an

  20. Single-molecule manipulation and detection.

    Science.gov (United States)

    Zhao, Deyu; Liu, Siyun; Gao, Ying

    2018-01-25

    Compared to conventional ensemble methods, studying macromolecules at single-molecule level can reveal extraordinary clear and even surprising views for a biological reaction. In the past 20 years, single-molecule techniques have been undergoing a very rapid development, and these cutting edge technologies have revolutionized the biological research by facilitating single-molecule manipulation and detection. Here we give a brief review about these advanced techniques, including optical tweezers, magnetic tweezers, atomic force microscopy (AFM), hydrodynamic flow-stretching assay, and single-molecule fluorescence resonance energy transfer (smFRET). We are trying to describe their basic principles and provide a few examples of applications for each technique. This review aims to give a rather introductory survey of single-molecule techniques for audiences with biological or biophysical background. © The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 2. Single-Molecule Spectroscopy: Every Molecule is Different! Kankan Bhattacharyya. General Article Volume 20 Issue 2 February 2015 pp 151-164. Fulltext. Click here to view fulltext PDF. Permanent link:

  2. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 2. Single-Molecule Spectroscopy: Every Molecule is Different! ... Author Affiliations. Kankan Bhattacharyya1. Department of Physical Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata 700 032 India.

  3. Measurements on MIMO-FRET Nano-Networks Based on Alexa Fluor Dyes

    Science.gov (United States)

    Wojcik, Krzysztof; Solarczyk, Kamil; Kulakowski, Pawel

    2015-05-01

    Nano-communication has gained significant attention in the last few years, as a means to establish information transfer between future nano-machines. Comparing with other communication techniques for nano-scale (calcium ions signaling, molecular or catalytic nanomotors, pheromones propagation, bacteria-based communication), the phenomenon called Forster Resonance Energy Transfer (FRET) offers significantly smaller propagation delays and high channel throughput. In this paper, we report our recent experiments on FRET-based nano-networks performed in the Laboratory of Cell Biophysics of the Jagiellonian University, Krakow. We propose to use Alexa Fluor dyes as nano transmitters and receivers, as they enable to create multiple-input multi-output (MIMO) FRET communication channels and thus enhance FRET efficiency. We measure FRET efficiency values, calculate bit error rates for the measured scenarios and extend the calculations to consider a general case of MIMO (n,m) FRET channels.

  4. Preface: Special Topic on Single-Molecule Biophysics.

    Science.gov (United States)

    Makarov, Dmitrii E; Schuler, Benjamin

    2018-03-28

    Single-molecule measurements are now almost routinely used to study biological systems and processes. The scope of this special topic emphasizes the physics side of single-molecule observations, with the goal of highlighting new developments in physical techniques as well as conceptual insights that single-molecule measurements bring to biophysics. This issue also comprises recent advances in theoretical physical models of single-molecule phenomena, interpretation of single-molecule signals, and fundamental areas of statistical mechanics that are related to single-molecule observations. A particular goal is to illustrate the increasing synergy between theory, simulation, and experiment in single-molecule biophysics.

  5. Single molecule studies of surface-induced secondary structure in a model peptide

    Science.gov (United States)

    English, Douglas S.; Cunningham, Joy A.; Wehri, Sarah C.; Petrik, Amy F.; Okamoto, Kenji

    2004-10-01

    We have proposed using single molecule fluorescence resonant energy transfer (SM-FRET) to investigate the induction of secondary structure in model, surface-active peptides upon binding at an interface. The ability for SM-FRET to distinguish structural heterogeneity will offer a distinct advantage over traditional biophysical methods in these types of studies. Ensemble methods mask heterogeneity and only provide an average measure of secondary structural features. Because secondary structure contributes greatly to the energetics of dehydrating the amide backbone, detailed information of conformational distributions is crucial to the understanding of the thermodynamic cycle involved. Here we present results from our first efforts at using SM-FRET to study an amphipathic α-helix forming peptide immobilized at the solid-liquid interface between an aqueous solution and an octadecylsilane modified glass surface. This system serves as a model for future studies of peptide partitioning to lipid bilayers and other relevant interfaces.

  6. Single molecule Studies of DNA Mismatch Repair

    Science.gov (United States)

    Erie, Dorothy A.; Weninger, Keith R.

    2015-01-01

    DNA mismatch repair involves is a widely conserved set of proteins that is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair. PMID:24746644

  7. Watching single molecules dance

    Science.gov (United States)

    Mehta, Amit Dinesh

    Molecular motors convert chemical energy, from ATP hydrolysis or ion flow, into mechanical motion. A variety of increasingly precise mechanical probes have been developed to monitor and perturb these motors at the single molecule level. Several outstanding questions can be best approached at the single molecule level. These include: how far does a motor progress per energy quanta consumed? how does its reaction cycle respond to load? how many productive catalytic cycles can it undergo per diffusional encounter with its track? and what is the mechanical stiffness of a single molecule connection? A dual beam optical trap, in conjunction with in vitro ensemble motility assays, has been used to characterize two members of the myosin superfamily: muscle myosin II and chick brain myosin V. Both move the helical polymer actin, but myosin II acts in large ensembles to drive muscle contraction or cytokinesis, while myosin V acts in small numbers to transport vesicles. An optical trapping apparatus was rendered sufficiently precise to identify a myosin working stroke with 1nm or so, barring systematic errors such as those perhaps due to random protein orientations. This and other light microscopic motility assays were used to characterize myosin V: unlike myosin II this vesicle transport protein moves through many increments of travel while remaining strongly bound to a single actin filament. The step size, stall force, and travel distance of myosin V reveal a remarkably efficient motor capable of moving along a helical track for over a micrometer without significantly spiraling around it. Such properties are fully consistent with the putative role of an organelle transport motor, present in small numbers to maintain movement over long ranges relative to cellular size scales. The contrast between myosin II and myosin V resembles that between a human running on the moon and one walking on earth, where the former allows for faster motion when in larger ensembles but for less

  8. A polypeptide-DNA hybrid with selective linking capability applied to single molecule nano-mechanical measurements using optical tweezers.

    Directory of Open Access Journals (Sweden)

    Fatemeh Moayed

    Full Text Available Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN and the peptide StrepTag II (ST. We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST. In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV linkage. It can be used in conjunction with Neutravidin (NTV-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications.

  9. The fabrication and characterization of adjustable nanogaps between gold electrodes on chip for electrical measurement of single molecules

    International Nuclear Information System (INIS)

    Tian Jinghua; Yang Yang; Liu Bo; Wu Deyin; Tian Zhongqun; Schoellhorn, Bernd; Maisonhaute, Emmanuel; Muns, Anna Serra; Chen Yong; Amatore, Christian; Tao, Nong-Jian

    2010-01-01

    This work reports on a new method to fabricate mechanically controllable break junctions (MCBJ) with finely adjustable nanogaps between two gold electrodes on solid state chips for characterizing electron transport properties of single molecules. The simple, low cost, robust and reproducible fabrication method combines conventional photolithography, chemical etching and electrodeposition to produce suspended electrodes separated with nanogaps. The MCBJ devices fabricated by the method can undergo many cycles in which the nanogap width can be precisely and repeatedly varied from zero to several nanometers. The method improves the success rate of the MCBJ experiments. Using these devices the electron transport properties of a typical molecular system, commercially available benzene-1,4-dithiol (BDT), have been studied. The I-V and G-V characteristic curves of BDT and the conductance value for a single BDT molecule established the excellent device suitability for molecular electronics research.

  10. The fabrication and characterization of adjustable nanogaps between gold electrodes on chip for electrical measurement of single molecules

    Energy Technology Data Exchange (ETDEWEB)

    Tian Jinghua; Yang Yang; Liu Bo; Wu Deyin; Tian Zhongqun [State Key Laboratory of Physical Chemistry of Solid Surfaces and LIA CNRS XiamENS ' NanoBioChem' , College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian (China); Schoellhorn, Bernd; Maisonhaute, Emmanuel; Muns, Anna Serra; Chen Yong; Amatore, Christian [UMR CNRS 8640 ' PASTEUR' and LIA CNRS XiamENS ' NanoBioChem' , Ecole Normale Superieure, Universite Pierre et Marie Curie-Paris 6, 24 rue Lhomond, 75231 Paris Cedex 05 (France); Tao, Nong-Jian, E-mail: zqtian@xmu.edu.cn, E-mail: christian.amatore@ens.fr [Ira A Fulton School of Engineering and Center for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University, Tempe, AZ 85287-6206 (United States)

    2010-07-09

    This work reports on a new method to fabricate mechanically controllable break junctions (MCBJ) with finely adjustable nanogaps between two gold electrodes on solid state chips for characterizing electron transport properties of single molecules. The simple, low cost, robust and reproducible fabrication method combines conventional photolithography, chemical etching and electrodeposition to produce suspended electrodes separated with nanogaps. The MCBJ devices fabricated by the method can undergo many cycles in which the nanogap width can be precisely and repeatedly varied from zero to several nanometers. The method improves the success rate of the MCBJ experiments. Using these devices the electron transport properties of a typical molecular system, commercially available benzene-1,4-dithiol (BDT), have been studied. The I-V and G-V characteristic curves of BDT and the conductance value for a single BDT molecule established the excellent device suitability for molecular electronics research.

  11. Lanthanide single molecule magnets

    CERN Document Server

    Tang, Jinkui

    2015-01-01

    This book begins by providing basic information on single-molecule magnets (SMMs), covering the magnetism of lanthanide, the characterization and relaxation dynamics of SMMs, and advanced means of studying lanthanide SMMs. It then systematically introduces lanthanide SMMs ranging from mononuclear and dinuclear to polynuclear complexes, classifying them and highlighting those SMMs with high barrier and blocking temperatures – an approach that provides some very valuable indicators for the structural features needed to optimize the contribution of an Ising type spin to a molecular magnet. The final chapter presents some of the newest developments in the lanthanide SMM field, such as the design of multifunctional and stimuli-responsive magnetic materials as well as the anchoring and organization of the SMMs on surfaces. In addition, the crystal structure and magnetic data are clearly presented with a wealth of illustrations in each chapter, helping newcomers and experts alike to better grasp ongoing trends and...

  12. Single-Molecule Nanomagnets

    Science.gov (United States)

    Friedman, Jonathan R.; Sarachik, Myriam P.

    2010-04-01

    Single-molecule magnets straddle the classical and quantum mechanical worlds, displaying many fascinating phenomena. They may have important technological applications in information storage and quantum computation. We review the physical properties of two prototypical molecular nanomagnets, Mn12-acetate and Fe8: Each behaves as a rigid, spin-10 object and exhibits tunneling between up and down directions. As temperature is lowered, the spin-reversal process evolves from thermal activation to pure quantum tunneling. At low temperatures, magnetic avalanches occur in which the magnetization of an entire sample rapidly reverses. We discuss the important role that symmetry-breaking fields play in driving tunneling and in producing Berry-phase interference. Recent experimental advances indicate that quantum coherence can be maintained on timescales sufficient to allow a meaningful number of quantum computing operations to be performed. Efforts are under way to create monolayers and to address and manipulate individual molecules.

  13. Lanthanide single molecule magnets

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jinkui; Zhang, Peng [Chinese Academy of Sciences, Changchun (China). Changchun Inst. of Applied Chemistry

    2015-10-01

    This book begins by providing basic information on single-molecule magnets (SMMs), covering the magnetism of lanthanide, the characterization and relaxation dynamics of SMMs and advanced means of studying lanthanide SMMs. It then systematically introduces lanthanide SMMs ranging from mononuclear and dinuclear to polynuclear complexes, classifying them and highlighting those SMMs with high barrier and blocking temperatures - an approach that provides some very valuable indicators for the structural features needed to optimize the contribution of an Ising type spin to a molecular magnet. The final chapter presents some of the newest developments in the lanthanide SMM field, such as the design of multifunctional and stimuli-responsive magnetic materials as well as the anchoring and organization of the SMMs on surfaces. In addition, the crystal structure and magnetic data are clearly presented with a wealth of illustrations in each chapter, helping newcomers and experts alike to better grasp ongoing trends and explore new directions.

  14. Single molecules in soft matter : a study of biomolecular conformation, heterogeneity and plasmon enhanced fluorescence

    NARCIS (Netherlands)

    Yuan, Haifeng

    2013-01-01

    We study the dynamics of single molecules and individual gold nanorods in glycerol at variable temperatures. We demonstrate temperature-cycle microscopy on FRET-labeled polyproline and double-stranded DNA molecules to access micro-second dynamics of single molecules, and reveal the influences of

  15. 8-spot smFRET analysis using two 8-pixel SPAD arrays.

    Science.gov (United States)

    Ingargiola, Antonino; Panzeri, Francesco; Sarkosh, Niusha; Gulinatti, Angelo; Rech, Ivan; Ghioni, Massimo; Weiss, Shimon; Michalet, Xavier

    2013-02-22

    Single-molecule Förster resonance energy transfer (smFRET) techniques are now widely used to address outstanding problems in biology and biophysics. In order to study freely diffusing molecules, current approaches consist in exciting a low concentration (pixel Single-Photon Avalanche Diodes (SPADs). A large enough number of single-molecule bursts must be accumulated in order to compute FRET efficiencies with sufficient statistics. As a result, the minimum timescale of observable phenomena is set by the minimum acquisition time needed for accurate measurements, typically a few minutes or more, limiting this approach mostly to equilibrium studies. Increasing smFRET analysis throughput would allow studying dynamics with shorter timescales. We recently demonstrated a new multi-spot excitation approach, employing a novel multi-pixel SPAD array, using a simplified dual-view setup in which a single 8-pixel SPAD array was used to collect FRET data from 4 independent spots. In this work we extend our results to 8 spots and use two 8-SPAD arrays to collect donor and acceptor photons and demonstrate the capabilities of this system by studying a series of doubly labeled dsDNA samples with different donor-acceptor distances ranging from low to high FRET efficiencies. Our results show that it is possible to enhance the throughput of smFRET measurements in solution by almost one order of magnitude, opening the way for studies of single-molecule dynamics with fast timescale once larger SPAD arrays become available.

  16. Determination of a Wear Initiation Cycle by using a Contact Resistance Measurement in Nuclear Fuel Fretting

    International Nuclear Information System (INIS)

    Lee, Young Ho; Kim, Hyung Kyu

    2008-01-01

    In nuclear fuel fretting, the improving of the contact condition with a modified spring shape is a useful method for increasing the wear resistance of the nuclear fuel rod. This is because the fretting wear resistance between the fuel rod and grid spring is mainly affected by the grid spring shape rather than the environment, the contact modes, etc. In addition, the wear resistance is affected by the wear debris behavior between contact surfaces. So, it is expected that the wear initiation of each spring shape should be determined in order to evaluate a wear resistance. However, it is almost impossible to measure the wear behavior in contact surfaces on a real time basis because the contact surfaces are always hidden. Besides, the results of the worn surface observation after the fretting wear tests are restricted to archive the information on the wear debris behavior and the formation mechanism of the wear scar. In order to evaluate the wear behavior during the fretting wear tests, it is proposed that the contact resistance measurement is a useful method for examining the wear initiation cycle and modes. Generally, fretting wear damages are rapidly progressed by a localized plastic deformation between the contact surfaces, crack initiation and fracture of the deformed surface with a strain hardening difference between a surface and a subsurface and finally a detachment of wear debris. After this, wear debris is easily oxidized by frictional heat, test environment, etc. At this time, a small amount of electric current applied between the contact surfaces will be influenced by the wear debris, which could be an obstacle to an electric current flow. So, it is possible to archive the information on the wear behavior by measuring the contact resistance. In order to determine the wear initiation cycle during the fretting wear tests, in this study, fretting wear tests have been performed by applying a constant electric current in room temperature air

  17. Single Molecule Spectroscopy of Electron Transfer

    International Nuclear Information System (INIS)

    Holman, Michael; Zang, Ling; Liu, Ruchuan; Adams, David M.

    2009-01-01

    The objectives of this research are threefold: (1) to develop methods for the study electron transfer processes at the single molecule level, (2) to develop a series of modifiable and structurally well defined molecular and nanoparticle systems suitable for detailed single molecule/particle and bulk spectroscopic investigation, (3) to relate experiment to theory in order to elucidate the dependence of electron transfer processes on molecular and electronic structure, coupling and reorganization energies. We have begun the systematic development of single molecule spectroscopy (SMS) of electron transfer and summaries of recent studies are shown. There is a tremendous need for experiments designed to probe the discrete electronic and molecular dynamic fluctuations of single molecules near electrodes and at nanoparticle surfaces. Single molecule spectroscopy (SMS) has emerged as a powerful method to measure properties of individual molecules which would normally be obscured in ensemble-averaged measurement. Fluctuations in the fluorescence time trajectories contain detailed molecular level statistical and dynamical information of the system. The full distribution of a molecular property is revealed in the stochastic fluctuations, giving information about the range of possible behaviors that lead to the ensemble average. In the case of electron transfer, this level of understanding is particularly important to the field of molecular and nanoscale electronics: from a device-design standpoint, understanding and controlling this picture of the overall range of possible behaviors will likely prove to be as important as designing ia the ideal behavior of any given molecule.

  18. Single molecule activity measurements of cytochrome P450 oxidoreductase reveal the existence of two discrete functional states

    DEFF Research Database (Denmark)

    Laursen, Tomas; Singha, Aparajita; Rantzau, Nicolai

    2014-01-01

    450 enzymes. Measurements and statistical analy-sis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date remained masked in bulk kinetics. To ensure that we...

  19. Electrophoresis- and FRET-Based Measures of Serpin Polymerization.

    Science.gov (United States)

    Faull, Sarah V; Brown, Anwen E; Haq, Imran; Irving, James A

    2017-01-01

    Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Förster resonance energy transfer (FRET) and gel-based approaches for their characterization.

  20. Current-voltage characteristics of single-molecule diarylethene junctions measured with adjustable gold electrodes in solution.

    Science.gov (United States)

    Briechle, Bernd M; Kim, Youngsang; Ehrenreich, Philipp; Erbe, Artur; Sysoiev, Dmytro; Huhn, Thomas; Groth, Ulrich; Scheer, Elke

    2012-01-01

    We report on an experimental analysis of the charge transport through sulfur-free photochromic molecular junctions. The conductance of individual molecules contacted with gold electrodes and the current-voltage characteristics of these junctions are measured in a mechanically controlled break-junction system at room temperature and in liquid environment. We compare the transport properties of a series of molecules, labeled TSC, MN, and 4Py, with the same switching core but varying side-arms and end-groups designed for providing the mechanical and electrical contact to the gold electrodes. We perform a detailed analysis of the transport properties of TSC in its open and closed states. We find rather broad distributions of conductance values in both states. The analysis, based on the assumption that the current is carried by a single dominating molecular orbital, reveals distinct differences between both states. We discuss the appearance of diode-like behavior for the particular species 4Py that features end-groups, which preferentially couple to the metal electrode by physisorption. We show that the energetic position of the molecular orbital varies as a function of the transmission. Finally, we show for the species MN that the use of two cyano end-groups on each side considerably enhances the coupling strength compared to the typical behavior of a single cyano group.

  1. Current–voltage characteristics of single-molecule diarylethene junctions measured with adjustable gold electrodes in solution

    Directory of Open Access Journals (Sweden)

    Bernd M. Briechle

    2012-11-01

    Full Text Available We report on an experimental analysis of the charge transport through sulfur-free photochromic molecular junctions. The conductance of individual molecules contacted with gold electrodes and the current–voltage characteristics of these junctions are measured in a mechanically controlled break-junction system at room temperature and in liquid environment. We compare the transport properties of a series of molecules, labeled TSC, MN, and 4Py, with the same switching core but varying side-arms and end-groups designed for providing the mechanical and electrical contact to the gold electrodes. We perform a detailed analysis of the transport properties of TSC in its open and closed states. We find rather broad distributions of conductance values in both states. The analysis, based on the assumption that the current is carried by a single dominating molecular orbital, reveals distinct differences between both states. We discuss the appearance of diode-like behavior for the particular species 4Py that features end-groups, which preferentially couple to the metal electrode by physisorption. We show that the energetic position of the molecular orbital varies as a function of the transmission. Finally, we show for the species MN that the use of two cyano end-groups on each side considerably enhances the coupling strength compared to the typical behavior of a single cyano group.

  2. Single-Molecule Stochastic Resonance

    Directory of Open Access Journals (Sweden)

    K. Hayashi

    2012-08-01

    Full Text Available Stochastic resonance (SR is a well-known phenomenon in dynamical systems. It consists of the amplification and optimization of the response of a system assisted by stochastic (random or probabilistic noise. Here we carry out the first experimental study of SR in single DNA hairpins which exhibit cooperatively transitions from folded to unfolded configurations under the action of an oscillating mechanical force applied with optical tweezers. By varying the frequency of the force oscillation, we investigate the folding and unfolding kinetics of DNA hairpins in a periodically driven bistable free-energy potential. We measure several SR quantifiers under varied conditions of the experimental setup such as trap stiffness and length of the molecular handles used for single-molecule manipulation. We find that a good quantifier of the SR is the signal-to-noise ratio (SNR of the spectral density of measured fluctuations in molecular extension of the DNA hairpins. The frequency dependence of the SNR exhibits a peak at a frequency value given by the resonance-matching condition. Finally, we carry out experiments on short hairpins that show how SR might be useful for enhancing the detection of conformational molecular transitions of low SNR.

  3. Single Molecule Electronics and Devices

    Science.gov (United States)

    Tsutsui, Makusu; Taniguchi, Masateru

    2012-01-01

    The manufacture of integrated circuits with single-molecule building blocks is a goal of molecular electronics. While research in the past has been limited to bulk experiments on self-assembled monolayers, advances in technology have now enabled us to fabricate single-molecule junctions. This has led to significant progress in understanding electron transport in molecular systems at the single-molecule level and the concomitant emergence of new device concepts. Here, we review recent developments in this field. We summarize the methods currently used to form metal-molecule-metal structures and some single-molecule techniques essential for characterizing molecular junctions such as inelastic electron tunnelling spectroscopy. We then highlight several important achievements, including demonstration of single-molecule diodes, transistors, and switches that make use of electrical, photo, and mechanical stimulation to control the electron transport. We also discuss intriguing issues to be addressed further in the future such as heat and thermoelectric transport in an individual molecule. PMID:22969345

  4. Two states or not two states: Single-molecule folding studies of protein L

    Science.gov (United States)

    Aviram, Haim Yuval; Pirchi, Menahem; Barak, Yoav; Riven, Inbal; Haran, Gilad

    2018-03-01

    Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ˜7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

  5. Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells.

    Science.gov (United States)

    Elliott, Amicia D; Bedard, Noah; Ustione, Alessandro; Baird, Michelle A; Davidson, Michael W; Tkaczyk, Tomasz; Piston, David W

    2017-01-01

    Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS's capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

  6. Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury.

    Directory of Open Access Journals (Sweden)

    David M Rissin

    Full Text Available We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA probe-containing a reactive amine instead of a nucleotide at a specific position in the sequence-for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase-containing an aldehyde group-that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG, the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122-an established biomarker of liver toxicity-extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use.

  7. Studies of G-quadruplex DNA structures at the single molecule level

    DEFF Research Database (Denmark)

    Kragh, Sofie Louise

    2015-01-01

    Folding of G-quaduplex structures adopted by the human telomeric repeat is here studied by single molecule FRET microscopy. This method allows for the investigation of G-quadruplex structures and their conformational dynamic. Telomeres are located at the ends of our chromosomes and end in a single...... with human telomeric repeat adopt several different G-quadruplex conformations in the presence of K+ ions. G-quadruplexes inhibit telomerase activity and are therefore potential targets for anti-cancer drugs, which can be small molecule ligands capable of stabilizing G-quadruplex structures. Understanding...... range. FRET spectroscopy can be performed on an ensemble of molecules, or on the single molecule level. In single molecule FRET experiments it is possible to follow the behaviour in time for each molecule independently, allowing insight into both dynamically and statistically heterogeneous molecular...

  8. Detailed single-crystal EPR line shape measurements for the single-molecule magnets Fe8Br and Mn12-acetate

    Science.gov (United States)

    Hill, S.; Maccagnano, S.; Park, Kyungwha; Achey, R. M.; North, J. M.; Dalal, N. S.

    2002-06-01

    It is shown that our multi-high-frequency (40-200 GHz) resonant cavity technique yields distortion-free high-field electron paramagnetic resonance (EPR) spectra for single-crystal samples of the uniaxial and biaxial spin S=10 single-molecule magnets (SMM's) [Mn12O12(CH3COO)16(H2O)4].2CH3COOH.4H2O and [Fe8O2(OH)12(tacn)6]Br8.9H2O. The observed line shapes exhibit a pronounced dependence on temperature, magnetic field, and the spin quantum numbers (MS values) associated with the levels involved in the transitions. Measurements at many frequencies allow us to separate various contributions to the EPR linewidths, including significant D strain, g strain, and broadening due to the random dipolar fields of neighboring molecules. We also identify asymmetry in some of the EPR line shapes for Fe8 and a previously unobserved fine structure to some of the EPR lines for both the Fe8 and Mn12 systems. These findings prove relevant to the mechanism of quantum tunneling of magnetization in these SMM's.

  9. The multi-state energy landscape of the SAM-I riboswitch: A single-molecule Förster resonance energy transfer spectroscopy study

    Science.gov (United States)

    Manz, Christoph; Kobitski, Andrei Yu.; Samanta, Ayan; Jäschke, Andres; Nienhaus, G. Ulrich

    2018-03-01

    RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs

  10. Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

    Science.gov (United States)

    Schmid, Sonja; Hugel, Thorsten

    2018-03-01

    Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings—such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

  11. Studying the structural dynamics of bipedal DNA motors with single-molecule fluorescence spectroscopy.

    Science.gov (United States)

    Masoud, Rula; Tsukanov, Roman; Tomov, Toma E; Plavner, Noa; Liber, Miran; Nir, Eyal

    2012-07-24

    We present a test case example of a detailed single-molecule fluorescence study of one of the most sophisticated and complex DNA devices introduced to date, a recently published autonomous bipedal DNA motor. We used the diffusion-based single-molecule Förster resonance energy transfer technique, coupled to alternating laser excitation (sm-FRET-ALEX), to monitor the motor assembly and operation. The study included verification of the formation of the correct structures, and of the correct motor operation, determination of the formation and stepping reaction yields, and identification of side products. Finally, the mechanisms of the motor assembly and operation were elucidated by measuring the reaction kinetics profile of track-walker binding and of lifting of the walker's leg upon fuel addition. The profiles revealed a fast phase, in which about half of the reaction was completed, followed by a slow phase which adds somewhat to the yield, reflecting the incomplete motor assembly and operation identified in the equilibrium experiments. Although further study is needed to fully understand the reasons for the incomplete assembly and operation, this work demonstrates that single-molecule fluorescence, based on its ability to provide detailed in situ structural dynamics information, inaccessible for traditional methods, constitutes an excellent tool for chaperoning the development of DNA-based technology.

  12. Single Molecule Nanoelectrochemistry in Electrical Junctions.

    Science.gov (United States)

    Nichols, Richard J; Higgins, Simon J

    2016-11-15

    It is now possible to reliably measure single molecule conductance in a wide variety of environments including organic liquids, ultrahigh vacuum, water, ionic liquids, and electrolytes. The most commonly used methods deploy scanning probe microscopes, mechanically formed break junctions, or lithographically formed nanogap contacts. Molecules are generally captured between a pair of facing electrodes, and the junction current response is measured as a function of bias voltage. Gating electrodes can also be added so that the electrostatic potential at the molecular bridge can be independently controlled by this third noncontacting electrode. This can also be achieved in an electrolytic environment using a four-electrode bipotentiostatic configuration, which allows independent electrode potential control of the two contacting electrodes. This is commonly realized using an electrochemical STM and enables single molecule electrical characterization as a function of electrode potential and redox state of the molecular bridge. This has emerged as a powerful tool in modern interfacial electrochemistry and nanoelectrochemistry for studying charge transport across single molecules as a function of electrode potential and the electrolytic environments. Such measurements are possible in electrolytes ranging from aqueous buffers to nonaqueous ionic liquids. In this Account, we illustrate a number of examples of single molecule electrical measurements under electrode potential control use a scanning tunneling microscope (STM) and demonstrate how these can help in the understanding of charge transport in single molecule junctions. Examples showing charge transport following phase coherent tunneling to incoherent charge hopping across redox active molecular bridges are shown. In the case of bipyridinium (or viologen) molecular wires, it is shown how electrochemical reduction leads to an increase of the single molecule conductance, which is controlled by the liquid electrochemical

  13. Single molecule transcription profiling with AFM

    Science.gov (United States)

    Reed, Jason; Mishra, Bud; Pittenger, Bede; Magonov, Sergei; Troke, Joshua; Teitell, Michael A.; Gimzewski, James K.

    2007-01-01

    Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations. Based on invited talk at the International Conference on Nanoscience and Technology 2006.

  14. Single molecule transcription profiling with AFM

    International Nuclear Information System (INIS)

    Reed, Jason; Mishra, Bud; Pittenger, Bede; Magonov, Sergei; Troke, Joshua; Teitell, Michael A; Gimzewski, James K

    2007-01-01

    Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations

  15. Single particle tracking and single molecule energy transfer

    CERN Document Server

    Bräuchle, Christoph; Michaelis, Jens

    2009-01-01

    Closing a gap in the literature, this handbook gathers all the information on single particle tracking and single molecule energy transfer. It covers all aspects of this hot and modern topic, from detecting virus entry to membrane diffusion, and from protein folding using spFRET to coupled dye systems, as well recent achievements in the field. Throughout, the first-class editors and top international authors present content of the highest quality, making this a must-have for physical chemists, spectroscopists, molecular physicists and biochemists.

  16. Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics

    Science.gov (United States)

    Dupuis, Nicholas F.; Holmstrom, Erik D.; Nesbitt, David J.

    2014-01-01

    The effects of “molecular crowding” on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop–receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high–molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ −2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop–receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple “hard-sphere” treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ −5 kcal/mol free-energy stabilization at excluded volume fractions of 30%. PMID:24850865

  17. Single-molecule nanopore enzymology

    Science.gov (United States)

    Wloka, Carsten; Maglia, Giovanni

    2017-01-01

    Biological nanopores are a class of membrane proteins that open nanoscale water-conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. More recently, proteins and enzymes have started being analysed with nanopores. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here we describe the approaches and challenges in nanopore enzymology. PMID:28630164

  18. Trapping and manipulating single molecules of DNA

    Science.gov (United States)

    Shon, Min Ju

    This thesis presents the development and application of nanoscale techniques to trap and manipulate biomolecules, with a focus on DNA. These methods combine single-molecule microscopy and nano- and micro-fabrication to study biophysical properties of DNA and proteins. The Dimple Machine is a lab-on-a-chip device that can isolate and confine a small number of molecules from a bulk solution. It traps molecules in nanofabricated chambers, or "dimples", and the trapped molecules are then studied on a fluorescence microscope at the single-molecule level. The sampling of bulk solution by dimples is representative, reproducible, and automated, enabling highthroughput single-molecule experiments. The device was applied to study hybridization of oligonucleotides, particularly in the context of reaction thermodynamics and kinetics in nanoconfinement. The DNA Pulley is a system to study protein binding and the local mechanical properties of DNA. A molecule of DNA is tethered to a surface on one end, and a superparamagnetic bead is attached to the other. A magnet pulls the DNA taut, and a silicon nitride knife with a nanoscale blade scans the DNA along its contour. Information on the local properties of the DNA is extracted by tracking the bead with nanometer precision in a white-light microscope. The system can detect proteins bound to DNA and localize their recognition sites, as shown with a model protein, EcoRI restriction enzyme. Progress on the measurements of nano-mechanical properties of DNA is included.

  19. Developing DNA nanotechnology using single-molecule fluorescence.

    Science.gov (United States)

    Tsukanov, Roman; Tomov, Toma E; Liber, Miran; Berger, Yaron; Nir, Eyal

    2014-06-17

    CONSPECTUS: An important effort in the DNA nanotechnology field is focused on the rational design and manufacture of molecular structures and dynamic devices made of DNA. As is the case for other technologies that deal with manipulation of matter, rational development requires high quality and informative feedback on the building blocks and final products. For DNA nanotechnology such feedback is typically provided by gel electrophoresis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). These analytical tools provide excellent structural information; however, usually they do not provide high-resolution dynamic information. For the development of DNA-made dynamic devices such as machines, motors, robots, and computers this constitutes a major problem. Bulk-fluorescence techniques are capable of providing dynamic information, but because only ensemble averaged information is obtained, the technique may not adequately describe the dynamics in the context of complex DNA devices. The single-molecule fluorescence (SMF) technique offers a unique combination of capabilities that make it an excellent tool for guiding the development of DNA-made devices. The technique has been increasingly used in DNA nanotechnology, especially for the analysis of structure, dynamics, integrity, and operation of DNA-made devices; however, its capabilities are not yet sufficiently familiar to the community. The purpose of this Account is to demonstrate how different SMF tools can be utilized for the development of DNA devices and for structural dynamic investigation of biomolecules in general and DNA molecules in particular. Single-molecule diffusion-based Förster resonance energy transfer and alternating laser excitation (sm-FRET/ALEX) and immobilization-based total internal reflection fluorescence (TIRF) techniques are briefly described and demonstrated. To illustrate the many applications of SMF to DNA nanotechnology, examples of SMF studies of DNA hairpins and

  20. A Brief Introduction to Single-Molecule Fluorescence Methods.

    Science.gov (United States)

    van den Wildenberg, Siet M J L; Prevo, Bram; Peterman, Erwin J G

    2018-01-01

    One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

  1. Single-Molecule Interfacial Electron Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Lu, H. Peter [Bowling Green State Univ., Bowling Green, OH (United States). Dept. of Chemistry and Center for Photochemical Sciences

    2017-11-28

    This project is focused on the use of single-molecule high spatial and temporal resolved techniques to study molecular dynamics in condensed phase and at interfaces, especially, the complex reaction dynamics associated with electron and energy transfer rate processes. The complexity and inhomogeneity of the interfacial ET dynamics often present a major challenge for a molecular level comprehension of the intrinsically complex systems, which calls for both higher spatial and temporal resolutions at ultimate single-molecule and single-particle sensitivities. Combined single-molecule spectroscopy and electrochemical atomic force microscopy approaches are unique for heterogeneous and complex interfacial electron transfer systems because the static and dynamic inhomogeneities can be identified and characterized by studying one molecule at a specific nanoscale surface site at a time. The goal of our project is to integrate and apply these spectroscopic imaging and topographic scanning techniques to measure the energy flow and electron flow between molecules and substrate surfaces as a function of surface site geometry and molecular structure. We have been primarily focusing on studying interfacial electron transfer under ambient condition and electrolyte solution involving both single crystal and colloidal TiO2 and related substrates. The resulting molecular level understanding of the fundamental interfacial electron transfer processes will be important for developing efficient light harvesting systems and broadly applicable to problems in fundamental chemistry and physics. We have made significant advancement on deciphering the underlying mechanism of the complex and inhomogeneous interfacial electron transfer dynamics in dyesensitized TiO2 nanoparticle systems that strongly involves with and regulated by molecule-surface interactions. We have studied interfacial electron transfer on TiO2 nanoparticle surfaces by using ultrafast single-molecule

  2. Single-molecule magnet engineering

    DEFF Research Database (Denmark)

    Pedersen, Kasper Steen; Bendix, Jesper; Clérac, Rodolphe

    2014-01-01

    Tailoring the specific magnetic properties of any material relies on the topological control of the constituent metal ion building blocks. Although this general approach does not seem to be easily applied to traditional inorganic bulk magnets, coordination chemistry offers a unique tool...... to delicately tune, for instance, the properties of molecules that behave as "magnets", the so-called single-molecule magnets (SMMs). Although many interesting SMMs have been prepared by a more or less serendipitous approach, the assembly of predesigned, isolatable molecular entities into higher nuclearity...... complexes constitutes an elegant and fascinating strategy. This Feature article focuses on the use of building blocks or modules (both terms being used indiscriminately) to direct the structure, and therefore also the magnetic properties, of metal ion complexes exhibiting SMM behaviour. This journal is...

  3. Single molecule insights on conformational selection and induced fit mechanism

    DEFF Research Database (Denmark)

    Hatzakis, Nikos

    2014-01-01

    of unsynchronized molecules, often masking intrinsic dynamic behavior of proteins and biologically significant transient intermediates. Single molecule measurements are emerging as a powerful tool for characterizing protein function. They offer the direct observation and quantification of the activity, abundance...... and lifetime of multiple states and transient intermediates in the energy landscape, that are typically averaged out in non-synchronized ensemble measurements. Here we survey new insights from single molecule studies that advance our understanding of the molecular mechanisms underlying biomolecular recognition....

  4. Detailed study of DNA hairpin dynamics using single-molecule fluorescence assisted by DNA origami.

    Science.gov (United States)

    Tsukanov, Roman; Tomov, Toma E; Masoud, Rula; Drory, Hagai; Plavner, Noa; Liber, Miran; Nir, Eyal

    2013-10-10

    The dynamics of two DNA hairpins (5'-TCGCCT-A31-AGGCGA-3' and 5'-TCGCCG-A31-CGGCGA-3') were studied using immobilization-based and diffusion-based single-molecule fluorescence techniques. The techniques enabled separated and detailed investigation of the states and of the transition reactions. Only two states, open and closed, were identified from analysis of the FRET histograms; metastable states with lifetimes longer than the technique resolution (0.3 ms) were not observed. The opening and closing reaction rates were determined directly from the FRET time trajectories, and the Gibbs free energies of these states and of the transition state were calculated using the Kramer theory. The rates, which are undoubtedly of transitions between the fully closed and the fully open states and ranged from 2 to 90 s(-1), were lower (∼10-fold) than the rates previously determined from fluorescence correlation spectroscopy. The heights of the barriers for closing were almost identical for the two hairpins. The barrier for opening the hairpin with the stronger stem was higher (4.3 kJ/mol) than that for the hairpin with the weaker stem, in very good agreement with the difference in stability calculated by the nearest-neighbor method. The barrier for closing the hairpin decreased (∼8 kJ/mol) and the barrier for opening increased (∼4 kJ/mol) with increasing NaCl concentration (10-100 mM), indicating that higher ionic strength stabilizes the folded state with respect to the transition state and stabilizes the transition state relative to the unfolded state. The very good agreements in the dynamics measured for free hairpins, for hairpins anchored to origami, and for hairpins anchored to the coverslip and the very good agreement between the two single-molecule techniques demonstrate that neither the origami nor the coverslip influence the hairpin dynamics, supporting a previous demonstration that origami can serve as a platform for biophysical investigations.

  5. Single molecule force spectroscopy: methods and applications in biology

    International Nuclear Information System (INIS)

    Shen Yi; Hu Jun

    2012-01-01

    Single molecule measurements have transformed our view of biomolecules. Owing to the ability of monitoring the activity of individual molecules, we now see them as uniquely structured, fluctuating molecules that stochastically transition between frequently many substrates, as two molecules do not follow precisely the same trajectory. Indeed, it is this discovery of critical yet short-lived substrates that were often missed in ensemble measurements that has perhaps contributed most to the better understanding of biomolecular functioning resulting from single molecule experiments. In this paper, we give a review on the three major techniques of single molecule force spectroscopy, and their applications especially in biology. The single molecular study of biotin-streptavidin interactions is introduced as a successful example. The problems and prospects of the single molecule force spectroscopy are discussed, too. (authors)

  6. Using FLIM-FRET to measure conformational changes of transglutaminase type 2 in live cells.

    Directory of Open Access Journals (Sweden)

    Nicholas S Caron

    Full Text Available Transglutaminase type 2 (TG2 is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that allows for quantitative assessment of TG2 conformational changes in live cells using Förster resonance energy transfer (FRET, as measured by fluorescence lifetime imaging microscopy (FLIM. Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible inhibitor of TG2, NC9, forces the enzyme into an open conformation, whereas the reversible inhibitor CP4d traps TG2 in the closed conformation. Thus, this biosensor provides new mechanistic insights into the action of two TG2 inhibitors and defines two new classes based on ability to alter TG2 conformation in addition to inhibiting transamidation activity. Future applications of this biosensor could be to discover small molecules that specifically alter TG2 conformation to affect GDP/GTP or calcium binding.

  7. Single-molecule studies using magnetic traps.

    Science.gov (United States)

    Lionnet, Timothée; Allemand, Jean-François; Revyakin, Andrey; Strick, Terence R; Saleh, Omar A; Bensimon, David; Croquette, Vincent

    2012-01-01

    In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to >100 pN. In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. In this article, we describe the principle of the magnetic trap, as well as its use in the measurement of the elastic properties of DNA and the study of DNA-protein interactions.

  8. Direct single-molecule dynamic detection of chemical reactions.

    Science.gov (United States)

    Guan, Jianxin; Jia, Chuancheng; Li, Yanwei; Liu, Zitong; Wang, Jinying; Yang, Zhongyue; Gu, Chunhui; Su, Dingkai; Houk, Kendall N; Zhang, Deqing; Guo, Xuefeng

    2018-02-01

    Single-molecule detection can reveal time trajectories and reaction pathways of individual intermediates/transition states in chemical reactions and biological processes, which is of fundamental importance to elucidate their intrinsic mechanisms. We present a reliable, label-free single-molecule approach that allows us to directly explore the dynamic process of basic chemical reactions at the single-event level by using stable graphene-molecule single-molecule junctions. These junctions are constructed by covalently connecting a single molecule with a 9-fluorenone center to nanogapped graphene electrodes. For the first time, real-time single-molecule electrical measurements unambiguously show reproducible large-amplitude two-level fluctuations that are highly dependent on solvent environments in a nucleophilic addition reaction of hydroxylamine to a carbonyl group. Both theoretical simulations and ensemble experiments prove that this observation originates from the reversible transition between the reactant and a new intermediate state within a time scale of a few microseconds. These investigations open up a new route that is able to be immediately applied to probe fast single-molecule physics or biophysics with high time resolution, making an important contribution to broad fields beyond reaction chemistry.

  9. Fretting of AISI 9310 and selected fretting resistant surface treatments

    Science.gov (United States)

    Bill, R. C.

    1977-01-01

    Fretting wear experiments were conducted with uncoated AISI 9310 mating surfaces, and with combinations incorporating a selected coating to one of the mating surfaces. Wear measurements and SEM observations indicated that surface fatigue, as made evident by spallation and surface crack formation, is an important mechanism in promoting fretting wear to uncoated 9310. Increasing humidity resulted in accelerated fretting, and a very noticeable difference in nature of the fretting debris. Of the coatings evaluated, alumimum bronze with a polyester additive was most effective at reducing wear and minimizing fretting damage to the mating uncoated surface, by means of a self-lubricating film that developed on the fretting surfaces. Chromium plate performed as an effective protective coating, itself resisting fretting and not accelerating damage to the uncoated surface.

  10. Construction of a laser combiner for dual fluorescent single molecule imaging of pRNA of phi29 DNA packaging motor.

    Science.gov (United States)

    Zhang, Hui; Shu, Dan; Browne, Mark; Guo, Peixuan

    2010-02-01

    A customized laser combiner was designed and constructed for dual channel single molecule imaging. The feasibility of a combiner-incorporated imaging system was demonstrated in studies of single molecule FRET. Distance rulers made of dual-labeled dsDNA were used to evaluate the system by determining the distance between one FRET pair. The results showed that the system is sensitive enough to distinguish between distances differing by two base pair and the distances calculated from FRET efficiencies are close to those documented in the literature. The single molecule FRET with the dual-color imaging system was also applied to reconstructed phi29 motor pRNA monomers. Finally, techniques for dual laser alignment and tuning of laser power for dual-color excitation are discussed.

  11. Single-Molecule Interfacial Electron Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ho, Wilson [Univ. of California, Irvine, CA (United States)

    2018-02-03

    Interfacial electron transfer (ET) plays an important role in many chemical and biological processes. Specifically, interfacial ET in TiO2-based systems is important to solar energy technology, catalysis, and environmental remediation technology. However, the microscopic mechanism of interfacial ET is not well understood with regard to atomic surface structure, molecular structure, bonding, orientation, and motion. In this project, we used two complementary methodologies; single-molecule fluorescence spectroscopy, and scanning-tunneling microscopy and spectroscopy (STM and STS) to address this scientific need. The goal of this project was to integrate these techniques and measure the molecular dependence of ET between adsorbed molecules and TiO2 semiconductor surfaces and the ET induced reactions such as the splitting of water. The scanning probe techniques, STM and STS, are capable of providing the highest spatial resolution but not easily time-resolved data. Single-molecule fluorescence spectroscopy is capable of good time resolution but requires further development to match the spatial resolution of the STM. The integrated approach involving Peter Lu at Bowling Green State University (BGSU) and Wilson Ho at the University of California, Irvine (UC Irvine) produced methods for time and spatially resolved chemical imaging of interfacial electron transfer dynamics and photocatalytic reactions. An integral aspect of the joint research was a significant exchange of graduate students to work at the two institutions. This project bridged complementary approaches to investigate a set of common problems by working with the same molecules on a variety of solid surfaces, but using appropriate techniques to probe under ambient (BGSU) and ultrahigh vacuum (UCI) conditions. The molecular level understanding of the fundamental interfacial electron transfer processes obtained in this joint project will be important for developing efficient light harvesting

  12. Massively Parallel Single-Molecule Manipulation Using Centrifugal Force

    Science.gov (United States)

    Wong, Wesley; Halvorsen, Ken

    2011-03-01

    Precise manipulation of single molecules has led to remarkable insights in physics, chemistry, biology, and medicine. However, two issues that have impeded the widespread adoption of these techniques are equipment cost and the laborious nature of making measurements one molecule at a time. To meet these challenges, we have developed an approach that enables massively parallel single- molecule force measurements using centrifugal force. This approach is realized in the centrifuge force microscope, an instrument in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force- field while their micro-to-nanoscopic motions are observed. We demonstrate high- throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Currently, we are taking steps to integrate high-resolution detection, fluorescence, temperature control and a greater dynamic range in force. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.

  13. Single-Molecule Electronics: Chemical and Analytical Perspectives.

    Science.gov (United States)

    Nichols, Richard J; Higgins, Simon J

    2015-01-01

    It is now possible to measure the electrical properties of single molecules using a variety of techniques including scanning probe microcopies and mechanically controlled break junctions. Such measurements can be made across a wide range of environments including ambient conditions, organic liquids, ionic liquids, aqueous solutions, electrolytes, and ultra high vacuum. This has given new insights into charge transport across molecule electrical junctions, and these experimental methods have been complemented with increasingly sophisticated theory. This article reviews progress in single-molecule electronics from a chemical perspective and discusses topics such as the molecule-surface coupling in electrical junctions, chemical control, and supramolecular interactions in junctions and gating charge transport. The article concludes with an outlook regarding chemical analysis based on single-molecule conductance.

  14. Single Molecule Applications of Quantum Dots

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Elmelund; Jauffred, Liselotte; Brewer, Jonathan R.

    2013-01-01

    Fluorescent nanocrystals composed of semiconductor materials were first introduced for biological applications in the late 1990s. The focus of this review is to give a brief survey of biological applications of quantum dots (QDs) at the single QD sensitivity level. These are described as follows:...... experiments held together with the prospects in localization microscopy and single molecule manipulation experiments gave QDs a promising future in single molecule research....

  15. Fluorescent Biosensors Based on Single-Molecule Counting.

    Science.gov (United States)

    Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

    2016-09-20

    Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the

  16. Single-molecule, structural and functional studies of Listeria monocytogenes Ca2+-ATPase

    DEFF Research Database (Denmark)

    Dyla, Mateusz

    -ion transport (e.g. H+ for Ca2+-ATPases). P-type ATPases undergo major conformational changes during their functional cycle, as has been learned from a wealth of atomic-resolution X-ray crystallographic structures (4). In this work, single-molecule, structural and functional studies were employed to investigate...... the dynamics and mechanism of the transport cycle of P-type ATPase at a single molecule level. A representative P-type ATPase, the Listeria monocytogenes Ca2+-ATPase (LMCA1) was engineered and characterized to facilitate smFRET studies. Pairs of cysteines were introduced and reacted with maleimide derivatives...... transitions to the E2 state triggered by ATP binding and phosphorylation were very brief, and could only be characterized in a dephosphorylation-deficient LMCA1 mutant. Owing to a spontaneous dephosphorylation of this mutant, full transport cycles at a single-molecule resolution were observed for the first...

  17. Extending Single-Molecule Microscopy Using Optical Fourier Processing

    Science.gov (United States)

    2015-01-01

    This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules. PMID:24745862

  18. Non-fluorescent Quenchers to Correlate Single-Molecule Conformational and Compositional Dynamics

    Science.gov (United States)

    Chen, Jin; Tsai, Albert; Petrov, Alexey; Puglisi, Joseph D.

    2015-01-01

    Single-molecule Förster resonance energy transfer (smFRET) is a powerful method to study the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging due to the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here, we report the use of a non-fluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently-labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of non-fluorescent quenchers in single-molecule correlation studies. PMID:22428667

  19. Studying σ 54-dependent transcription at the single-molecule level using alternating-laser excitation (ALEX) spectroscopy

    Science.gov (United States)

    Heilemann, M.; Lymperopoulos, K.; Wigneshweraraj, S. R.; Buck, M.; Kapanidis, A. N.

    2007-07-01

    We present single-molecule fluorescence studies of σ 54-dependent gene-transcription complexes using singlemolecule fluorescence resonance energy transfer (smFRET) and alternating-laser excitation (ALEX) spectroscopy. The ability to study one biomolecule at the time allowed us to resolve and analyze sample heterogeneities and extract structural information on subpopulations and transient intermediates of transcription; such information is hidden in bulk experiments. Using site-specifically labeled σ 54 derivatives and site-specifically labeled promoter-DNA fragments, we demonstrate that we can observe single diffusing σ 54-DNA and transcription-initiation RNA polymerase-σ 54- DNA complexes, and that we can measure distances within such complexes; the identity of the complexes has been confirmed using electrophoretic-mobility-shift assays. Our studies pave the way for understanding the mechanism of abortive initiation and promoter escape in σ 54-dependent transcription.

  20. Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

    Science.gov (United States)

    2015-01-01

    Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

  1. A large collapsed-state RNA can exhibit simple exponential single-molecule dynamics.

    Science.gov (United States)

    Smith, Glenna J; Lee, Kang Taek; Qu, Xiaohui; Xie, Zheng; Pesic, Jelena; Sosnick, Tobin R; Pan, Tao; Scherer, Norbert F

    2008-05-09

    The process of large RNA folding is believed to proceed from many collapsed structures to a unique functional structure requiring precise organization of nucleotides. The diversity of possible structures and stabilities of large RNAs could result in non-exponential folding kinetics (e.g. stretched exponential) under conditions where the molecules have not achieved their native state. We describe a single-molecule fluorescence resonance energy transfer (FRET) study of the collapsed-state region of the free energy landscape of the catalytic domain of RNase P RNA from Bacillus stearothermophilus (C(thermo)). Ensemble measurements have shown that this 260 residue RNA folds cooperatively to its native state at >or=1 mM Mg(2+), but little is known about the conformational dynamics at lower ionic strength. Our measurements of equilibrium conformational fluctuations reveal simple exponential kinetics that reflect a small number of discrete states instead of the expected inhomogeneous dynamics. The distribution of discrete dwell times, collected from an "ensemble" of 300 single molecules at each of a series of Mg(2+) concentrations, fit well to a double exponential, which indicates that the RNA conformational changes can be described as a four-state system. This finding is somewhat unexpected under [Mg(2+)] conditions in which this RNA does not achieve its native state. Observation of discrete well-defined conformations in this large RNA that are stable on the seconds timescale at low [Mg(2+)] (<0.1 mM) suggests that even at low ionic strength, with a tremendous number of possible (weak) interactions, a few critical interactions may produce deep energy wells that allow for rapid averaging of motions within each well, and yield kinetics that are relatively simple.

  2. Single Molecule Analysis of Resection Tracks.

    Science.gov (United States)

    Huertas, Pablo; Cruz-García, Andrés

    2018-01-01

    Homologous recombination is initiated by the so-called DNA end resection, the 5'-3' nucleolytic degradation of a single strand of the DNA at each side of the break. The presence of resected DNA is an obligatory step for homologous recombination. Moreover, the amount of resected DNA modulates the prevalence of different recombination pathways. In different model organisms, there are several published ways to visualize and measure with more or less detail the amount of DNA resected. In human cells, however, technical constraints hampered the study of resection at high resolution. Some information might be gathered from the study of endonuclease-created DSBs, in which the resection of breaks at known sites can be followed by PCR or ChIP. In this chapter, we describe in detail a novel assay to study DNA end resection in breaks located on unknown positions. Here, we use ionizing radiation to induce double-strand breaks, but the same approach can be used to monitor resection induced by different DNA damaging agents. By modifying the DNA-combing technique, used for high-resolution replication analyses, we can measure resection progression at the level of individual DNA fibers. Thus, we named the method Single Molecule Analysis of Resection Tracks (SMART). We use human cells in culture as a model system, but in principle the same approach would be feasible to any model organism adjusting accordingly the DNA isolation part of the protocol.

  3. Investigation on Single-Molecule Junctions Based on Current–Voltage Characteristics

    Directory of Open Access Journals (Sweden)

    Yuji Isshiki

    2018-02-01

    Full Text Available The relationship between the current through an electronic device and the voltage across its terminals is a current–voltage characteristic (I–V that determine basic device performance. Currently, I–V measurement on a single-molecule scale can be performed using break junction technique, where a single molecule junction can be prepared by trapping a single molecule into a nanogap between metal electrodes. The single-molecule I–Vs provide not only the device performance, but also reflect information on energy dispersion of the electronic state and the electron-molecular vibration coupling in the junction. This mini review focuses on recent representative studies on I–Vs of the single molecule junctions that cover investigation on the single-molecule diode property, the molecular vibration, and the electronic structure as a form of transmission probability, and electronic density of states, including the spin state of the single-molecule junctions. In addition, thermoelectronic measurements based on I–Vs and identification of the charged carriers (i.e., electrons or holes are presented. The analysis in the single-molecule I–Vs provides fundamental and essential information for a better understanding of the single-molecule science, and puts the single molecule junction to more practical use in molecular devices.

  4. Single-molecule magnets ``without'' intermolecular interactions

    Science.gov (United States)

    Wernsdorfer, W.; Vergnani, L.; Rodriguez-Douton, M. J.; Cornia, A.; Neugebauer, P.; Barra, A. L.; Sorace, L.; Sessoli, R.

    2012-02-01

    Intermolecular magnetic interactions (dipole-dipole and exchange) affect strongly the magnetic relaxation of crystals of single-molecule magnets (SMMs), especially at low temperature, where quantum tunneling of the magnetization (QTM) dominates. This leads to complex many-body problems [l]. Measurements on magnetically diluted samples are desirable to clearly sort out the behaviour of magnetically-isolated SMMs and to reveal, by comparison, the effect of intermolecular interactions. Here, we diluted a Fe4 SMM into a diamagnetic crystal lattice, affording arrays of independent and iso-oriented magnetic units. We found that the resonant tunnel transitions are much sharper, the tunneling efficiency changes significantly, and two-body QTM transitions disappear. These changes have been rationalized on the basis of a dipolar shuffling mechanism and of transverse dipolar fields, whose effect has been analyzed using a multispin model. Our findings directly prove the impact of intermolecular magnetic couplings on the SMM behaviour and disclose the magnetic response of truly-isolated giant spins in a diamagnetic crystalline environment.[4pt] [1] W. Wernsdorfer, at al, PRL 82, 3903 (1999); PRL 89, 197201 (2002); Nature 416, 406 (2002); IS Tupitsyn, PCE Stamp, NV Prokof'ev, PRB 69, 132406 (2004).

  5. Magnetization reversal in single molecule magnets

    Science.gov (United States)

    Bokacheva, Louisa

    2002-09-01

    I have studied the magnetization reversal in single molecule magnets (SMMs). SMMs are Van der Waals crystals, consisting of identical molecules containing transition metal ions, with high spin and large uniaxial magnetic anisotropy. They can be considered as ensembles of identical, iso-oriented nanomagnets. At high temperature, these materials behave as superparamagnets and their magnetization reversal occurs by thermal activation. At low temperature they become blocked, and their magnetic relaxation occurs via thermally assisted tunneling or pure quantum tunneling through the anisotropy barrier. We have conducted detailed experimental studies of the magnetization reversal in SMM material Mn12-acetate (Mn12) with S = 10. Low temperature measurements were conducted using micro-Hall effect magnetometry. We performed hysteresis and relaxation studies as a function of temperature, transverse field, and magnetization state of the sample. We identified magnetic sublevels that dominate the tunneling at a given field, temperature and magnetization. We observed a crossover between thermally assisted and pure quantum tunneling. The form of this crossover depends on the magnitude and direction of the applied field. This crossover is abrupt (first-order) and occurs in a narrow temperature interval (tunneling mechanisms in Mn12.

  6. Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions

    Energy Technology Data Exchange (ETDEWEB)

    Laurence, Ted Alfred [Univ. of California, Berkeley, CA (United States)

    2002-01-01

    Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors to detect bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract fluorescence resonance energy transfer (FRET) efficiency. Advances in data acquisition and analysis techniques that are providing a more complete picture of the accessible molecular information are discussed. Photon Arrival-time Interval Distribution (PAID) analysis is a new method for monitoring macromolecular interactions by fluorescence detection with simultaneous determination of coincidence, brightness, diffusion time, and occupancy (proportional to concentration) of fluorescently-labeled molecules undergoing diffusion in a confocal detection volume. This method is based on recording the time of arrival of all detected photons, and then plotting the two-dimensional histogram of photon pairs, where one axis is the time interval between each pair of photons 1 and 2, and the second axis is the number of other photons detected in the time interval between photons 1 and 2. PAID is related to Fluorescence Correlation Spectroscopy (FCS) by a collapse of this histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS

  7. Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions

    International Nuclear Information System (INIS)

    Laurence, Ted Alfred

    2002-01-01

    Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors to detect bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract fluorescence resonance energy transfer (FRET) efficiency. Advances in data acquisition and analysis techniques that are providing a more complete picture of the accessible molecular information are discussed. Photon Arrival-time Interval Distribution (PAID) analysis is a new method for monitoring macromolecular interactions by fluorescence detection with simultaneous determination of coincidence, brightness, diffusion time, and occupancy (proportional to concentration) of fluorescently-labeled molecules undergoing diffusion in a confocal detection volume. This method is based on recording the time of arrival of all detected photons, and then plotting the two-dimensional histogram of photon pairs, where one axis is the time interval between each pair of photons 1 and 2, and the second axis is the number of other photons detected in the time interval between photons 1 and 2. PAID is related to Fluorescence Correlation Spectroscopy (FCS) by a collapse of this histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS

  8. Single Molecule Biophysics Experiments and Theory

    CERN Document Server

    Komatsuzaki, Tamiki; Takahashi, Satoshi; Yang, Haw; Silbey, Robert J; Rice, Stuart A; Dinner, Aaron R

    2011-01-01

    Discover the experimental and theoretical developments in optical single-molecule spectroscopy that are changing the ways we think about molecules and atoms The Advances in Chemical Physics series provides the chemical physics field with a forum for critical, authoritative evaluations of advances in every area of the discipline. This latest volume explores the advent of optical single-molecule spectroscopy, and how atomic force microscopy has empowered novel experiments on individual biomolecules, opening up new frontiers in molecular and cell biology and leading to new theoretical approaches

  9. Regulation of membrane protein function by lipid bilayer elasticity—a single molecule technology to measure the bilayer properties experienced by an embedded protein

    DEFF Research Database (Denmark)

    Lundbæk, Jens August

    2008-01-01

    -dependent sodium channels, N-type calcium channels and GABAA receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic......Membrane protein function is generally regulated by the molecular composition of the host lipid bilayer. The underlying mechanisms have long remained enigmatic. Some cases involve specific molecular interactions, but very often lipids and other amphiphiles, which are adsorbed to lipid bilayers......, regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes...

  10. Regulation of membrane protein function by lipid bilayer elasticity-a single molecule technology to measure the bilayer properties experienced by an embedded protein

    International Nuclear Information System (INIS)

    Lundbaek, Jens August

    2006-01-01

    Membrane protein function is generally regulated by the molecular composition of the host lipid bilayer. The underlying mechanisms have long remained enigmatic. Some cases involve specific molecular interactions, but very often lipids and other amphiphiles, which are adsorbed to lipid bilayers, regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes, in the general regulation of membrane protein function, is unclear. This is to a large extent due to lack of a generally accepted framework in which to understand the many observations. The present review summarizes studies which have demonstrated that the hydrophobic interactions between a membrane protein and the host lipid bilayer provide an energetic coupling, whereby protein function can be regulated by the bilayer elasticity. The feasibility of this 'hydrophobic coupling mechanism' has been demonstrated using the gramicidin channel, a model membrane protein, in planar lipid bilayers. Using voltage-dependent sodium channels, N-type calcium channels and GABA A receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic properties experienced by an embedded protein has been developed. A theoretical and technological framework, to study the regulation of membrane protein function by lipid bilayer elasticity, has been established

  11. Single-Molecule Analysis of Protein Large-Amplitude Conformational Transitions

    Science.gov (United States)

    Yang, Haw

    2011-03-01

    Proteins have evolved to harness thermal fluctuations, rather than frustrated by them, to carry out chemical transformations and mechanical work. What are, then, the operation and design principles of protein machines? To frame the problem in a tractable way, several basic questions have been formulated to guide the experimental design: (a) How many conformational states can a protein sample on the functionally important timescale? (b) What are the inter-conversion rates between states? (c) How do ligand binding or interactions with other proteins modulate the motions? (d) What are the structural basis of flexibility and its underlying molecular mechanics? Guided by this framework, we have studied protein tyrosine phosphatase B, PtpB, from M. tuberculosis (a virulence factor of tuberculosis and a potential drug target) and adenylate kinase, AK, from E. coli (a ubiquitous energy-balancing enzyme in cells). These domain movements have been followed in real time on their respective catalytic timescales using high-resolution single-molecule Förster resonance energy transfer (FRET) spectroscopy. It is shown quantitatively that both PtpB and AK are capable of dynamically sampling two distinct states that correlate well with those observed by x-ray crystallography. Integrating these microscopic dynamics into macroscopic kinetics allows us to place the experimentally measured free-energy landscape in the context of enzymatic turnovers.

  12. Automated imaging system for single molecules

    Science.gov (United States)

    Schwartz, David Charles; Runnheim, Rodney; Forrest, Daniel

    2012-09-18

    There is provided a high throughput automated single molecule image collection and processing system that requires minimal initial user input. The unique features embodied in the present disclosure allow automated collection and initial processing of optical images of single molecules and their assemblies. Correct focus may be automatically maintained while images are collected. Uneven illumination in fluorescence microscopy is accounted for, and an overall robust imaging operation is provided yielding individual images prepared for further processing in external systems. Embodiments described herein are useful in studies of any macromolecules such as DNA, RNA, peptides and proteins. The automated image collection and processing system and method of same may be implemented and deployed over a computer network, and may be ergonomically optimized to facilitate user interaction.

  13. Handbook of Single-Molecule Biophysics

    CERN Document Server

    Hinterdorfer, Peter

    2009-01-01

    The last decade has seen the development of a number of novel biophysical methods that allow the manipulation and study of individual biomolecules. The ability to monitor biological processes at this fundamental level of sensitivity has given rise to an improved understanding of the underlying molecular mechanisms. Through the removal of ensemble averaging, distributions and fluctuations of molecular properties can be characterized, transient intermediates identified, and catalytic mechanisms elucidated. By applying forces on biomolecules while monitoring their activity, important information can be obtained on how proteins couple function to structure. The Handbook of Single-Molecule Biophysics provides an introduction to these techniques and presents an extensive discussion of the new biological insights obtained from them. Coverage includes: Experimental techniques to monitor and manipulate individual biomolecules The use of single-molecule techniques in super-resolution and functional imaging Single-molec...

  14. Single molecule SERS: Perspectives of analytical applications

    Czech Academy of Sciences Publication Activity Database

    Vlčková, B.; Pavel, I.; Sládková, M.; Šišková, K.; Šlouf, Miroslav

    834-836, - (2007), s. 42-47 ISSN 0022-2860. [European Congress on Molecular Spectroscopy /28./. Istanbul, 03.09.2006-08.09.2006] R&D Projects: GA ČR GA203/04/0688 Institutional research plan: CEZ:AV0Z40500505 Keywords : surface-enhanced Raman scattering (SERS) * surface-enhanced resonance Raman (SERRS) * single molecule SERS Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.486, year: 2007

  15. In silico FRET from simulated dye dynamics

    Science.gov (United States)

    Hoefling, Martin; Grubmüller, Helmut

    2013-03-01

    Single molecule fluorescence resonance energy transfer (smFRET) experiments probe molecular distances on the nanometer scale. In such experiments, distances are recorded from FRET transfer efficiencies via the Förster formula, E=1/(1+(). The energy transfer however also depends on the mutual orientation of the two dyes used as distance reporter. Since this information is typically inaccessible in FRET experiments, one has to rely on approximations, which reduce the accuracy of these distance measurements. A common approximation is an isotropic and uncorrelated dye orientation distribution. To assess the impact of such approximations, we present the algorithms and implementation of a computational toolkit for the simulation of smFRET on the basis of molecular dynamics (MD) trajectory ensembles. In this study, the dye orientation dynamics, which are used to determine dynamic FRET efficiencies, are extracted from MD simulations. In a subsequent step, photons and bursts are generated using a Monte Carlo algorithm. The application of the developed toolkit on a poly-proline system demonstrated good agreement between smFRET simulations and experimental results and therefore confirms our computational method. Furthermore, it enabled the identification of the structural basis of measured heterogeneity. The presented computational toolkit is written in Python, available as open-source, applicable to arbitrary systems and can easily be extended and adapted to further problems. Catalogue identifier: AENV_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENV_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GPLv3, the bundled SIMD friendly Mersenne twister implementation [1] is provided under the SFMT-License. No. of lines in distributed program, including test data, etc.: 317880 No. of bytes in distributed program, including test data, etc.: 54774217 Distribution format: tar.gz Programming language

  16. Single-Molecule Imaging of GPCR Interactions.

    Science.gov (United States)

    Calebiro, Davide; Sungkaworn, Titiwat

    2018-02-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of great interest as pharmacological targets. Although the occurrence of GPCR signaling nanodomains has long been hypothesized based on indirect evidence, this and other fundamental aspects of GPCR signaling have been difficult to prove. The advent of single-molecule microscopy methods, which allow direct visualization of individual membrane proteins with unprecedented spatiotemporal resolution, provides unique opportunities to address several of these open questions. Indeed, recent single-molecule studies have revealed that GPCRs and G proteins transiently interact with each other as well as with structural components of the plasma membrane, leading to the formation of dynamic complexes and 'hot spots' for GPCR signaling. Whereas we are only beginning to understand the implications of this unexpected level of complexity, single-molecule approaches are likely to play a crucial role to further dissect the protein-protein interactions that are at the heart of GPCR signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Controlling single-molecule junction conductance by molecular interactions

    Science.gov (United States)

    Kitaguchi, Y.; Habuka, S.; Okuyama, H.; Hatta, S.; Aruga, T.; Frederiksen, T.; Paulsson, M.; Ueba, H.

    2015-01-01

    For the rational design of single-molecular electronic devices, it is essential to understand environmental effects on the electronic properties of a working molecule. Here we investigate the impact of molecular interactions on the single-molecule conductance by accurately positioning individual molecules on the electrode. To achieve reproducible and precise conductivity measurements, we utilize relatively weak π-bonding between a phenoxy molecule and a STM-tip to form and cleave one contact to the molecule. The anchoring to the other electrode is kept stable using a chalcogen atom with strong bonding to a Cu(110) substrate. These non-destructive measurements permit us to investigate the variation in single-molecule conductance under different but controlled environmental conditions. Combined with density functional theory calculations, we clarify the role of the electrostatic field in the environmental effect that influences the molecular level alignment. PMID:26135251

  18. Dysprosium Acetylacetonato Single-Molecule Magnet Encapsulated in Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Ryo Nakanishi

    2016-12-01

    Full Text Available Dy single-molecule magnets (SMMs, which have several potential uses in a variety of applications, such as quantum computing, were encapsulated in multi-walled carbon nanotubes (MWCNTs by using a capillary method. Encapsulation was confirmed by using transmission electron microscopy (TEM. In alternating current magnetic measurements, the magnetic susceptibilities of the Dy acetylacetonato complexes showed clear frequency dependence even inside the MWCNTs, meaning that this hybrid can be used as magnetic materials in devices.

  19. Lattice diffusion of a single molecule in solution

    Science.gov (United States)

    Ruggeri, Francesca; Krishnan, Madhavi

    2017-12-01

    The ability to trap a single molecule in an electrostatic potential well in solution has opened up new possibilities for the use of molecular electrical charge to study macromolecular conformation and dynamics at the level of the single entity. Here we study the diffusion of a single macromolecule in a two-dimensional lattice of electrostatic traps in solution. We report the ability to measure both the size and effective electrical charge of a macromolecule by observing single-molecule transport trajectories, typically a few seconds in length, using fluorescence microscopy. While, as shown previously, the time spent by the molecule in a trap is a strong function of its effective charge, we demonstrate here that the average travel time between traps in the landscape yields its hydrodynamic radius. Tailoring the pitch of the lattice thus yields two different experimentally measurable time scales that together uniquely determine both the size and charge of the molecule. Since no information is required on the location of the molecule between consecutive departure and arrival events at lattice sites, the technique is ideally suited to measurements on weakly emitting entities such as single molecules.

  20. Assessing FRET using Spectral Techniques

    Science.gov (United States)

    Leavesley, Silas J.; Britain, Andrea L.; Cichon, Lauren K.; Nikolaev, Viacheslav O.; Rich, Thomas C.

    2015-01-01

    Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein–protein interactions, protein folding, and intracellular signaling events. These techniques have traditionally been implemented with the use of one or more fluorescence band-pass filters, either as fluorescence microscopy filter cubes, or as dichroic mirrors and band-pass filters in flow cytometry. In addition, new approaches for measuring FRET, such as fluorescence lifetime and acceptor photobleaching, have been developed. Hyperspectral techniques for imaging and flow cytometry have also shown to be promising for performing FRET measurements. In this study, we have compared traditional (filter-based) FRET approaches to three spectral-based approaches: the ratio of acceptor-to-donor peak emission, linear spectral unmixing, and linear spectral unmixing with a correction for direct acceptor excitation. All methods are estimates of FRET efficiency, except for one-filter set and three-filter set FRET indices, which are included for consistency with prior literature. In the first part of this study, spectrofluorimetric data were collected from a CFP–Epac–YFP FRET probe that has been used for intracellular cAMP measurements. All comparisons were performed using the same spectrofluorimetric datasets as input data, to provide a relevant comparison. Linear spectral unmixing resulted in measurements with the lowest coefficient of variation (0.10) as well as accurate fits using the Hill equation. FRET efficiency methods produced coefficients of variation of less than 0.20, while FRET indices produced coefficients of variation greater than 8.00. These results demonstrate that spectral FRET measurements provide improved response over standard, filter-based measurements. Using spectral approaches, single-cell measurements were conducted through hyperspectral confocal microscopy, linear unmixing, and cell segmentation with quantitative image analysis

  1. Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

    Science.gov (United States)

    Ha, Taekjip; Tinnefeld, Philip

    2012-05-01

    Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own “personalities” regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.

  2. A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.

    Directory of Open Access Journals (Sweden)

    Jakobus van Unen

    Full Text Available G-protein coupled receptors (GPCRs can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.

  3. Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics

    KAUST Repository

    Sobhy, M. A.

    2011-11-07

    Single-molecule fluorescence imaging is at the forefront of tools applied to study biomolecular dynamics both in vitro and in vivo. The ability of the single-molecule fluorescence microscope to conduct simultaneous multi-color excitation and detection is a key experimental feature that is under continuous development. In this paper, we describe in detail the design and the construction of a sophisticated and versatile multi-color excitation and emission fluorescence instrument for studying biomolecular dynamics at the single-molecule level. The setup is novel, economical and compact, where two inverted microscopes share a laser combiner module with six individual laser sources that extend from 400 to 640 nm. Nonetheless, each microscope can independently and in a flexible manner select the combinations, sequences, and intensities of the excitation wavelengths. This high flexibility is achieved by the replacement of conventional mechanical shutters with acousto-optic tunable filter (AOTF). The use of AOTF provides major advancement by controlling the intensities, duration, and selection of up to eight different wavelengths with microsecond alternation time in a transparent and easy manner for the end user. To our knowledge this is the first time AOTF is applied to wide-field total internal reflection fluorescence (TIRF) microscopy even though it has been commonly used in multi-wavelength confocal microscopy. The laser outputs from the combiner module are coupled to the microscopes by two sets of four single-mode optic fibers in order to allow for the optimization of the TIRF angle for each wavelength independently. The emission is split into two or four spectral channels to allow for the simultaneous detection of up to four different fluorophores of wide selection and using many possible excitation and photoactivation schemes. We demonstrate the performance of this new setup by conducting two-color alternating excitation single-molecule fluorescence resonance energy

  4. Deep learning for single-molecule science

    Science.gov (United States)

    Albrecht, Tim; Slabaugh, Gregory; Alonso, Eduardo; Al-Arif, SM Masudur R.

    2017-10-01

    Exploring and making predictions based on single-molecule data can be challenging, not only due to the sheer size of the datasets, but also because a priori knowledge about the signal characteristics is typically limited and poor signal-to-noise ratio. For example, hypothesis-driven data exploration, informed by an expectation of the signal characteristics, can lead to interpretation bias or loss of information. Equally, even when the different data categories are known, e.g., the four bases in DNA sequencing, it is often difficult to know how to make best use of the available information content. The latest developments in machine learning (ML), so-called deep learning (DL) offer interesting, new avenues to address such challenges. In some applications, such as speech and image recognition, DL has been able to outperform conventional ML strategies and even human performance. However, to date DL has not been applied much in single-molecule science, presumably in part because relatively little is known about the ‘internal workings’ of such DL tools within single-molecule science as a field. In this Tutorial, we make an attempt to illustrate in a step-by-step guide how one of those, a convolutional neural network (CNN), may be used for base calling in DNA sequencing applications. We compare it with a SVM as a more conventional ML method, and discuss some of the strengths and weaknesses of the approach. In particular, a ‘deep’ neural network has many features of a ‘black box’, which has important implications on how we look at and interpret data.

  5. Single molecule microscopy and spectroscopy: concluding remarks.

    Science.gov (United States)

    van Hulst, Niek F

    2015-01-01

    Chemistry is all about molecules: control, synthesis, interaction and reaction of molecules. All too easily on a blackboard, one draws molecules, their structures and dynamics, to create an insightful picture. The dream is to see these molecules in reality. This is exactly what "Single Molecule Detection" provides: a look at molecules in action at ambient conditions; a breakthrough technology in chemistry, physics and biology. Within the realms of the Royal Society of Chemistry, the Faraday Discussion on "Single Molecule Microscopy and Spectroscopy" was a very appropriate topic for presentation, deliberation and debate. Undoubtedly, the Faraday Discussions have a splendid reputation in stimulating scientific debates along the traditions set by Michael Faraday. Interestingly, back in the 1830's, Faraday himself pursued an experiment that led to the idea that atoms in a compound were joined by an electrical component. He placed two opposite electrodes in a solution of water containing a dissolved compound, and observed that one of the elements of the compound accumulated on one electrode, while the other was deposited on the opposite electrode. Although Faraday was deeply opposed to atomism, he had to recognize that electrical forces were responsible for the joining of atoms. Probably a direct view on the atoms or molecules in his experiment would have convinced him. As such, Michael Faraday might have liked the gathering at Burlington House in September 2015 (). Surely, with the questioning eyes of his bust on the 1st floor corridor, the non-believer Michael Faraday has incited each passer-by to enter into discussion and search for deeper answers at the level of single molecules. In these concluding remarks, highlights of the presented papers and discussions are summarized, complemented by a conclusion on future perspectives.

  6. Single Molecule Electrochemical Detection in Aqueous Solutions and Ionic Liquids.

    Science.gov (United States)

    Byers, Joshua C; Paulose Nadappuram, Binoy; Perry, David; McKelvey, Kim; Colburn, Alex W; Unwin, Patrick R

    2015-10-20

    Single molecule electrochemical detection (SMED) is an extremely challenging aspect of electroanalytical chemistry, requiring unconventional electrochemical cells and measurements. Here, SMED is reported using a "quad-probe" (four-channel probe) pipet cell, fabricated by depositing carbon pyrolytically into two diagonally opposite barrels of a laser-pulled quartz quadruple-barreled pipet and filling the open channels with electrolyte solution, and quasi-reference counter electrodes. A meniscus forms at the end of the probe covering the two working electrodes and is brought into contact with a substrate working electrode surface. In this way, a nanogap cell is produced whereby the two carbon electrodes in the pipet can be used to promote redox cycling of an individual molecule with the substrate. Anticorrelated currents generated at the substrate and tip electrodes, at particular distances (typically tens of nanometers), are consistent with the detection of single molecules. The low background noise realized in this droplet format opens up new opportunities in single molecule electrochemistry, including the use of ionic liquids, as well as aqueous solution, and the quantitative assessment and analysis of factors influencing redox cycling currents, due to a precisely known gap size.

  7. Mechanoenzymatics and Nanoassembly of Single Molecules

    Science.gov (United States)

    Puchner, Elias M.; Gaub, Hermann E.

    We investigated the muscle enzyme, titin kinase, by means of single-molecule force spectroscopy. Our results show that the binding of ATP, which is the first step of its signaling cascade controlling the muscle gene expression and protein turnover, is mechanically induced. The detailed determination of barrier positions in the mechanical activation pathway and the corresponding functional states allow structural insight, by comparing the experiment with molecular dynamics simulations. From our results, we conclude that titin kinase acts as a natural force sensor controlling the muscle build-up. To study the interplay of functional units, we developed the single-molecule cut-and-paste technique, which combines the precision of AFM with the selectivity of DNA hybridization. Functional units can be assembled one-by-one in an arbitrarily predefined pattern, with an accuracy that is better than 11 nm. The cyclic assembly process is optically monitored and mechanically recorded by force-extension traces. Using biotin as a functional unit attached to the transported DNA, patterns of binding sites may be created, to which streptavidin-modified nanoobjects like fluorescent nanoparticles can specifically self-assemble in a second step.

  8. Fluorescence resonance energy transfer (FRET) in chemistry and ...

    Indian Academy of Sciences (India)

    Abstract. Fluorescence resonance energy transfer (FRET) is a popular tool to study equilibrium and dynamical properties of polymers and biopolymers in condensed phases and is now widely used in conjunc- tion with single molecule spectroscopy. In the data analysis, one usually employs the Förster expression.

  9. Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

    Science.gov (United States)

    Isbaner, Sebastian; Karedla, Narain; Kaminska, Izabela; Ruhlandt, Daja; Raab, Mario; Bohlen, Johann; Chizhik, Alexey; Gregor, Ingo; Tinnefeld, Philip; Enderlein, Jörg; Tsukanov, Roman

    2018-03-27

    Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

  10. A brief introduction to single-molecule fluorescence methods

    NARCIS (Netherlands)

    Wildenberg, S.M.J.L.; Prevo, B.; Peterman, E.J.G.; Peterman, EJG; Wuite, GJL

    2011-01-01

    One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow

  11. A brief introduction to single-molecule fluorescence methods

    NARCIS (Netherlands)

    van den Wildenberg, Siet M.J.L.; Prevo, Bram; Peterman, Erwin J.G.

    2018-01-01

    One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also

  12. Spin-Spin Cross Relaxation in Single-Molecule Magnets

    Science.gov (United States)

    Wernsdorfer, W.; Bhaduri, S.; Tiron, R.; Hendrickson, D. N.; Christou, G.

    2002-10-01

    The one-body tunnel picture of single-molecule magnets (SMMs) is not always sufficient to explain the measured tunnel transitions. An improvement to the picture is proposed by including also two-body tunnel transitions such as spin-spin cross relaxation (SSCR) which are mediated by dipolar and weak superexchange interactions between molecules. A Mn4 SMM is used as a model system. At certain external fields, SSCRs lead to additional quantum resonances which show up in hysteresis loop measurements as well-defined steps. A simple model is used to explain quantitatively all observed transitions.

  13. Development of new microscope unit for single molecule spectroscopy under various ambient conditions

    International Nuclear Information System (INIS)

    Yamada, T; Kaji, T; Ueda, R; Otomo, A

    2013-01-01

    This paper introduces techniques we previously developed for single molecule spectroscopy and continues on to describe our studies on dipole orientation imaging of single molecules under various ambient conditions. In these studies, we successfully obtained defocused images of single perylene diimide (PDI) molecules under air, high-vacuum, and pure N 2 gas conditions by utilizing the advantages of our new microscope unit. The studies are positioned as one of the important applications of our microscope unit for single molecule spectroscopy. We expect a wide range of applications for this unit for various microscope measurements for many types of materials.

  14. Nonequilibrium Energetics of Single Molecule Motor, Kinesin-1

    Science.gov (United States)

    Ariga, Takayuki; Tomishige, Michio; Mizuno, Daisuke

    2018-02-01

    Molecular motors are nonequilibrium open systems that convert chemical energy to mechanical work. Here we investigate the nonequilibrium energetics of a single molecule kinesin by measuring the motion of an attached probe particle and its response to external forces with optical tweezers. The sum of the heat dissipation estimated from the violation of the fluctuation-response relation and the output power was inconsistent with the input free energy rate, implying that internal dissipation is dominant. By using a two-state Markov model, we discuss the energy flow of the kinesin motor.

  15. Electron transfer dynamics of bistable single-molecule junctions

    DEFF Research Database (Denmark)

    Danilov, A.V; Kubatkin, S.; Kafanov, S. G.

    2006-01-01

    We present transport measurements of single-molecule junctions bridged by a molecule with three benzene rings connected by two double bonds and with thiol end-groups that allow chemical binding to gold electrodes. The I-V curves show switching behavior between two distinct states. By statistical ...... analysis of the switching events, we show that a 300 meV mode mediates the transition between the two states. We propose that breaking and reformation of a S-H bond in the contact zone between molecule and electrode explains the observed bistability....

  16. DNA heterogeneity and phosphorylation unveiled by single-molecule electrophoresis

    Science.gov (United States)

    Wang, Hui; Dunning, James E.; Huang, Albert P.-H.; Nyamwanda, Jacqueline A.; Branton, Daniel

    2004-09-01

    Broad-spectrum analysis of DNA and RNA samples is of increasing importance in the growing field of biotechnology. We show that nanopore measurements may be used to assess the purity, phosphorylation state, and chemical integrity of nucleic acid preparations. In contrast with gel electrophoresis and mass spectrometry, an unprecedented dynamic range of DNA sizes and concentrations can be evaluated in a single data acquisition process that spans minutes. Because the molecule information is quantized and digitally recorded with single-molecule resolution, the sensitivity of the system can be adjusted in real time to detect trace amounts of a particular DNA species.

  17. Toward The Reconstitution of the Maturation of Okazaki Fragments Multiprotein Complex in Human At The Single Molecule Level

    KAUST Repository

    Joudeh, Luay

    2017-04-01

    The maturation of Okazaki fragments on the lagging strand in eukaryotes is mediated by a highly coordinated multistep process involving several proteins that ensure the accurate and efficient replication of genomic DNA. Human proliferating cell nuclear antigen (PCNA) that slides on double-stranded DNA is the key player that coordinates the access of various proteins to the different intermediary steps in this process. In this study, I am focusing on characterizing how PCNA recruits and stimulates the structure specific flap endonuclease 1 (FEN1) to process the aberrant double flap (DF) structures that are produced during maturation of Okazaki fragments. FEN1 distorts the DF structures into a bent conformer to place the scissile phosphate into the active site for cleavage. The product is a nick substrate that can be sealed by DNA ligase I whose recruitment is also mediated by its interaction with PCNA. Using single-molecule Förster resonance energy transfer (smFRET) measurements that simultaneously monitored bending and cleavage of various DF substrates by FEN1 alone or in the presence of PCNA, we found that FEN1 and PCNA bends cognate and non-cognate substrates but display remarkable selectivity to stabilize the bent conformer in cognate substrate while promoting the dissociation of non-cognate substrates. This mechanism provides efficiency and accuracy for FEN1 and PCNA to cleave the correct substrate while avoiding the deleterious cleavage of incorrect substrates. This work provides a true molecular level understanding of the key step during the maturation of Okazaki fragment and contributes towards the reconstitution of its entire activity at the single molecule level.

  18. Development of the Measurement Technology of Fretting Wear Test Parameters under the Environment of High Temperature and Pressure - Final Report -

    International Nuclear Information System (INIS)

    Kim, Hyung Kyu; Lee, Young Ho; Song, Ju Sun

    2006-01-01

    This report is dealing with the results of the development procedure of a load cell that could be applied to the fretting wear tester in high temperature and pressure water condition in order to measure the contact force between the nuclear fuel rods and their supports. The strain gage of WK-06-062TT-350 was used with considering the temperature limits and the economical concerns in this study and two rosette pairs were bonded on opposite sides with a 180 .deg. C angle between each other to establish the Wheatstone full bridge circuit. To design bi-axial load cells with good performance in a high temperature and pressure water condition, we should preferentially consider the shape and size of a force sensing element and the waterproofing method at the same time. Because of a limited test section within the autoclave of the fretting wear tester, the load cell should have a relatively small size. In addition, strain gages and their lead wire should be shielded against high temperature water. A simple cylinder type is adopted for the shape of the force sensing element in consideration of easy waterproofing. This force measurement system consists of a force sensing element, a cylinder type cap with a hole, a base case, a force transfer screw, two metallic sealants, tube connector and metallic tube. In this study, a fluoro plastic-coated Nickel-plated Copper wire was used as signal wires for the purpose of the cable shield against the metallic tube in which the wires are inserted. Also, a metallic sealant that was made from Silver-coated Copper was used for waterproofing between the force sensing element and the cylinder type cap in the high temperature and pressure water condition. This enables an easy fabrication without a welding process for waterproofing. From the strain analysis result of the sensing element, the estimated coupling error between the normal and shear component is less than 4%. We determined the location of the strain gages based on the above result

  19. Biological Nanopores: Confined Spaces for Electrochemical Single-Molecule Analysis.

    Science.gov (United States)

    Cao, Chan; Long, Yi-Tao

    2018-02-20

    Nanopore sensing is developing into a powerful single-molecule approach to investigate the features of biomolecules that are not accessible by studying ensemble systems. When a target molecule is transported through a nanopore, the ions occupying the pore are excluded, resulting in an electrical signal from the intermittent ionic blockade event. By statistical analysis of the amplitudes, duration, frequencies, and shapes of the blockade events, many properties of the target molecule can be obtained in real time at the single-molecule level, including its size, conformation, structure, charge, geometry, and interactions with other molecules. With the development of the use of α-hemolysin to characterize individual polynucleotides, nanopore technology has attracted a wide range of research interest in the fields of biology, physics, chemistry, and nanoscience. As a powerful single-molecule analytical method, nanopore technology has been applied for the detection of various biomolecules, including oligonucleotides, peptides, oligosaccharides, organic molecules, and disease-related proteins. In this Account, we highlight recent developments of biological nanopores in DNA-based sensing and in studying the conformational structures of DNA and RNA. Furthermore, we introduce the application of biological nanopores to investigate the conformations of peptides affected by charge, length, and dipole moment and to study disease-related proteins' structures and aggregation transitions influenced by an inhibitor, a promoter, or an applied voltage. To improve the sensing ability of biological nanopores and further extend their application to a wider range of molecular sensing, we focus on exploring novel biological nanopores, such as aerolysin and Stable Protein 1. Aerolysin exhibits an especially high sensitivity for the detection of single oligonucleotides both in current separation and duration. Finally, to facilitate the use of nanopore measurements and statistical analysis

  20. Grafting single molecule magnets on gold nanoparticles.

    Science.gov (United States)

    Perfetti, Mauro; Pineider, Francesco; Poggini, Lorenzo; Otero, Edwige; Mannini, Matteo; Sorace, Lorenzo; Sangregorio, Claudio; Cornia, Andrea; Sessoli, Roberta

    2014-01-29

    The chemical synthesis and characterization of the first hybrid material composed by gold nanoparticles and single molecule magnets (SMMs) are described. Gold nanoparticles are functionalized via ligand exchange using a tetrairon(III) SMM containing two 1,2-dithiolane end groups. The grafting is evidenced by the shift of the plasmon resonance peak recorded with a UV-vis spectrometer, by the suppression of nuclear magnetic resonance signals, by X-ray photoemission spectroscopy peaks, and by transmission electron microscopy images. The latter evidence the formation of aggregates of nanoparticles as a consequence of the cross-linking ability of Fe4 through the two 1,2-dithiolane rings located on opposite sides of the metal core. The presence of intact Fe4 molecules is directly proven by synchrotron-based X-ray absorption spectroscopy and X-ray magnetic circular dichroism spectroscopy, while a detailed magnetic characterization, obtained using electron paramagnetic resonance and alternating-current susceptibility, confirms the persistence of SMM behavior in this new hybrid nanostructure. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines.

    Science.gov (United States)

    Babcock, Hazen P; Zhuang, Xiaowei

    2017-04-03

    The resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2-3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed.

  2. Voltage-Driven Conformational Switching with Distinct Raman Signature in a Single-Molecule Junction.

    Science.gov (United States)

    Bi, Hai; Palma, Carlos-Andres; Gong, Yuxiang; Hasch, Peter; Elbing, Mark; Mayor, Marcel; Reichert, Joachim; Barth, Johannes V

    2018-04-11

    Precisely controlling well-defined, stable single-molecule junctions represents a pillar of single-molecule electronics. Early attempts to establish computing with molecular switching arrays were partly challenged by limitations in the direct chemical characterization of metal-molecule-metal junctions. While cryogenic scanning probe studies have advanced the mechanistic understanding of current- and voltage-induced conformational switching, metal-molecule-metal conformations are still largely inferred from indirect evidence. Hence, the development of robust, chemically sensitive techniques is instrumental for advancement in the field. Here we probe the conformation of a two-state molecular switch with vibrational spectroscopy, while simultaneously operating it by means of the applied voltage. Our study emphasizes measurements of single-molecule Raman spectra in a room-temperature stable single-molecule switch presenting a signal modulation of nearly 2 orders of magnitude.

  3. Single-Molecule Plasmon Sensing: Current Status and Future Prospects.

    Science.gov (United States)

    Taylor, Adam B; Zijlstra, Peter

    2017-08-25

    Single-molecule detection has long relied on fluorescent labeling with high quantum-yield fluorophores. Plasmon-enhanced detection circumvents the need for labeling by allowing direct optical detection of weakly emitting and completely nonfluorescent species. This review focuses on recent advances in single molecule detection using plasmonic metal nanostructures as a sensing platform, particularly using a single particle-single molecule approach. In the past decade two mechanisms for plasmon-enhanced single-molecule detection have been demonstrated: (1) by plasmonically enhancing the emission of weakly fluorescent biomolecules, or (2) by monitoring shifts of the plasmon resonance induced by single-molecule interactions. We begin with a motivation regarding the importance of single molecule detection, and advantages plasmonic detection offers. We describe both detection mechanisms and discuss challenges and potential solutions. We finalize by highlighting the exciting possibilities in analytical chemistry and medical diagnostics.

  4. New Antifouling Platform Characterized by Single-Molecule Imaging

    Science.gov (United States)

    2015-01-01

    Antifouling surfaces have been widely studied for their importance in medical devices and industry. Antifouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm2 which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e., an ultimate resolution) to study antifouling properties of functionalized surfaces. We found that about 600 immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ∼5 pg/cm2 adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new antifouling platform that exhibits improved antifouling performance that shows only 78 IgG molecules adsorbed (∼0.5 pg/cm2). The antifouling platform consists of forming 1 nm TiO2 thin layer, on which peptidomimetic antifouling polymer (PMAP) is robustly anchored. The unprecedented antifouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others. PMID:24503420

  5. Single molecule study of a processivity clamp sliding on DNA

    Energy Technology Data Exchange (ETDEWEB)

    Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A; Barsky, D

    2007-07-05

    Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.

  6. A new microcavity design for single molecule detection

    International Nuclear Information System (INIS)

    Steiner, M.; Schleifenbaum, F.; Stupperich, C.; Failla, A.V.; Hartschuh, A.; Meixner, A.J.

    2006-01-01

    We present a new microcavity design which allows for efficient detection of single molecules by measuring the molecular fluorescence emission coupled into a resonant cavity mode. The Fabry-Perot-type microresonator consists of two silver mirrors separated by a thin polymer film doped with dye molecules in ultralow concenctration. By slightly tilting one of the mirrors different cavity lengths can be selected within the same sample. Locally, on a μm scale, the microcavity still acts as a planar Fabry-Perot resonator. Using scanning confocal fluorescence microscopy, single emitters on resonance with a single mode of the microresonator can be spatially addressed. Our microcavity is demonstrated to be well-suited for investigating the coupling mechanism between single quantum emitters and single modes of the electromagnetic field. The microcavity layout could be integrated in a lab-on-a-microchip design for ultrasensitive microfluidic analytics and can be considered as an important improvement for single photon sources based on single molecules operating at room temperature

  7. Probing spatial heterogeneity in supercooled glycerol and temporal heterogeneity with single-molecule FRET in polyprolines

    NARCIS (Netherlands)

    Xia, Ted

    2010-01-01

    This thesis presents two lines of research. On the one hand, we investigate heterogeneity in supercooled glycerol by means of rheometry, small-angle neutron scattering, and fluorescence imaging. We find from the rheological experiments that supercooled glycerol can behave like weak solids at

  8. Förster resonance energy transfer: Role of diffusion of fluorophore orientation and separation in observed shifts of FRET efficiency.

    Directory of Open Access Journals (Sweden)

    Bram Wallace

    Full Text Available Förster resonance energy transfer (FRET is a widely used single-molecule technique for measuring nanoscale distances from changes in the non-radiative transfer of energy between donor and acceptor fluorophores. For macromolecules and complexes this observed transfer efficiency is used to infer changes in molecular conformation under differing experimental conditions. However, sometimes shifts are observed in the FRET efficiency even when there is strong experimental evidence that the molecular conformational state is unchanged. We investigate ways in which such discrepancies can arise from kinetic effects. We show that significant shifts can arise from the interplay between excitation kinetics, orientation diffusion of fluorophores, separation diffusion of fluorophores, and non-emitting quenching.

  9. Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling

    Science.gov (United States)

    Das, Sulagna; Yin, Taofei; Yang, Qingfen; Zhang, Jingqiao; Wu, Yi I.; Yu, Ji

    2015-01-01

    Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of single-molecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P3, instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction. PMID:25561548

  10. Optics clustered to output unique solutions: a multi-laser facility for combined single molecule and ensemble microscopy.

    Science.gov (United States)

    Clarke, David T; Botchway, Stanley W; Coles, Benjamin C; Needham, Sarah R; Roberts, Selene K; Rolfe, Daniel J; Tynan, Christopher J; Ward, Andrew D; Webb, Stephen E D; Yadav, Rahul; Zanetti-Domingues, Laura; Martin-Fernandez, Marisa L

    2011-09-01

    Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena. © 2011 American Institute of Physics

  11. SINGLE MOLECULE APPROACHES TO BIOLOGY, 2010 GORDON RESEARCH CONFERENCE, JUNE 27-JULY 2, 2010, ITALY

    Energy Technology Data Exchange (ETDEWEB)

    Professor William Moerner

    2010-07-09

    The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues to emerge. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of biomolecular machines: what they do, how they work individually, how they work together, and finally, how they work inside live cells. The burgeoning use of single-molecule methods to elucidate biological problems is a highly multidisciplinary pursuit, involving both force- and fluorescence-based methods, the most up-to-date advances in microscopy, innovative biological and chemical approaches, and nanotechnology tools. This conference seeks to bring together top experts in molecular and cell biology with innovators in the measurement and manipulation of single molecules, and will provide opportunities for junior scientists and graduate students to present their work in poster format and to exchange ideas with leaders in the field. A number of excellent poster presenters will be selected for short oral talks. Topics as diverse as single-molecule sequencing, DNA/RNA/protein interactions, folding machines, cellular biophysics, synthetic biology and bioengineering, force spectroscopy, new method developments, superresolution imaging in cells, and novel probes for single-molecule imaging will be on the program. Additionally, the collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings in the beauty of the Il Ciocco site in

  12. Single-molecule-sensitive fluorescence resonance energy transfer in freely-diffusing attoliter droplets

    Energy Technology Data Exchange (ETDEWEB)

    Rahmanseresht, Sheema; Ramos, Kieran P.; Gamari, Ben D.; Goldner, Lori S., E-mail: lgoldner@physics.umass.edu [Department of Physics, University of Massachusetts, Amherst, Massachusetts 01003 (United States); Milas, Peker [Department of Neuroscience, University of Wisconsin, Madison, Wisconsin 53705 (United States)

    2015-05-11

    Fluorescence resonance energy transfer (FRET) from individual, dye-labeled RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is carefully compared to FRET from unconfined RNA in solution. The use of freely-diffusing droplets is a remarkably simple and high-throughput technique that facilitates a substantial increase in signal-to-noise for single-molecular-pair FRET measurements. We show that there can be dramatic differences between FRET in solution and in droplets, which we attribute primarily to an altered pH in the confining environment. We also demonstrate that a sufficient concentration of a non-ionic surfactant mitigates this effect and restores FRET to its neutral-pH solution value. At low surfactant levels, even accounting for pH, we observe differences between the distribution of FRET values in solution and in droplets which remain unexplained. Our results will facilitate the use of nanoemulsion droplets as attoliter volume reactors for use in biophysical and biochemical assays, and also in applications such as protein crystallization or nanoparticle synthesis, where careful attention to the pH of the confined phase is required.

  13. Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

    Science.gov (United States)

    Yan, Rui; Moon, Seonah; Kenny, Samuel J; Xu, Ke

    2018-03-20

    As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with

  14. Rotation of a single molecule within a supramolecular bearing

    DEFF Research Database (Denmark)

    Gimzewski, J.K.; Joachim, C.; Schlittler, R.R.

    1998-01-01

    Experimental visualization and verification of a single-molecule rotor operating within a supramolecular bearing is reported. Using a scanning tunneling microscope, single molecules were observed to exist in one of two spatially defined states Laterally separated by 0.26 nanometers. One...

  15. Single-molecule probes in organic field-effect transistors

    NARCIS (Netherlands)

    Nicolet, Aurélien Armel Louis

    2007-01-01

    The goal of this thesis is to study charge transport phenomena in organic materials. This is done optically by means of single-molecule spectroscopy in a field-effect transistor based on a molecular crystal. We present (in Chapter 2) a fundamental requirement for single-molecule spectroscopy

  16. Single-molecule mechanics of protein-labelled DNA handles

    Directory of Open Access Journals (Sweden)

    Vivek S. Jadhav

    2016-01-01

    Full Text Available DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular

  17. Extending single molecule fluorescence observation time by amplitude-modulated excitation

    Science.gov (United States)

    Kisley, Lydia; Chang, Wei-Shun; Cooper, David; Mansur, Andrea P.; Landes, Christy F.

    2013-09-01

    We present a hardware-based method that can improve single molecule fluorophore observation time by up to 1500% and super-localization by 47% for the experimental conditions used. The excitation was modulated using an acousto-optic modulator (AOM) synchronized to the data acquisition and inherent data conversion time of the detector. The observation time and precision in super-localization of four commonly used fluorophores were compared under modulated and traditional continuous excitation, including direct total internal reflectance excitation of Alexa 555 and Cy3, non-radiative Förster resonance energy transfer (FRET) excited Cy5, and direct epi-fluorescence wide field excitation of Rhodamine 6G. The proposed amplitude-modulated excitation does not perturb the chemical makeup of the system or sacrifice signal and is compatible with multiple types of fluorophores. Amplitude-modulated excitation has practical applications for any fluorescent study utilizing an instrumental setup with time-delayed detectors.

  18. Extending single molecule fluorescence observation time by amplitude-modulated excitation

    International Nuclear Information System (INIS)

    Kisley, Lydia; Chang, Wei-Shun; Cooper, David; Mansur, Andrea P; Landes, Christy F

    2013-01-01

    We present a hardware-based method that can improve single molecule fluorophore observation time by up to 1500% and super-localization by 47% for the experimental conditions used. The excitation was modulated using an acousto-optic modulator (AOM) synchronized to the data acquisition and inherent data conversion time of the detector. The observation time and precision in super-localization of four commonly used fluorophores were compared under modulated and traditional continuous excitation, including direct total internal reflectance excitation of Alexa 555 and Cy3, non-radiative Förster resonance energy transfer (FRET) excited Cy5, and direct epi-fluorescence wide field excitation of Rhodamine 6G. The proposed amplitude-modulated excitation does not perturb the chemical makeup of the system or sacrifice signal and is compatible with multiple types of fluorophores. Amplitude-modulated excitation has practical applications for any fluorescent study utilizing an instrumental setup with time-delayed detectors. (technical note)

  19. Robust Magnetic Properties of a Sublimable Single-Molecule Magnet.

    Science.gov (United States)

    Kiefl, Evan; Mannini, Matteo; Bernot, Kevin; Yi, Xiaohui; Amato, Alex; Leviant, Tom; Magnani, Agnese; Prokscha, Thomas; Suter, Andreas; Sessoli, Roberta; Salman, Zaher

    2016-06-28

    The organization of single-molecule magnets (SMMs) on surfaces via thermal sublimation is a prerequisite for the development of future devices for spintronics exploiting the richness of properties offered by these magnetic molecules. However, a change in the SMM properties due to the interaction with specific surfaces is usually observed. Here we present a rare example of an SMM system that can be thermally sublimated on gold surfaces while maintaining its intact chemical structure and magnetic properties. Muon spin relaxation and ac susceptibility measurements are used to demonstrate that, unlike other SMMs, the magnetic properties of this system in thin films are very similar to those in the bulk, throughout the full volume of the film, including regions near the metal and vacuum interfaces. These results exhibit the robustness of chemical and magnetic properties of this complex and provide important clues for the development of nanostructures based on SMMs.

  20. An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

    Science.gov (United States)

    Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

    2017-09-22

    Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

  1. Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

    DEFF Research Database (Denmark)

    Krüger, Asger Christian; Raarup, Merete Krog; Nielsen, Morten Muhlig

    2010-01-01

    G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate...... the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G...... to the previously reported crystal structures of UP1-telomere DNA complexes where the DNA oligo within the protein-DNA complex is in a fully open conformation....

  2. Effect of the HIV-1 nucleocapsid protein on reverse transcriptase pause sites revealed by single molecule microscopy

    Science.gov (United States)

    Jouonang, A.; Przybilla, F.; Godet, J.; Sharma, K. K.; Restlé, T.; de Rocquigny, H.; Darlix, J.-L.; Kenfack, C.; Didier, P.; Mély, Y.

    2013-02-01

    During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into the NCp7 molecular mechanism in reverse transcription.

  3. SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoliang Sunney [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology

    2017-03-13

    Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly, even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher molecular

  4. DNA origami as biocompatible surface to match single-molecule and ensemble experiments

    Science.gov (United States)

    Gietl, Andreas; Holzmeister, Phil; Grohmann, Dina; Tinnefeld, Philip

    2012-01-01

    Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements. PMID:22523083

  5. Action spectroscopy for single-molecule reactions - Experiments and theory

    Science.gov (United States)

    Kim, Y.; Motobayashi, K.; Frederiksen, T.; Ueba, H.; Kawai, M.

    2015-05-01

    We review several representative experimental results of action spectroscopy (AS) of single molecules on metal surfaces using a scanning tunneling microscope (STM) by M. Kawai's group over last decade. The experimental procedures to observe STM-AS are described. A brief description of a low-temperature STM and experimental setup are followed by key experimental techniques of how to determine an onset bias voltage of a reaction and how to measure a current change associated with reactions and finally how to observe AS for single molecule reactions. The experimental results are presented for vibrationally mediated chemical transformation of trans-2-butene to 1.3-butadiene molecule and rotational motion of a single cis-2-butene molecule among four equivalent orientations on Pd(1 1 0). The AS obtained from the motion clearly detects more vibrational modes than inelastic electron tunneling spectroscopy with an STM. AS is demonstrated as a useful and novel single molecule vibrational spectroscopy. The AS for a lateral hopping of water dimer on Pt(1 1 1) is presented as an example of novelty. Several distinct vibrational modes are detected as the thresholds in the AS. The assignment of the vibrational modes determined from the analysis of the AS is made from a view of the adsorption geometry of hydrogen-bond donor or acceptor molecules in water dimer. A generic theory of STM-AS, i.e., a reaction rate or yield as a function of bias voltage, is presented using a single adsorbate resonance model for single molecule reactions induced by the inelastic tunneling current. Formulas for the reaction rate R (V) and Y (V) , i.e., reaction yield per electron Y (V) = eR (V) / I are derived. It provides a versatile framework to analyze any vibrationally mediated reactions of single adsorbates on metal surfaces. Numerical examples are presented to demonstrate generic features of the vibrational generation rate and Y (V) at different levels of approximations and to show how the effective

  6. Multicolour single molecule imaging in cells with near infra-red dyes.

    Directory of Open Access Journals (Sweden)

    Christopher J Tynan

    Full Text Available The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging.A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells.We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470-1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.

  7. Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

    Science.gov (United States)

    Tynan, Christopher J.; Clarke, David T.; Coles, Benjamin C.; Rolfe, Daniel J.; Martin-Fernandez, Marisa L.; Webb, Stephen E. D.

    2012-01-01

    Background The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. Methodology/Principal Findings A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells. Conclusions/Significance We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging. PMID:22558412

  8. Modulation and Control of Charge Transport Through Single-Molecule Junctions.

    Science.gov (United States)

    Wang, Kun; Xu, Bingqian

    2017-02-01

    The ability to modulate and control charge transport though single-molecule junction devices is crucial to achieving the ultimate goal of molecular electronics: constructing real-world-applicable electronic components from single molecules. This review aims to highlight the progress made in single-molecule electronics, emphasizing the development of molecular junction electronics in recent years. Among many techniques that attempt to wire a molecule to metallic electrodes, the single-molecule break junction (SMBJ) technique is one of the most reliable and tunable experimental platforms for achieving metal-molecule-metal configurations. It also provides great freedom to tune charge transport through the junction. Soon after the SMBJ technique was introduced, it was extensively used to measure the conductances of individual molecules; however, different conductances were obtained for the same molecule, and it proved difficult to interpret this wide distribution of experimental data. This phenomenon was later found to be mainly due to a lack of precise experimental control and advanced data analysis methods. In recent years, researchers have directed considerable effort into advancing the SMBJ technique by gaining a deeper physical understanding of charge transport through single molecules and thus enhancing its potential applicability in functional molecular-scale electronic devices, such as molecular diodes and molecular transistors. In parallel with that research, novel data analysis methods and approaches that enable the discovery of hidden yet important features in the data are being developed. This review discusses various aspects of molecular junction electronics, from the initial goal of molecular electronics, the development of experimental techniques for creating single-molecule junctions and determining single-molecule conductance, to the characterization of functional current-voltage features and the investigation of physical properties other than charge

  9. Revealing Two-State Protein-Protein Interaction of Calmodulin by Single-Molecule Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ruchuan; Hu, Dehong; Tan, Xin; Lu, H PETER.

    2006-08-09

    We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of 28 amino-acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM-C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (F1AsH) that enables our unambiguously probing the CaM N-terminal target-binding domain motions at a millisecond timescale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between F1AsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal slow (at sub-second time scale) binding-unbinding motions of the N-terminal domain of the CaM in CaM-C28W complexes, which is a strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

  10. Stochastic single-molecule dynamics of synaptic membrane protein domains

    Science.gov (United States)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  11. A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex.

    Science.gov (United States)

    Arsenovic, Paul T; Bathula, Kranthidhar; Conway, Daniel E

    2017-04-11

    The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a Förster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

  12. Shedding light on protein folding, structural and functional dynamics by single molecule studies

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Hatzakis, Nikos

    2014-01-01

    The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean...... property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out...

  13. Single-molecule imaging and manipulation of biomolecular machines and systems.

    Science.gov (United States)

    Iino, Ryota; Iida, Tatsuya; Nakamura, Akihiko; Saita, Ei-Ichiro; You, Huijuan; Sako, Yasushi

    2018-02-01

    Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems. Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines. Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Supramolecular Systems and Chemical Reactions in Single-Molecule Break Junctions.

    Science.gov (United States)

    Li, Xiaohui; Hu, Duan; Tan, Zhibing; Bai, Jie; Xiao, Zongyuan; Yang, Yang; Shi, Jia; Hong, Wenjing

    2017-04-01

    The major challenges of molecular electronics are the understanding and manipulation of the electron transport through the single-molecule junction. With the single-molecule break junction techniques, including scanning tunneling microscope break junction technique and mechanically controllable break junction technique, the charge transport through various single-molecule and supramolecular junctions has been studied during the dynamic fabrication and continuous characterization of molecular junctions. This review starts from the charge transport characterization of supramolecular junctions through a variety of noncovalent interactions, such as hydrogen bond, π-π interaction, and electrostatic force. We further review the recent progress in constructing highly conductive molecular junctions via chemical reactions, the response of molecular junctions to external stimuli, as well as the application of break junction techniques in controlling and monitoring chemical reactions in situ. We suggest that beyond the measurement of single molecular conductance, the single-molecule break junction techniques provide a promising access to study molecular assembly and chemical reactions at the single-molecule scale.

  15. DNA analysis by single molecule stretching in nanofluidic biochips

    DEFF Research Database (Denmark)

    Abad, E.; Juarros, A.; Retolaza, A.

    2011-01-01

    Stretching single DNA molecules by confinement in nanofluidic channels has attracted a great interest during the last few years as a DNA analysis tool. We have designed and fabricated a sealed micro/nanofluidic device for DNA stretching applications, based on the use of the high throughput Nano......Imprint Lithography (NIL) technology combined with a conventional anodic bonding of the silicon base and Pyrex cover. Using this chip, we have performed single molecule imaging on a bench-top fluorescent microscope system. Lambda phage DNA was used as a model sample to characterize the chip. Single molecules of λ...... a method to determining DNA size. The results of this work prove that the developed fabrication process is a good alternative for the fabrication of single molecule DNA biochips and it allows developing a variety of innovative bio/chemical sensors based on single-molecule DNA sequencing devices....

  16. Single Molecule Spectroscopy in Chemistry, Physics and Biology Nobel Symposium

    CERN Document Server

    Gräslund, Astrid; Widengren, Jerker

    2010-01-01

    Written by the leading experts in the field, this book describes the development and current state-of-the-art in single molecule spectroscopy. The application of this technique, which started 1989, in physics, chemistry and biosciences is displayed.

  17. Single Molecule Scanning of DNA Radiation Oxidative Damage Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

  18. Single-molecule fluorescence microscopy in living Caenorhabditis elegans

    NARCIS (Netherlands)

    van Krugten, Jaap; Peterman, Erwin J.G.

    2018-01-01

    Transportation of organelles and biomolecules is vital for many cellular processes. Single-molecule (SM) fluorescence microscopy can expose molecular aspects of the dynamics that remain unresolved in ensemble experiments. For example, trajectories of individual, moving biomolecules can reveal

  19. Single Molecule Scanning of DNA Radiation Oxidative Damage, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

  20. OPE3 : A model system for single-molecule transport

    NARCIS (Netherlands)

    Frisenda, R.

    2016-01-01

    In this dissertation, charge-transport through individual organic molecules is investigated. The single molecules are contacted with two-terminal mechanically controllable break junction gold electrodes and their electrical and mechanical behavior studied at room and low temperature.

  1. Computer systems for annotation of single molecule fragments

    Science.gov (United States)

    Schwartz, David Charles; Severin, Jessica

    2016-07-19

    There are provided computer systems for visualizing and annotating single molecule images. Annotation systems in accordance with this disclosure allow a user to mark and annotate single molecules of interest and their restriction enzyme cut sites thereby determining the restriction fragments of single nucleic acid molecules. The markings and annotations may be automatically generated by the system in certain embodiments and they may be overlaid translucently onto the single molecule images. An image caching system may be implemented in the computer annotation systems to reduce image processing time. The annotation systems include one or more connectors connecting to one or more databases capable of storing single molecule data as well as other biomedical data. Such diverse array of data can be retrieved and used to validate the markings and annotations. The annotation systems may be implemented and deployed over a computer network. They may be ergonomically optimized to facilitate user interactions.

  2. Single Molecule 3D Orientation in Time and Space

    NARCIS (Netherlands)

    Börner, Richard; Ehrlich, Nicky; Hohlbein, Johannes; Hübner, Christian G.

    2016-01-01

    Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection

  3. Transition paths in single-molecule force spectroscopy.

    Science.gov (United States)

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2018-03-28

    In a typical single-molecule force spectroscopy experiment, the ends of the molecule of interest are connected by long polymer linkers to a pair of mesoscopic beads trapped in the focus of two laser beams. At constant force load, the total extension, i.e., the end-to-end distance of the molecule plus linkers, is measured as a function of time. In the simplest systems, the measured extension fluctuates about two values characteristic of folded and unfolded states, with occasional transitions between them. We have recently shown that molecular (un)folding rates can be recovered from such trajectories, with a small linker correction, as long as the characteristic time of the bead fluctuations is shorter than the residence time in the unfolded (folded) state. Here, we show that accurate measurements of the molecular transition path times require an even faster apparatus response. Transition paths, the trajectory segments in which the molecule (un)folds, are properly resolved only if the beads fluctuate more rapidly than the end-to-end distance of the molecule. Therefore, over a wide regime, the measured rates may be meaningful but not the transition path times. Analytic expressions for the measured mean transition path times are obtained for systems diffusing anisotropically on a two-dimensional free energy surface. The transition path times depend on the properties both of the molecule and of the pulling device.

  4. Single-Molecule Spectroscopic Investigations of Amphipathic Helix Formation

    Science.gov (United States)

    Cunningham, Joy Ann; Okamoto, Kenji; English, Douglas

    2004-03-01

    We are using single molecule spectroscopy to examine surface-induced conformational states occurring through interaction of a polypeptide with an interface. Specifically, we investigate the folding of amphipathic helices by using single-molecule fluorescence resonance energy transfer to construct peptide conformational distributions in solution and at interfaces. Analysis of the conformational distributions and kinetics of peptides in different environments reveals properties of the free energy surface for helix formation at an interface relative to formation in solution.

  5. Zero-mode waveguide nanophotonic structures for single molecule characterization

    Science.gov (United States)

    Crouch, Garrison M.; Han, Donghoon; Bohn, Paul W.

    2018-05-01

    Single-molecule characterization has become a crucial research tool in the chemical and life sciences, but limitations, such as limited concentration range, inability to control molecular distributions in space, and intrinsic phenomena, such as photobleaching, present significant challenges. Recent developments in non-classical optics and nanophotonics offer promising routes to mitigating these restrictions, such that even low affinity (K D ~ mM) biomolecular interactions can be studied. Here we introduce and review specific nanophotonic devices used to support single molecule studies. Optical nanostructures, such as zero-mode waveguides (ZMWs), are usually fabricated in thin gold or aluminum films and serve to confine the observation volume of optical microspectroscopy to attoliter to zeptoliter volumes. These simple nanostructures allow individual molecules to be isolated for optical and electrochemical analysis, even when the molecules of interest are present at high concentration (µM–mM) in bulk solution. Arrays of ZMWs may be combined with optical probes such as single molecule fluorescence, single molecule fluorescence resonance energy transfer, and fluorescence correlation spectroscopy for distributed analysis of large numbers of single-molecule reactions or binding events in parallel. Furthermore, ZMWs may be used as multifunctional devices, for example by combining optical and electrochemical functions in a single discrete architecture to achieve electrochemical ZMWs. In this review, we will describe the optical properties, fabrication, and applications of ZMWs for single-molecule studies, as well as the integration of ZMWs into systems for chemical and biochemical analysis.

  6. Spectrally resolved single-molecule electrometry

    Science.gov (United States)

    Ruggeri, F.; Krishnan, M.

    2018-03-01

    Escape-time electrometry is a recently developed experimental technique that offers the ability to measure the effective electrical charge of a single biomolecule in solution with sub-elementary charge precision. The approach relies on measuring the average escape-time of a single charged macromolecule or molecular species transiently confined in an electrostatic fluidic trap. Comparing the experiments with the predictions of a mean-field model of molecular electrostatics, we have found that the measured effective charge even reports on molecular conformation, e.g., folded or disordered state, and non-uniform charge distribution in disordered proteins or polyelectrolytes. Here we demonstrate the ability to use the spectral dimension to distinguish minute differences in electrical charge between individual molecules or molecular species in a single simultaneous measurement, under identical experimental conditions. Using one spectral channel for referenced measurement, this kind of photophysical distinguishability essentially eliminates the need for accurate knowledge of key experimental parameters, otherwise obtained through intensive characterization of the experimental setup. As examples, we demonstrate the ability to detect small differences (˜5%) in the length of double-stranded DNA fragments as well as single amino acid exchange in an intrinsically disordered protein, prothymosin α.

  7. Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

    Science.gov (United States)

    Seyfert, K.; Oosaka, T.; Yaginuma, H.; Ernst, S.; Noji, H.; Iino, R.; Börsch, M.

    2011-03-01

    FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

  8. Single-Molecule Sensing with Nanopore Confinement: from Chemical Reactions to Biological Interactions.

    Science.gov (United States)

    Lin, Yao; Ying, Yi-Lun; Gao, Rui; Long, Yi-Tao

    2018-03-25

    The nanopore can generate an electrochemical confinement for single-molecule sensing which help understand the fundamental chemical principle in nanoscale dimensions. By observing the generated ionic current, individual bond-making and bond-breaking steps, single biomolecule dynamic conformational changes and electron transfer processes that occur within pore can be monitored with high temporal and current resolution. These single-molecule studies in nanopore confinement are revealing information about the fundamental chemical and biological processes that cannot be extracted from ensemble measurements. In this concept, we introduce and discuss the electrochemical confinement effects on single-molecule covalent reactions, conformational dynamics of individual molecules and host-guest interactions in protein nanopores. Then, we extend the concept of nanopore confinement effects to confine electrochemical redox reactions in solid-state nanopores for developing new sensing mechanisms. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Polymerase specific error rates and profiles identified by single molecule sequencing.

    Science.gov (United States)

    Hestand, Matthew S; Van Houdt, Jeroen; Cristofoli, Francesca; Vermeesch, Joris R

    2016-01-01

    DNA polymerases have an innate error rate which is polymerase and DNA context specific. Historically the mutational rate and profiles have been measured using a variety of methods, each with their own technical limitations. Here we used the unique properties of single molecule sequencing to evaluate the mutational rate and profiles of six DNA polymerases at the sequence level. In addition to accurately determining mutations in double strands, single molecule sequencing also captures direction specific transversions and transitions through the analysis of heteroduplexes. Not only did the error rates vary, but also the direction specific transitions differed among polymerases. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Single-molecule theory of enzymatic inhibition.

    Science.gov (United States)

    Robin, Tal; Reuveni, Shlomi; Urbakh, Michael

    2018-02-22

    The classical theory of enzymatic inhibition takes a deterministic, bulk based approach to quantitatively describe how inhibitors affect the progression of enzymatic reactions. Catalysis at the single-enzyme level is, however, inherently stochastic which could lead to strong deviations from classical predictions. To explore this, we take the single-enzyme perspective and rebuild the theory of enzymatic inhibition from the bottom up. We find that accounting for multi-conformational enzyme structure and intrinsic randomness should strongly change our view on the uncompetitive and mixed modes of inhibition. There, stochastic fluctuations at the single-enzyme level could make inhibitors act as activators; and we state-in terms of experimentally measurable quantities-a mathematical condition for the emergence of this surprising phenomenon. Our findings could explain why certain molecules that inhibit enzymatic activity when substrate concentrations are high, elicit a non-monotonic dose response when substrate concentrations are low.

  11. Giant magnetoresistance through a single molecule.

    Science.gov (United States)

    Schmaus, Stefan; Bagrets, Alexei; Nahas, Yasmine; Yamada, Toyo K; Bork, Annika; Bowen, Martin; Beaurepaire, Eric; Evers, Ferdinand; Wulfhekel, Wulf

    2011-03-01

    Magnetoresistance is a change in the resistance of a material system caused by an applied magnetic field. Giant magnetoresistance occurs in structures containing ferromagnetic contacts separated by a metallic non-magnetic spacer, and is now the basis of read heads for hard drives and for new forms of random access memory. Using an insulator (for example, a molecular thin film) rather than a metal as the spacer gives rise to tunnelling magnetoresistance, which typically produces a larger change in resistance for a given magnetic field strength, but also yields higher resistances, which are a disadvantage for real device operation. Here, we demonstrate giant magnetoresistance across a single, non-magnetic hydrogen phthalocyanine molecule contacted by the ferromagnetic tip of a scanning tunnelling microscope. We measure the magnetoresistance to be 60% and the conductance to be 0.26G(0), where G(0) is the quantum of conductance. Theoretical analysis identifies spin-dependent hybridization of molecular and electrode orbitals as the cause of the large magnetoresistance.

  12. Photoinduced nuclear spin conversion of methyl groups of single molecules

    International Nuclear Information System (INIS)

    Sigl, A.

    2007-01-01

    A methyl group is an outstanding quantum system due to its special symmetry properties. The threefold rotation around one of its bond is isomorphic to the group of even permutations of the remaining protons, a property which imposes severe quantum restrictions on the system, for instance a strict correlation of rotational states with nuclear spin states. The resulting long lifetimes of the rotational tunneling states of the methyl group can be exploited for applying certain high resolution optical techniques, like hole burning or single molecule spectroscopy to optically switch the methyl group from one tunneling state to another therebye changing the nuclear spin of the protons. One goal of the thesis was to perform this switching in single methyl groups. To this end the methyl group was attached to a chromophoric system, in the present case terrylene, which is well suited for single molecule spectroscopy as well as for hole burning. Experiments were performed with the bare terrylene molecule in a hexadecane lattice which served as a reference system, with alphamethyl terrylene and betamethyl terrylene, both embedded in hexadecane, too. A single molecular probe is a highly sensitive detector for dynamic lattice instabilities. Already the bare terrylene probe showed a wealth of interesting local dynamic effects of the hexadecane lattice which could be well acounted for by the assumption of two nearly degenerate sites with rather different optical and thermal properties, all of which could be determined in a quantitative fashion. As to the methylated terrylene systems, the experiments verified that for betamethyl terrylene it is indeed possible to measure rotational tunneling events in single methyl groups. However, the spectral patterns obtained was much more complicated than expected pointing to the presence of three spectroscopically different methyl groups. In order to achieve a definite assignement, molecular mechanics simulations of the terrylene probes in the

  13. Radiation- and phonon-bottleneck--induced tunneling in the Fe8 single-molecule magnet

    Science.gov (United States)

    Bal, M.; Friedman, Jonathan R.; Chen, W.; Tuominen, M. T.; Beedle, C. C.; Rumberger, E. M.; Hendrickson, D. N.

    2008-04-01

    We measure magnetization changes in a single crystal of the single-molecule magnet Fe8 when exposed to intense, short (spin dynamics, allowing observation of thermally assisted resonant tunneling between spin states at the 100 ns time scale. Detailed numerical simulations quantitatively reproduce the data and yield a spin-phonon relaxation time T1~40 ns.

  14. A single molecule switch based on two Pd nanocrystals linked by a ...

    Indian Academy of Sciences (India)

    Tunneling spectroscopy measurements have been carried out on a single molecule device formed by two Pd nanocrystals (dia. ∼ 5 nm) electronically coupled by a conducting molecule, dimercaptodiphenylacetylene. The – data, obtained by positioning the tip over a nanocrystal electrode, exhibit negative differential ...

  15. Electrochemically-gated single-molecule electrical devices

    International Nuclear Information System (INIS)

    Guo, Shaoyin; Artés, Juan Manuel; Díez-Pérez, Ismael

    2013-01-01

    In the last decade, single-molecule electrical contacts have emerged as a new experimental platform that allows exploring charge transport phenomena in individual molecular blocks. This novel tool has evolved into an essential element within the Molecular Electronics field to understand charge transport processes in hybrid (bio)molecule/electrode interfaces at the nanoscale, and prospect the implementation of active molecular components into functional nanoscale optoelectronic devices. Within this area, three-terminal single-molecule devices have been sought, provided that they are highly desired to achieve full functionality in logic electronic circuits. Despite the latest experimental developments offer consistent methods to bridge a molecule between two electrodes (source and drain in a transistor notation), placing a third electrode (gate) close to the single-molecule electrical contact is still technically challenging. In this vein, electrochemically-gated single-molecule devices have emerged as an experimentally affordable alternative to overcome these technical limitations. In this review, the operating principle of an electrochemically-gated single-molecule device is presented together with the latest experimental methodologies to built them and characterize their charge transport characteristics. Then, an up-to-date comprehensive overview of the most prominent examples will be given, emphasizing on the relationship between the molecular structure and the final device electrical behaviour

  16. Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells.

    Science.gov (United States)

    Horiguchi, Mika; Fujioka, Mari; Kondo, Takeshi; Fujioka, Yoichiro; Li, Xinxin; Horiuchi, Kosui; O Satoh, Aya; Nepal, Prabha; Nishide, Shinya; Nanbo, Asuka; Teshima, Takanori; Ohba, Yusuke

    2017-02-02

    Although the co-development of companion diagnostics with molecular targeted drugs is desirable, truly efficient diagnostics are limited to diseases in which chromosomal translocations or overt mutations are clearly correlated with drug efficacy. Moreover, even for such diseases, few methods are available to predict whether drug administration is effective for each individual patient whose disease is expected to respond to the drug(s). We have previously developed a biosensor based on the principle of Förster resonance energy transfer to measure the activity of the tyrosine kinase BCR-ABL and its response to drug treatment in patient-derived chronic myeloid leukemia cells. The biosensor harbors CrkL, one of the major substrates of BCR-ABL, and is therefore named Pickles after phosphorylation indicator of CrkL en substrate. The efficacy of this technique as a clinical test has been demonstrated, but the number of cells available for analysis is limited in a case-dependent manner, owing to the cleavage of the biosensor in patient-derived leukemia cells. Here, we describe an improved biosensor with an amino acid substitution and a nuclear export signal being introduced. Of the two predicted cleavage positions in CrkL, the mutations inhibited one cleavage completely and the other cleavage partially, thus collectively increasing the number of cells available for drug evaluation. This improved version of the biosensor holds promise in the future development of companion diagnostics to predict responses to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.

  17. Investigation of polyelectrolyte desorption by single molecule force spectroscopy

    International Nuclear Information System (INIS)

    Friedsam, C; Seitz, M; Gaub, H E

    2004-01-01

    Single molecule force spectroscopy has evolved into a powerful method for the investigation of intra- and intermolecular interactions at the level of individual molecules. Many examples, including the investigation of the dynamic properties of complex biological systems as well as the properties of covalent bonds or intermolecular transitions within individual polymers, are reported in the literature. The technique has recently been extended to the systematic investigation of desorption processes of individual polyelectrolyte molecules adsorbed on generic surfaces. The stable covalent attachment of polyelectrolyte molecules to the AFM-tip provides the possibility of performing long-term measurements with the same set of molecules and therefore allows the in situ observation of the impact of environmental changes on the adsorption behaviour of individual molecules. Different types of interactions, e.g. electrostatic or hydrophobic interactions, that determine the adsorption process could be identified and characterized. The experiments provided valuable details that help to understand the nature and the properties of non-covalent interactions, which is helpful with regard to biological systems as well as for technical applications. Apart from this, desorption experiments can be utilized to characterize the properties of surfaces or polymer coatings. Therefore they represent a versatile tool that can be further developed in terms of various aspects

  18. A theoretical justification for single molecule peptide sequencing.

    Directory of Open Access Journals (Sweden)

    Jagannath Swaminathan

    2015-02-01

    Full Text Available The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology.

  19. Light-Induced Switching of Tunable Single-Molecule Junctions

    KAUST Repository

    Sendler, Torsten

    2015-04-16

    A major goal of molecular electronics is the development and implementation of devices such as single-molecular switches. Here, measurements are presented that show the controlled in situ switching of diarylethene molecules from their nonconductive to conductive state in contact to gold nanoelectrodes via controlled light irradiation. Both the conductance and the quantum yield for switching of these molecules are within a range making the molecules suitable for actual devices. The conductance of the molecular junctions in the opened and closed states is characterized and the molecular level E 0, which dominates the current transport in the closed state, and its level broadening Γ are identified. The obtained results show a clear light-induced ring forming isomerization of the single-molecule junctions. Electron withdrawing side-groups lead to a reduction of conductance, but do not influence the efficiency of the switching mechanism. Quantum chemical calculations of the light-induced switching processes correlate these observations with the fundamentally different low-lying electronic states of the opened and closed forms and their comparably small modification by electron-withdrawing substituents. This full characterization of a molecular switch operated in a molecular junction is an important step toward the development of real molecular electronics devices.

  20. Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

    Directory of Open Access Journals (Sweden)

    Krutika Bavishi

    2014-11-01

    Full Text Available The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

  1. Electrochemical single-molecule conductivity of duplex and quadruplex DNA

    DEFF Research Database (Denmark)

    Zhang, Ling; Zhang, Jingdong; Ulstrup, Jens

    2017-01-01

    Photoinduced and electrochemical charge transport in DNA (oligonucleotides, OGNs) and the notions “hopping”, superexchange, polaron, and vibrationally gated charge transport have been in focus over more than two decades. In recent years mapping of electrochemical charge transport of pure and redox...... marked single- and double-strand OGNs has reached the single-molecule level based i.a. on electrochemical in situ scanning tunnelling microscopy (STM) and break-junction (B-J) STM. There are much fewer such reports on “non-canonical” OGN structures such as G-quadruplexes. We discuss first single......-molecule electrochemical conductivity of pure and redox marked duplex OGNs, and address next electrochemistry and electrochemical conductivity in the few reported monolayer and single-molecule G-quadruplex studies. Facile electrochemical electron transfer of iron protoporphyrin IX stacked onto three-quartet 12-guanine...

  2. Single-Molecule Protein Folding: A Study of the Surface-Mediated Conformational Dynamics of a Model Amphipathic Peptide

    Science.gov (United States)

    Cunningham, Joy; English, Douglas

    2004-03-01

    Most surface-active polypeptides, composed of 10-50 amino acids, are devoid of well-defined tertiary structure. The conformation of these proteins is greatly dependent upon their environment and may assume totally different characteristics in an aqueous environment, in a detergent micelle, or in an organic solvent. Most antimicrobial peptides are helix-forming and are activated upon interaction with a membrane-mimicking environment. We are seeking to physically characterize the mechanism of membrane-peptide interaction through studying a simple model peptide, MT-1. MT-1 was designed as a nonhomologous analogue of melittin, the principle component in bee venom. We are using single molecule spectroscopy to examine the induction of secondary structure upon interaction of MT-1 with various membrane-mimicking interfaces. Specifically, we monitor coil-to-helix transition through single molecule fluorescence resonance energy transfer (sm-FRET) to determine conformational distributions of folded and unfolded peptides at an interface. Studies with MT-1 will focus upon the biologically relevant issues of orientation, aggregation, and folding at surfaces using both ensemble and single molecule experiments.

  3. A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer.

    Science.gov (United States)

    Murakoshi, Hideji; Shibata, Akihiro C E; Nakahata, Yoshihisa; Nabekura, Junichi

    2015-10-15

    Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.

  4. Electrochemical Single-Molecule Transistors with Optimized Gate Coupling

    DEFF Research Database (Denmark)

    Osorio, Henrry M.; Catarelli, Samantha; Cea, Pilar

    2015-01-01

    . These data are rationalized in terms of a two-step electrochemical model for charge transport across the redox bridge. In this model the gate coupling in the ionic liquid is found to be fully effective with a modeled gate coupling parameter, ξ, of unity. This compares to a much lower gate coupling parameter......Electrochemical gating at the single molecule level of viologen molecular bridges in ionic liquids is examined. Contrary to previous data recorded in aqueous electrolytes, a clear and sharp peak in the single molecule conductance versus electrochemical potential data is obtained in ionic liquids...

  5. Novel approaches for single molecule activation and detection

    CERN Document Server

    Benfenati, Fabio; Torre, Vincent

    2014-01-01

    How can we obtain tools able to process and exchange information at the molecular scale In order to do this, it is necessary to activate and detect single molecules under controlled conditions. This book focuses on the generation of biologically-inspired molecular devices. These devices are based on the developments of new photonic tools able to activate and stimulate single molecule machines. Additionally, new light sensitive molecules can be selectively activated by photonic tools. These technological innovations will provide a way to control activation of single light-sensitive molecules, a

  6. Single-Molecule Analysis of Pre-mRNA Splicing with Colocalization Single-Molecule Spectroscopy (CoSMoS).

    Science.gov (United States)

    Braun, Joerg E; Serebrov, Victor

    2017-01-01

    Recent development of single-molecule techniques to study pre-mRNA splicing has provided insights into the dynamic nature of the spliceosome. Colocalization single-molecule spectroscopy (CoSMoS) allows following spliceosome assembly in real time at single-molecule resolution in the full complexity of cellular extracts. A detailed protocol of CoSMoS has been published previously (Anderson and Hoskins, Methods Mol Biol 1126:217-241, 2014). Here, we provide an update on the technical advances since the first CoSMoS studies including slide surface treatment, data processing, and representation. We describe various labeling strategies to generate RNA reporters with multiple dyes (or other moieties) at specific locations.

  7. Quantifying and optimizing single-molecule switching nanoscopy at high speeds.

    Directory of Open Access Journals (Sweden)

    Yu Lin

    Full Text Available Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria - localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5-25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.

  8. Towards single molecule biosensors using super-resolution fluorescence microscopy.

    Science.gov (United States)

    Lu, Xun; Nicovich, Philip R; Gaus, Katharina; Gooding, J Justin

    2017-07-15

    Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Molecular electronics: the single molecule switch and transistor

    NARCIS (Netherlands)

    Sotthewes, Kai; Geskin, Victor; Heimbuch, Rene; Kumar, Avijit; Zandvliet, Henricus J.W.

    2014-01-01

    In order to design and realize single-molecule devices it is essential to have a good understanding of the properties of an individual molecule. For electronic applications, the most important property of a molecule is its conductance. Here we show how a single octanethiol molecule can be connected

  10. Single-molecule experiments in biophysics: Exploring the thermal ...

    Indian Academy of Sciences (India)

    cules contributes to our understanding of the nonequilibrium thermal behavior of small systems. Keywords. Biophysics; single-molecule experiments; fluctuation theorems. PACS Nos 05.70.Ln; 82.37.Rs; 87.10.+e; 87.15.Ya. 1. Biomolecules, molecular demons and statistical physics. Biophysics is a relatively young discipline ...

  11. Computing magnetic anisotropy constants of single molecule magnets

    Indian Academy of Sciences (India)

    Administrator

    Abstract. We present here a theoretical approach to compute the molecular magnetic anisotropy parameters, DM and EM for single molecule magnets in any given spin eigenstate of exchange spin Hami- ltonian. We first describe a hybrid constant MS-valence bond (VB) technique of solving spin Hamilto- nians employing ...

  12. Computing magnetic anisotropy constants of single molecule magnets

    Indian Academy of Sciences (India)

    We present here a theoretical approach to compute the molecular magnetic anisotropy parameters, and for single molecule magnets in any given spin eigenstate of exchange spin Hamiltonian. We first describe a hybrid constant -valence bond (VB) technique of solving spin Hamiltonians employing full spatial ...

  13. Electrical and mechanical effects in single-molecule junctions

    NARCIS (Netherlands)

    Seldenthuis, J.S.

    2011-01-01

    In single-molecule junctions, the behavior of a device is determined by the properties of an individual molecule. In this thesis we develop several models to describe both electrical and mechanical effects in such devices, which can be used to design molecules with a specific functionality. We show

  14. Single Molecule Study of Cellulase Hydrolysis of Crystalline Cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.-S.; Luo, Y.; Baker, J. O.; Zeng, Y.; Himmel, M. E.; Smith, S.; Ding, S.-Y.

    2009-12-01

    This report seeks to elucidate the role of cellobiohydrolase-I (CBH I) in the hydrolysis of crystalline cellulose. A single-molecule approach uses various imaging techniques to investigate the surface structure of crystalline cellulose and changes made in the structure by CBH I.

  15. Large negative differential conductance in single-molecule break junctions

    NARCIS (Netherlands)

    Perrin, Mickael L.; Frisenda, Riccardo; Koole, Max; Seldenthuis, Johannes S.; Gil, Jose A. Celis; Valkenier, Hennie; Hummelen, Jan C.; Renaud, Nicolas; Grozema, Ferdinand C.; Thijssen, Joseph M.; Dulic, Diana; van der Zant, Herre S. J.

    2014-01-01

    Molecular electronics aims at exploiting the internal structure and electronic orbitals of molecules to construct functional building blocks(1). To date, however, the overwhelming majority of experimentally realized single-molecule junctions can be described as single quantum dots, where transport

  16. Single-molecule denaturation mapping of DNA in nanofluidic channels

    DEFF Research Database (Denmark)

    Reisner, Walter; Larsen, Niels Bent; Silahtaroglu, Asli

    2010-01-01

    Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO (R)-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips...

  17. Investigating single molecule adhesion by atomic force spectroscopy.

    Science.gov (United States)

    Stetter, Frank W S; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

    2015-02-27

    Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

  18. An RNA toolbox for single-molecule force spectroscopy studies

    NARCIS (Netherlands)

    Vilfan, I.D.; Kamping, W.; Van den Hout, M.; Candelli, A.; Hage, S.; Dekker, N.H.

    2007-01-01

    Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNAenzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct.

  19. Atomic-Scale Control of Electron Transport through Single Molecules

    DEFF Research Database (Denmark)

    Wang, Y. F.; Kroger, J.; Berndt, R.

    2010-01-01

    Tin-phthalocyanine molecules adsorbed on Ag(111) were contacted with the tip of a cryogenic scanning tunneling microscope. Orders-of-magnitude variations of the single-molecule junction conductance were achieved by controllably dehydrogenating the molecule and by modifying the atomic structure...

  20. BRCA Testing by Single-Molecule Molecular Inversion Probes

    NARCIS (Netherlands)

    Neveling, K.; Mensenkamp, A.R.; Derks, R; Kwint, M.P.; Ouchene, H.; Steehouwer, M.; Lier, L.A. van; Bosgoed, E.A.J.; Rikken, A.; Tychon, M.W.J.; Zafeiropoulou, D.; Castelein, S.; Hehir-Kwa, J.Y.; Thung, G.W.; Hofste, T.; Lelieveld, S.H.; Bertens, S.M.; Adan, I.B.; Eijkelenboom, A.; Tops, B.B.J.; Yntema, H.G.; Stokowy, T.; Knappskog, P.M.; Hoberg-Vetti, H.; Steen, V.M.; Boyle, E.; Martin, B.; Ligtenberg, M.J.L.; Shendure, J.; Nelen, M.R.; Hoischen, A.

    2017-01-01

    BACKGROUND: Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the

  1. A single molecule DNA flow stretching microscope for undergraduates

    NARCIS (Netherlands)

    Williams, Kelly; Grafe, Brendan; Burke, Kathryn M.; Tanner, Nathan; van Oijen, Antoine M.; Loparo, Joseph; Price, Allen C.

    2011-01-01

    The design of a simple, safe, and inexpensive single molecule flow stretching instrument is presented. The instrument uses a low cost upright microscope coupled to a webcam for imaging single DNA molecules that are tethered in an easy to construct microfluidic flow cell. The system requires no

  2. Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

    Science.gov (United States)

    Stetter, Frank W. S.; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

    2015-01-01

    Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment. PMID:25867282

  3. Efficient unfolding pattern recognition in single molecule force spectroscopy data

    Directory of Open Access Journals (Sweden)

    Labudde Dirk

    2011-06-01

    Full Text Available Abstract Background Single-molecule force spectroscopy (SMFS is a technique that measures the force necessary to unfold a protein. SMFS experiments generate Force-Distance (F-D curves. A statistical analysis of a set of F-D curves reveals different unfolding pathways. Information on protein structure, conformation, functional states, and inter- and intra-molecular interactions can be derived. Results In the present work, we propose a pattern recognition algorithm and apply our algorithm to datasets from SMFS experiments on the membrane protein bacterioRhodopsin (bR. We discuss the unfolding pathways found in bR, which are characterised by main peaks and side peaks. A main peak is the result of the pairwise unfolding of the transmembrane helices. In contrast, a side peak is an unfolding event in the alpha-helix or other secondary structural element. The algorithm is capable of detecting side peaks along with main peaks. Therefore, we can detect the individual unfolding pathway as the sequence of events labeled with their occurrences and co-occurrences special to bR's unfolding pathway. We find that side peaks do not co-occur with one another in curves as frequently as main peaks do, which may imply a synergistic effect occurring between helices. While main peaks co-occur as pairs in at least 50% of curves, the side peaks co-occur with one another in less than 10% of curves. Moreover, the algorithm runtime scales well as the dataset size increases. Conclusions Our algorithm satisfies the requirements of an automated methodology that combines high accuracy with efficiency in analyzing SMFS datasets. The algorithm tackles the force spectroscopy analysis bottleneck leading to more consistent and reproducible results.

  4. Force feedback effects on single molecule hopping and pulling experiments

    Science.gov (United States)

    Rico-Pasto, M.; Pastor, I.; Ritort, F.

    2018-03-01

    Single-molecule experiments with optical tweezers have become an important tool to study the properties and mechanisms of biological systems, such as cells and nucleic acids. In particular, force unzipping experiments have been used to extract the thermodynamics and kinetics of folding and unfolding reactions. In hopping experiments, a molecule executes transitions between the unfolded and folded states at a preset value of the force [constant force mode (CFM) under force feedback] or trap position [passive mode (PM) without feedback] and the force-dependent kinetic rates extracted from the lifetime of each state (CFM) and the rupture force distributions (PM) using the Bell-Evans model. However, hopping experiments in the CFM are known to overestimate molecular distances and folding free energies for fast transitions compared to the response time of the feedback. In contrast, kinetic rate measurements from pulling experiments have been mostly done in the PM while the CFM is seldom implemented in pulling protocols. Here, we carry out hopping and pulling experiments in a short DNA hairpin in the PM and CFM at three different temperatures (6 °C, 25 °C, and 45 °C) exhibiting largely varying kinetic rates. As expected, we find that equilibrium hopping experiments in the CFM and PM perform well at 6 °C (where kinetics are slow), whereas the CFM overestimates molecular parameters at 45 °C (where kinetics are fast). In contrast, nonequilibrium pulling experiments perform well in both modes at all temperatures. This demonstrates that the same kind of feedback algorithm in the CFM leads to more reliable determination of the folding reaction parameters in irreversible pulling experiments.

  5. Inferring properties of disordered chains from FRET transfer efficiencies

    Science.gov (United States)

    Zheng, Wenwei; Zerze, Gül H.; Borgia, Alessandro; Mittal, Jeetain; Schuler, Benjamin; Best, Robert B.

    2018-03-01

    Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely, the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble properties from FRET data, in which the polymer scaling exponent is allowed to vary with solution conditions. In its simplest form, it requires either the mean FRET efficiency or fluorescence lifetime information. In order to test the accuracy of the method, we have utilized both synthetic FRET data from implicit and explicit solvent simulations for 30 different protein sequences, and experimental single-molecule FRET data for an intrinsically disordered and a denatured protein. In all cases, we find that the inferred radii of gyration are within 10% of the true values, thus providing higher accuracy than simpler polymer models. In addition, the scaling exponents obtained by our procedure are in good agreement with those determined directly from the molecular ensemble. Our approach can in principle be generalized to treating other ensemble-averaged functions of intramolecular distances from experimental data.

  6. Localization microscopy: mapping cellular dynamics with single molecules.

    Science.gov (United States)

    Nelson, A J; Hess, S T

    2014-04-01

    Resolution describes the smallest details within a sample that can be recovered by a microscope lens system. For optical microscopes detecting visible light, diffraction limits the resolution to ∼200-250 nm. In contrast, localization measures the position of an isolated object using its image. Single fluorescent molecules can be localized with an uncertainty of a few tens of nanometres, and in some cases less than one nanometre. Superresolution fluorescence localization microscopy (SRFLM) images and localizes fluorescent molecules in a sample. By controlling the visibility of the fluorescent molecules with light, it is possible to cause a sparse subset of the tags to fluoresce and be spatially separated from each other. A movie is acquired with a camera, capturing images of many sets of visible fluorescent tags over a period of time. The movie is then analysed by a computer whereby all of the single molecules are independently measured, and their positions are recorded. When the coordinates of a sufficient number of molecules are collected, an image can be rendered by plotting the coordinates of the localized molecules. The spatial resolution of these rendered images can be better than 20 nm, roughly an order of magnitude better than the diffraction limited resolution. The invention of SRFLM has led to an explosion of related techniques. Through the use of specialized optics, the fluorescent signal can be split into multiple detection channels. These channels can capture additional information such as colour (emission wavelength), orientation and three-dimensional position of the detected molecules. Measurement of the colour of the detected fluorescence can allow researchers to distinguish multiple types of fluorescent tags and to study the interaction between multiple molecules of interest. Three-dimensional imaging and determination of molecular orientations offer insight into structural organization of the sample. SRFLM is compatible with living samples and

  7. Single Molecule Junctions: Probing Contact Chemistry and Fundamental Circuit Laws

    Energy Technology Data Exchange (ETDEWEB)

    Hybertsen M. S.

    2013-04-11

    By exploiting selective link chemistry, formation of single molecule junctions with reproducible conductance has become established. Systematic studies reveal the structure-conductance relationships for diverse molecules. I will draw on experiments from my collaborators at Columbia University, atomic-scale calculations and theory to describe progress in two areas. First, I will describe a novel route to form single molecule junctions, based on SnMe3 terminated molecules, in which gold directly bonds to carbon in the molecule backbone resulting in near ideal contact resistance [1]. Second, comparison of the conductance of junctions formed with molecular species containing either one backbone or two backbones in parallel allows demonstration of the role of quantum interference in the conductance superposition law at the molecular scale [2].

  8. Precision control of single-molecule electrical junctions.

    Science.gov (United States)

    Haiss, Wolfgang; Wang, Changsheng; Grace, Iain; Batsanov, Andrei S; Schiffrin, David J; Higgins, Simon J; Bryce, Martin R; Lambert, Colin J; Nichols, Richard J

    2006-12-01

    There is much discussion of molecules as components for future electronic devices. However, the contacts, the local environment and the temperature can all affect their electrical properties. This sensitivity, particularly at the single-molecule level, may limit the use of molecules as active electrical components, and therefore it is important to design and evaluate molecular junctions with a robust and stable electrical response over a wide range of junction configurations and temperatures. Here we report an approach to monitor the electrical properties of single-molecule junctions, which involves precise control of the contact spacing and tilt angle of the molecule. Comparison with ab initio transport calculations shows that the tilt-angle dependence of the electrical conductance is a sensitive spectroscopic probe, providing information about the position of the Fermi energy. It is also shown that the electrical properties of flexible molecules are dependent on temperature, whereas those of molecules designed for their rigidity are not.

  9. A Single Molecule Investigation of the Photostability of Quantum Dots

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Kulatunga, Pasad; Lagerholm, B. Christoffer

    2012-01-01

    Quantum dots (QDs) are very attractive probes for multi-color fluorescence applications. We report here however that single QDs that are subject to continuous blue excitation from a 100W mercury arc lamp will undergo a continuous blue-switching of the emission wavelength eventually reaching a per...... is especially detrimental for multi-color single molecule applications, as we regularly observe spectral blue-shifts of 50 nm, or more even after only ten seconds of illumination....

  10. Berry-phase blockade in single-molecule magnets

    OpenAIRE

    Gonzalez, Gabriel; Leuenberger, Michael N.

    2006-01-01

    We formulate the problem of electron transport through a single-molecule magnet (SMM) in the Coulomb blockade regime taking into account topological interference effects for the tunneling of the large spin of a SMM. The interference originates from spin Berry phases associated with different tunneling paths. We show that in the case of incoherent spin states it is essential to place the SMM between oppositely spin-polarized source and drain leads in order to detect the spin tunneling in the s...

  11. Rationale for single molecule detection by means of Raman spectroscopy

    International Nuclear Information System (INIS)

    Gaponenko, S.V.; Guzatov, D.V.

    2009-01-01

    A consistent quantum electrodynamical description is proposed of Raman scattering of light by a molecule in a medium with a modified photon density of states. Enhanced local density of states near a metal nanobody is shown to increase a scattering rate by several orders of magnitude, thus providing a rationale for experimental detection of single molecules by means of Raman spectroscopy. For an ellipsoidal particle 10 14 -fold enhancement of the Raman scattering cross-section is obtained. (authors)

  12. Viruses and Tetraspanins: Lessons from Single Molecule Approaches

    Science.gov (United States)

    Dahmane, Selma; Rubinstein, Eric; Milhiet, Pierre-Emmanuel

    2014-01-01

    Tetraspanins are four-span membrane proteins that are widely distributed in multi-cellular organisms and involved in several infectious diseases. They have the unique property to form a network of protein-protein interaction within the plasma membrane, due to the lateral associations with one another and with other membrane proteins. Tracking tetraspanins at the single molecule level using fluorescence microscopy has revealed the membrane behavior of the tetraspanins CD9 and CD81 in epithelial cell lines, providing a first dynamic view of this network. Single molecule tracking highlighted that these 2 proteins can freely diffuse within the plasma membrane but can also be trapped, permanently or transiently, in tetraspanin-enriched areas. More recently, a similar strategy has been used to investigate tetraspanin membrane behavior in the context of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection. In this review we summarize the main results emphasizing the relationship in terms of membrane partitioning between tetraspanins, some of their partners such as Claudin-1 and EWI-2, and viral proteins during infection. These results will be analyzed in the context of other membrane microdomains, stressing the difference between raft and tetraspanin-enriched microdomains, but also in comparison with virus diffusion at the cell surface. New advanced single molecule techniques that could help to further explore tetraspanin assemblies will be also discussed. PMID:24800676

  13. Application of Recognition Tunneling in Single Molecule Identification

    Science.gov (United States)

    Zhao, Yanan

    Single molecule identification is one essential application area of nanotechnology. The application areas including DNA sequencing, peptide sequencing, early disease detection and other industrial applications such as quantitative and quantitative analysis of impurities, etc. The recognition tunneling technique we have developed shows that after functionalization of the probe and substrate of a conventional Scanning Tunneling Microscope with recognition molecules ("tethered molecule-pair" configuration), analyte molecules trapped in the gap that is formed by probe and substrate will bond with the reagent molecules. The stochastic bond formation/breakage fluctuations give insight into the nature of the intermolecular bonding at a single molecule-pair level. The distinct time domain and frequency domain features of tunneling signals were extracted from raw signals of analytes such as amino acids and their enantiomers. The Support Vector Machine (a machine-learning method) was used to do classification and predication based on the signal features generated by analytes, giving over 90% accuracy of separation of up to seven analytes. This opens up a new interface between chemistry and electronics with immediate implications for rapid Peptide/DNA sequencing and molecule identification at single molecule level.

  14. Single-molecule imaging towards precise detection of individual photophysics

    International Nuclear Information System (INIS)

    Tani, Toshiro; Oda, Masaru; Mashimo, Kei; Tachibana, Fumi; Horiuchi, Hiromi

    2006-01-01

    We present our recent study of single fluorescent molecules with specific structure, i.e. tetramethylrhodamine derivative linked with a propyl chain onto silica glass surface. For fluorescent reagent in its synthesis, we used a mixture of two kinds of isomers, which provides a sample with single molecules photophysically different each other even if chemically the same. The isomeric structural difference so introduced in the molecules will provide rather small but probably distinctive photophysical difference, for example, in non-radiative relaxation rates, which we try to detect out with our improved single-molecule microscope imaging technique. To make clear the detectability of such weak inter- or intra-molecular interactions microscopically is significant for versatile applications of single-molecule detections in life science. Our present observation at room temperatures shows so far that such decoupled contributions can be discriminated in the histograms of the intensities of the observed fluorescent spots as broader but separated multi-component structures in the distribution under specific experimental configurations. We will discuss some of the prerequisite for such detections; suitable spatio-temporal resolutions with sufficient S/N ratio, algorithms for data analysis, etc. but also precise sample operations are inevitable

  15. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Manifestation of Spin Selection Rules on the Quantum Tunneling of Magnetization in a Single Molecule Magnet

    OpenAIRE

    Henderson, J. J.; Koo, C.; Feng, P. L.; del Barco, E.; Hill, S.; Tupitsyn, I. S.; Stamp, P. C. E.; Hendrickson, D. N.

    2009-01-01

    We present low temperature magnetometry measurements on a new Mn3 single-molecule magnet (SMM) in which the quantum tunneling of magnetization (QTM) displays clear evidence for quantum mechanical selection rules. A QTM resonance appearing only at elevated temperatures demonstrates tunneling between excited states with spin projections differing by a multiple of three: this is dictated by the C3 symmetry of the molecule, which forbids pure tunneling from the lowest metastable state. Resonances...

  17. Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection

    OpenAIRE

    Ke, Rongqin; Nong, Rachel Yuan; Fredriksson, Simon; Landegren, Ulf; Nilsson, Mats

    2013-01-01

    Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection...

  18. Aberration-accounting calibration for 3D single-molecule localization microscopy

    Science.gov (United States)

    Cabriel, Clément; Bourg, Nicolas; Dupuis, Guillaume; Lévêque-Fort, Sandrine

    2018-01-01

    We propose a straightforward sample-based technique to calibrate the axial detection in 3D single molecule localization microscopy (SMLM). Using microspheres coated with fluorescent molecules, the calibration curves of PSF-shaping- or intensity-based measurements can be obtained for any required depth range from a few hundreds of nanometers to several tens of microns. This experimental method takes into account the effect of the spherical aberration without requiring computational correction.

  19. Single-molecule studies of unconventional motor protein myosin VI

    Science.gov (United States)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  20. Single Molecule Study of DNA Organization and Recombination

    Science.gov (United States)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  1. The role of molecule–electrode contact in single-molecule electronics

    International Nuclear Information System (INIS)

    Hihath, Joshua; Tao, Nongjian

    2014-01-01

    Creating complex electronic systems from individual molecular components is one of the most formidable challenges in nanotechnology today. To achieve this goal it is necessary not only to design the functionality of the molecular system to create devices, but also to control the interface between bulk contacts and molecular systems. In this brief perspective we discuss the role of molecule–electrode contact in single-molecule systems. This contact is responsible for making the system mechanically stable enough to perform measurements, and can also have profound impacts on both the contact resistance and the energy level alignment in the system. We will discuss a variety of different linker groups that have been explored, note the advantages and disadvantages of various contact chemistries, and discuss new methodologies used for understanding the impact that the molecule–electrode contact has on the energetics of single-molecule devices. (paper)

  2. Mapping Nanoscale Hotspots with Single-Molecule Emitters Assembled into Plasmonic Nanocavities Using DNA Origami

    Science.gov (United States)

    Chikkaraddy, Rohit; Turek, V. A.; Kongsuwan, Nuttawut; Benz, Felix; Carnegie, Cloudy; van de Goor, Tim; de Nijs, Bart; Demetriadou, Angela; Hess, Ortwin; Keyser, Ulrich F.; Baumberg, Jeremy J.

    2018-01-01

    Fabricating nanocavities in which optically-active single quantum emitters are precisely positioned, is crucial for building nanophotonic devices. Here we show that self-assembly based on robust DNA-origami constructs can precisely position single molecules laterally within sub-5nm gaps between plasmonic substrates that support intense optical confinement. By placing single-molecules at the center of a nanocavity, we show modification of the plasmon cavity resonance before and after bleaching the chromophore, and obtain enhancements of $\\geq4\\times10^3$ with high quantum yield ($\\geq50$%). By varying the lateral position of the molecule in the gap, we directly map the spatial profile of the local density of optical states with a resolution of $\\pm1.5$ nm. Our approach introduces a straightforward non-invasive way to measure and quantify confined optical modes on the nanoscale.

  3. Applications of a single-molecule detection in early disease diagnosis and enzymatic reaction study

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jiangwei [Iowa State Univ., Ames, IA (United States)

    2008-01-01

    Various single-molecule techniques were utilized for ultra-sensitive early diagnosis of viral DNA and antigen and basic mechanism study of enzymatic reactions. DNA of human papilloma virus (HPV) served as the screening target in a flow system. Alexa Fluor 532 (AF532) labeled single-stranded DNA probes were hybridized to the target HPV-16 DNA in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell and had a linear dynamic range of over 6 orders of magnitude. In the dual-color mode, fluorescence resonance energy transfer (FRET) was employed to achieve zero false-positive count. We also showed that DNA extracts from Pap test specimens did not interfere with the system. A surface-based method was used to improve the throughput of the flow system. HPV-16 DNA was hybridized to probes on a glass surface and detected with a total internal reflection fluorescence (TIRF) microscope. In the single-probe mode, the whole genome and target DNA were fluorescently labeled before hybridization, and the detection limit is similar to the flow system. In the dual-probe mode, a second probe was introduced. The linear dynamic range covers 1.44-7000 copies/cell, which is typical of early infection to near-cancer stages. The dual-probe method was tested with a crudely prepared sample. Even with reduced hybridization efficiency caused by the interference of cellular materials, we were still able to differentiate infected cells from healthy cells. Detection and quantification of viral antigen with a novel single-molecule immunosorbent assay (SMISA) was achieved. Antigen from human immunodeficiency virus type 1(HIV-1) was chosen to be the target in this study. The target was sandwiched between a monoclonal capture antibody and a

  4. Equivalent configurations for notch and fretting fatigue

    Directory of Open Access Journals (Sweden)

    J. A. Araújo

    2015-07-01

    Full Text Available Under the typical partial slip conditions under which fretting fatigue takes place, the amount of superficial damage is small. Therefore, the substantial reduction in fatigue life caused by fretting, when compared to plain fatigue, may well be more associated with the stress concentration and the stress gradient phenomena generated by the contact problem than to the superficial loss of material. In this setting, notch stress-based methodologies could, in principle, be applied to fretting in the medium/high cycle fatigue regime. The aim of this work was to investigate whether it is possible to design fretting and notch fatigue configurations, which are nominally identical in terms of damage measured by a multiaxial fatigue model. The methodology adopted to carry out this search considered a cylindrical on flat contact and a V-notch. Load and geometry dimensions of both configurations were adjusted in order to try to obtain the “same” decay of the Multiaxial Fatigue Index from the hot spot up to a critical distance. Positive results of such simulations can lead us to design an experimental program that can bring more firm conclusions on the use of pure stress-based approaches, which do not include the wear damage, in the modeling of fretting fatigue.

  5. Vibrationally coupled electron transport through single-molecule junctions

    Energy Technology Data Exchange (ETDEWEB)

    Haertle, Rainer

    2012-04-26

    Single-molecule junctions are among the smallest electric circuits. They consist of a molecule that is bound to a left and a right electrode. With such a molecular nanocontact, the flow of electrical currents through a single molecule can be studied and controlled. Experiments on single-molecule junctions show that a single molecule carries electrical currents that can even be in the microampere regime. Thereby, a number of transport phenomena have been observed, such as, for example, diode- or transistor-like behavior, negative differential resistance and conductance switching. An objective of this field, which is commonly referred to as molecular electronics, is to relate these transport phenomena to the properties of the molecule in the contact. To this end, theoretical model calculations are employed, which facilitate an understanding of the underlying transport processes and mechanisms. Thereby, one has to take into account that molecules are flexible structures, which respond to a change of their charge state by a profound reorganization of their geometrical structure or may even dissociate. It is thus important to understand the interrelation between the vibrational degrees of freedom of a singlemolecule junction and the electrical current flowing through the contact. In this thesis, we investigate vibrational effects in electron transport through singlemolecule junctions. For these studies, we calculate and analyze transport characteristics of both generic and first-principles based model systems of a molecular contact. To this end, we employ a master equation and a nonequilibrium Green's function approach. Both methods are suitable to describe this nonequilibrium transport problem and treat the interactions of the tunneling electrons on the molecular bridge non-perturbatively. This is particularly important with respect to the vibrational degrees of freedom, which may strongly interact with the tunneling electrons. We show in detail that the resulting

  6. Simple test system for single molecule recognition force microscopy

    International Nuclear Information System (INIS)

    Riener, Christian K.; Stroh, Cordula M.; Ebner, Andreas; Klampfl, Christian; Gall, Alex A.; Romanin, Christoph; Lyubchenko, Yuri L.; Hinterdorfer, Peter; Gruber, Hermann J.

    2003-01-01

    We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG 800 diamine was glutarylated, the mono-adduct NH 2 -PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy

  7. Single Molecule Studies on Dynamics in Liquid Crystals

    Directory of Open Access Journals (Sweden)

    Daniela Täuber

    2013-09-01

    Full Text Available Single molecule (SM methods are able to resolve structure related dynamics of guest molecules in liquid crystals (LC. Highly diluted small dye molecules on the one hand explore structure formation and LC dynamics, on the other hand they report about a distortion caused by the guest molecules. The anisotropic structure of LC materials is used to retrieve specific conformation related properties of larger guest molecules like conjugated polymers. This in particular sheds light on organization mechanisms within biological cells, where large molecules are found in nematic LC surroundings. This review gives a short overview related to the application of highly sensitive SM detection schemes in LC.

  8. Tunneling anisotropic magnetoresistance in single-molecule magnet junctions

    Science.gov (United States)

    Xie, Haiqing; Wang, Qiang; Jiao, Hujun; Liang, J.-Q.

    2012-08-01

    We theoretically investigate quantum transport through single-molecule magnet (SMM) junctions with ferromagnetic and normal-metal leads in the sequential regime. The current obtained by means of the rate-equation gives rise to the tunneling anisotropic magnetoresistance (TAMR), which varies with the angle between the magnetization direction of ferromagnetic lead and the easy axis of SMM. The angular dependence of TAMR can serve as a probe to determine experimentally the easy axis of SMM. Moreover, it is demonstrated that both the magnitude and the sign of TAMR are tunable by the bias voltage, suggesting a new spin-valve device with only one magnetic electrode in molecular spintronics.

  9. Berry-Phase Blockade in Single-Molecule Magnets

    Science.gov (United States)

    González, Gabriel; Leuenberger, Michael N.

    2007-06-01

    We formulate the problem of electron transport through a single-molecule magnet (SMM) in the Coulomb blockade regime taking into account topological interference effects for the tunneling of the large spin of a SMM. The interference originates from spin Berry phases associated with different tunneling paths. We show that, in the case of incoherent spin states, it is essential to place the SMM between oppositely spin-polarized source and drain leads in order to detect the spin tunneling in the stationary current, which exhibits topological zeros as a function of the transverse magnetic field.

  10. Single molecule studies on dynamics in liquid crystals.

    Science.gov (United States)

    Täuber, Daniela; von Borczyskowski, Christian

    2013-09-26

    Single molecule (SM) methods are able to resolve structure related dynamics of guest molecules in liquid crystals (LC). Highly diluted small dye molecules on the one hand explore structure formation and LC dynamics, on the other hand they report about a distortion caused by the guest molecules. The anisotropic structure of LC materials is used to retrieve specific conformation related properties of larger guest molecules like conjugated polymers. This in particular sheds light on organization mechanisms within biological cells, where large molecules are found in nematic LC surroundings. This review gives a short overview related to the application of highly sensitive SM detection schemes in LC.

  11. Electrochemical proton relay at the single-molecule level

    DEFF Research Database (Denmark)

    Kuznetsov, A. M.; Medvedev, I. G.; Ulstrup, Jens

    2009-01-01

    A scheme for the experimental study of single-proton transfer events, based on proton-coupled two-electron transfer between a proton donor and a proton acceptor molecule confined in the tunneling gap between two metal leads in electrolyte solution is suggested. Expressions for the electric current...... are derived and compared with formalism for electron tunneling through redox molecules. The scheme allows studying the kinetics of proton and hydrogen atom transfer as well as kinetic isotope effects at the single-molecule level under electrochemical potential control....

  12. Pulsed IR Heating Studies of Single-Molecule DNA Duplex Dissociation Kinetics and Thermodynamics

    Science.gov (United States)

    Holmstrom, Erik D.; Dupuis, Nicholas F.; Nesbitt, David J.

    2014-01-01

    Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10−11 liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20–100°C); 2), substantially superior spatial (≤20 μm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation. PMID:24411254

  13. Research Update: Molecular electronics: The single-molecule switch and transistor

    Directory of Open Access Journals (Sweden)

    Kai Sotthewes

    2014-01-01

    Full Text Available In order to design and realize single-molecule devices it is essential to have a good understanding of the properties of an individual molecule. For electronic applications, the most important property of a molecule is its conductance. Here we show how a single octanethiol molecule can be connected to macroscopic leads and how the transport properties of the molecule can be measured. Based on this knowledge we have realized two single-molecule devices: a molecular switch and a molecular transistor. The switch can be opened and closed at will by carefully adjusting the separation between the electrical contacts and the voltage drop across the contacts. This single-molecular switch operates in a broad temperature range from cryogenic temperatures all the way up to room temperature. Via mechanical gating, i.e., compressing or stretching of the octanethiol molecule, by varying the contact's interspace, we are able to systematically adjust the conductance of the electrode-octanethiol-electrode junction. This two-terminal single-molecule transistor is very robust, but the amplification factor is rather limited.

  14. Massively Parallel Haplotyping on Microscopic Beads for the High-Throughput Phase Analysis of Single Molecules

    Science.gov (United States)

    Tiemann-Boege, Irene

    2012-01-01

    In spite of the many advances in haplotyping methods, it is still very difficult to characterize rare haplotypes in tissues and different environmental samples or to accurately assess the haplotype diversity in large mixtures. This would require a haplotyping method capable of analyzing the phase of single molecules with an unprecedented throughput. Here we describe such a haplotyping method capable of analyzing in parallel hundreds of thousands single molecules in one experiment. In this method, multiple PCR reactions amplify different polymorphic regions of a single DNA molecule on a magnetic bead compartmentalized in an emulsion drop. The allelic states of the amplified polymorphisms are identified with fluorescently labeled probes that are then decoded from images taken of the arrayed beads by a microscope. This method can evaluate the phase of up to 3 polymorphisms separated by up to 5 kilobases in hundreds of thousands single molecules. We tested the sensitivity of the method by measuring the number of mutant haplotypes synthesized by four different commercially available enzymes: Phusion, Platinum Taq, Titanium Taq, and Phire. The digital nature of the method makes it highly sensitive to detecting haplotype ratios of less than 1∶10,000. We also accurately quantified chimera formation during the exponential phase of PCR by different DNA polymerases. PMID:22558329

  15. Single molecule force spectroscopy at high data acquisition: A Bayesian nonparametric analysis

    Science.gov (United States)

    Sgouralis, Ioannis; Whitmore, Miles; Lapidus, Lisa; Comstock, Matthew J.; Pressé, Steve

    2018-03-01

    Bayesian nonparametrics (BNPs) are poised to have a deep impact in the analysis of single molecule data as they provide posterior probabilities over entire models consistent with the supplied data, not just model parameters of one preferred model. Thus they provide an elegant and rigorous solution to the difficult problem encountered when selecting an appropriate candidate model. Nevertheless, BNPs' flexibility to learn models and their associated parameters from experimental data is a double-edged sword. Most importantly, BNPs are prone to increasing the complexity of the estimated models due to artifactual features present in time traces. Thus, because of experimental challenges unique to single molecule methods, naive application of available BNP tools is not possible. Here we consider traces with time correlations and, as a specific example, we deal with force spectroscopy traces collected at high acquisition rates. While high acquisition rates are required in order to capture dwells in short-lived molecular states, in this setup, a slow response of the optical trap instrumentation (i.e., trapped beads, ambient fluid, and tethering handles) distorts the molecular signals introducing time correlations into the data that may be misinterpreted as true states by naive BNPs. Our adaptation of BNP tools explicitly takes into consideration these response dynamics, in addition to drift and noise, and makes unsupervised time series analysis of correlated single molecule force spectroscopy measurements possible, even at acquisition rates similar to or below the trap's response times.

  16. Simultaneous quantitative live cell imaging of multiple FRET-based biosensors.

    Science.gov (United States)

    Woehler, Andrew

    2013-01-01

    We have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/emission spectral fingerprint of FRET from reference measurements of the donor and acceptor alone. By unmixing the 3D spectrum of the FRET sample, the total relative concentrations of the fluorophores and their scaled FRET efficiencies are directly measured, from which apparent FRET efficiencies can be computed. If the FRET sample is composed of intramolecular FRET sensors it is possible to determine the total relative concentration of the sensors and then estimate absolute FRET efficiency of each sensor. Using multiple tandem constructs with fixed FRET efficiency as well as FRET-based calcium sensors with novel fluorescent protein combinations we demonstrate that the computed FRET efficiencies are accurate and changes in these quantities occur without crosstalk. We provide an example of this method's potential by demonstrating simultaneous imaging of spatially colocalized changes in [Ca(2+)], [cAMP], and PKA activity.

  17. Simultaneous quantitative live cell imaging of multiple FRET-based biosensors.

    Directory of Open Access Journals (Sweden)

    Andrew Woehler

    Full Text Available We have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/emission spectral fingerprint of FRET from reference measurements of the donor and acceptor alone. By unmixing the 3D spectrum of the FRET sample, the total relative concentrations of the fluorophores and their scaled FRET efficiencies are directly measured, from which apparent FRET efficiencies can be computed. If the FRET sample is composed of intramolecular FRET sensors it is possible to determine the total relative concentration of the sensors and then estimate absolute FRET efficiency of each sensor. Using multiple tandem constructs with fixed FRET efficiency as well as FRET-based calcium sensors with novel fluorescent protein combinations we demonstrate that the computed FRET efficiencies are accurate and changes in these quantities occur without crosstalk. We provide an example of this method's potential by demonstrating simultaneous imaging of spatially colocalized changes in [Ca(2+], [cAMP], and PKA activity.

  18. Single molecule transcription factor dynamics in the syncytial Drosophila embryo

    Science.gov (United States)

    Darzacq, Xavier

    During early development in the Drosophila embryo, cell fates are determined over the course of just 2 hours with exquisite spatio-temoral precision. One of the key regulators of this process is the transcription factor Bicoid which forms a concentration gradient across the long axis of the embryo. Although Bicoids' primary role is activation at the anterior, where concentrations are highest, it is also known to play a role in the posterior where there are only 100s of molecules per nucleus. Understanding how Bicoid can find its target at such low concentrations has remained intractable, largely due to the inability to perform single molecule imaging in the context of the developing embryo. Here we use lattice light sheet microscopy to overcome the technical barriers of sample thickness and auto-fluorescence to characterize the single molecule dynamics of Bicoid. We find that off-rates do not vary across the embryo and that instead the on-rates are modulated through the formation of clusters that enrich local concentration. This data is contrary to the current concentration dependent model of Bicoid function since local concentration within the nucleus is now a regulated parameter and suggests a previously unknown mechanism for regulation at extremely low concentrations.

  19. Single-molecule correlated chemical probing of RNA.

    Science.gov (United States)

    Homan, Philip J; Favorov, Oleg V; Lavender, Christopher A; Kursun, Olcay; Ge, Xiyuan; Busan, Steven; Dokholyan, Nikolay V; Weeks, Kevin M

    2014-09-23

    Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify through-space interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA.

  20. Dual-Colored DNA Comb Polymers for Single Molecule Rheology

    Science.gov (United States)

    Mai, Danielle; Marciel, Amanda; Schroeder, Charles

    2014-03-01

    We report the synthesis and characterization of branched biopolymers for single molecule rheology. In our work, we utilize a hybrid enzymatic-synthetic approach to graft ``short'' DNA branches to ``long'' DNA backbones, thereby producing macromolecular DNA comb polymers. The branches and backbones are synthesized via polymerase chain reaction with chemically modified deoxyribonucleotides (dNTPs): ``short'' branches consist of Cy5-labeled dNTPs and a terminal azide group, and ``long'' backbones contain dibenzylcyclooctyne-modified (DBCO) dNTPs. In this way, we utilize strain-promoted, copper-free cycloaddition ``click'' reactions for facile grafting of azide-terminated branches at DBCO sites along backbones. Copper-free click reactions are bio-orthogonal and nearly quantitative when carried out under mild conditions. Moreover, comb polymers can be labeled with an intercalating dye (e.g., YOYO) for dual-color fluorescence imaging. We characterized these materials using gel electrophoresis, HPLC, and optical microscopy, with atomic force microscopy in progress. Overall, DNA combs are suitable for single molecule dynamics, and in this way, our work holds the potential to improve our understanding of topologically complex polymer melts and solutions.

  1. Machine learning approach for single molecule localisation microscopy.

    Science.gov (United States)

    Colabrese, Silvia; Castello, Marco; Vicidomini, Giuseppe; Del Bue, Alessio

    2018-04-01

    Single molecule localisation (SML) microscopy is a fundamental tool for biological discoveries; it provides sub-diffraction spatial resolution images by detecting and localizing "all" the fluorescent molecules labeling the structure of interest. For this reason, the effective resolution of SML microscopy strictly depends on the algorithm used to detect and localize the single molecules from the series of microscopy frames. To adapt to the different imaging conditions that can occur in a SML experiment, all current localisation algorithms request, from the microscopy users, the choice of different parameters. This choice is not always easy and their wrong selection can lead to poor performance. Here we overcome this weakness with the use of machine learning. We propose a parameter-free pipeline for SML learning based on support vector machine (SVM). This strategy requires a short supervised training that consists in selecting by the user few fluorescent molecules (∼ 10-20) from the frames under analysis. The algorithm has been extensively tested on both synthetic and real acquisitions. Results are qualitatively and quantitatively consistent with the state of the art in SML microscopy and demonstrate that the introduction of machine learning can lead to a new class of algorithms competitive and conceived from the user point of view.

  2. Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop–Receptor Tertiary Interaction†

    Science.gov (United States)

    Fiore, Julie L.; Kraemer, Benedikt; Koberling, Felix; Edmann, Rainer; Nesbitt, David J.

    2010-01-01

    RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop–receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21–47 °C), from which a van’t Hoff analysis yields the enthalpy (ΔH°) and entropy (ΔS°) of docking. Tetraloop–receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl2 and 100 mM NaCl, with excellent agreement between immobilized (ΔH° = −17.4 ± 1.6 kcal/mol, and ΔS° = −56.2 ± 5.4 cal mol−1 K−1) and freely diffusing (ΔH° = −17.2 ± 1.6 kcal/mol, and ΔS° = −55.9 ± 5.2 cal mol−1 K−1) species. Kinetic heterogeneity in the tetraloop–receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop–receptor interaction can account for ~60% of the ΔH° and ΔS° of P4–P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop–receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions. PMID:19186984

  3. Manifestation of spin selection rules on the quantum tunneling of magnetization in a single-molecule magnet.

    Science.gov (United States)

    Henderson, J J; Koo, C; Feng, P L; del Barco, E; Hill, S; Tupitsyn, I S; Stamp, P C E; Hendrickson, D N

    2009-07-03

    We present low temperature magnetometry measurements on a new Mn3 single-molecule magnet in which the quantum tunneling of magnetization (QTM) displays clear evidence for quantum mechanical selection rules. A QTM resonance appearing only at high temperatures demonstrates tunneling between excited states with spin projections differing by a multiple of three. This is dictated by the C3 molecular symmetry, which forbids pure tunneling from the lowest metastable state. Transverse field resonances are understood by correctly orienting the Jahn-Teller axes of the individual manganese ions and including transverse dipolar fields. These factors are likely to be important for QTM in all single-molecule magnets.

  4. Two-body tunnel transitions in a Mn 4 single-molecule magnet

    Science.gov (United States)

    Wernsdorfer, W.; Bhaduri, S.; Tiron, R.; Hendrickson, D. N.; Christou, G.

    2004-05-01

    The one-body tunnel picture of single-molecule magnets (SMMs) is not always sufficient to explain the measured tunnel transitions. An improvement to the picture is proposed by including also two-body tunnel transitions such as spin-spin cross-relaxation (SSCR) which are mediated by dipolar and weak superexchange interactions between molecules. A Mn 4 SMM is used as a model system. At certain external fields, SSCRs lead to additional quantum resonances which show up in hysteresis loop measurements as well-defined steps.

  5. Single molecule DNA detection with an atomic vapor notch filter

    Energy Technology Data Exchange (ETDEWEB)

    Uhland, Denis; Rendler, Torsten; Widmann, Matthias; Lee, Sang-Yun [University of Stuttgart and Stuttgart Research Center of Photonic Engineering (SCoPE) and IQST, 3rd Physics Institute, Stuttgart (Germany); Wrachtrup, Joerg; Gerhardt, Ilja [University of Stuttgart and Stuttgart Research Center of Photonic Engineering (SCoPE) and IQST, 3rd Physics Institute, Stuttgart (Germany); Max Planck Institute for Solid State Research, Stuttgart (Germany)

    2015-12-01

    The detection of single molecules has facilitated many advances in life- and material-science. Commonly the fluorescence of dye molecules is detected, which are attached to a non-fluorescent structure under study. For fluorescence microscopy one desires to maximize the detection efficiency together with an efficient suppression of undesired laser leakage. Here we present the use of the narrow-band filtering properties of hot atomic sodium vapor to selectively filter the excitation light from the red-shifted fluorescence of dye labeled single-stranded DNA molecules. A statistical analysis proves an enhancement in detection efficiency of more than 15% in a confocal and in a wide-field configuration. (orig.)

  6. Single-Molecule Electrochemical Gating in Ionic Liquids

    DEFF Research Database (Denmark)

    Kay, Nicola J.; Higgins, Simon J.; Jeppesen, Jan O.

    2012-01-01

    The single-molecular conductance of a redox active molecular bridge has been studied in an electrochemical single-molecule transistor configuration in a room-temperature ionic liquid (RTIL). The redox active pyrrolo-tetrathiafulvalene (pTTF) moiety was attached to gold contacts at both ends through...... and decreases again as the second redox process is passed. This is described as an “off–on–off–on–off” conductance switching behavior. This molecular conductance vs electrochemical potential relation could be modeled well as a sequential two-step charge transfer process with full or partial vibrational...... relaxation. Using this view, reorganization energies of ∼1.2 eV have been estimated for both the first and second redox transitions for the pTTF bridge in the 1-butyl-3-methylimidazolium trifluoromethanesulfonate (BMIOTf) ionic liquid environment. By contrast, in aqueous environments, a much smaller...

  7. Single Molecule Raman Detection of Enkephalin on Silver Colloidal Particles

    DEFF Research Database (Denmark)

    Kneipp, Katrin; Kneipp, Holger; Abdali, Salim

    2004-01-01

    Enkephalin, an endogeneous substance in the human brain showing morphine-like biological functions, has been detected at the single molecule level based on the surface-enhanced Raman signal of the ring breathing mode of phenylalanine, which is one building block of the molecule. For enhancing...... the Raman signal the enkephalin molecules have been attached to silver colloidal cluster structures. The experiments demonstrate that the SERS signal of the strongly enhanced ring breathing vibration of phenylalanine at 1000 cm-1 can be used as “intrinsic marker” for detecting a single enkephalin molecule...... and for monitoring its diffusion on the surface of the silver colloidal cluster without using a specific label molecule....

  8. Mechanisms of Cellular Proteostasis: Insights from Single-Molecule Approaches

    Science.gov (United States)

    Bustamante, Carlos J.; Kaiser, Christian M.; Maillard, Rodrigo A.; Goldman, Daniel H.; Wilson, Christian A.M.

    2015-01-01

    Cells employ a variety of strategies to maintain proteome homeostasis. Beginning during protein biogenesis, the translation machinery and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state. Polypeptides that are no longer needed or pose a threat to the cell, such as misfolded proteins and aggregates, are removed in an efficient and timely fashion by ATP-dependent proteases. In this review, we describe how applications of single-molecule manipulation methods, in particular optical tweezers, are shedding new light on the molecular mechanisms of quality control during the life cycles of proteins. PMID:24895851

  9. Quantum turnstile operation of single-molecule magnets

    International Nuclear Information System (INIS)

    Moldoveanu, V; Dinu, I V; Tanatar, B; Moca, C P

    2015-01-01

    The time-dependent transport through single-molecule magnets coupled to magnetic or non-magnetic electrodes is studied in the framework of the generalized master equation method. We investigate the transient regime induced by the periodic switching of the source and drain contacts. If the electrodes have opposite magnetizations the quantum turnstile operation allows the stepwise writing of intermediate excited states. In turn, the transient currents provide a way to read these states. Within our approach we take into account both the uniaxial and transverse anisotropy. The latter may induce additional quantum tunneling processes which affect the efficiency of the proposed read-and-write scheme. An equally weighted mixture of molecular spin states can be prepared if one of the electrodes is ferromagnetic. (paper)

  10. Kondo effect in single-molecule magnet transistors

    Science.gov (United States)

    Gonzalez, Gabriel; Leuenberger, Michael; Mucciolo, Eduardo

    2009-03-01

    We present a careful and thorough microscopic derivation of the anisotropic Kondo Hamiltonian for single-molecule magnet (SMM) transistors. When the molecule is strongly coupled to metallic leads, we show that by applying a transverse magnetic field it is possible to topologically induce or quench the Kondo effect in the conductance of a SMM with either an integer or a half-integer spin S>1/2. This topological Kondo effect is due to the Berry-phase interference between multiple quantum tunneling paths of the spin. We calculate the renormalized Berry-phase oscillations of the two Kondo peaks as a function of a transverse magnetic field by means of the poor man's scaling approach. We illustrate our findings with the SMM Ni4, which we propose as a possible candidate for the experimental observation of the conductance oscillations.

  11. Quantum Tunneling of Magnetization in Trigonal Single-Molecule Magnets

    Science.gov (United States)

    Liu, Junjie; Del Barco, Enrique; Hill, Stephen

    2012-02-01

    We perform a numerical analysis of the quantum tunneling of magnetization (QTM) that occurs in a spin S = 6 single-molecule magnet (SMM) with idealized C3 symmetry. The deconstructive points in the QTM are located by following the Berry-phase interference (BPI) oscillations. We find that the O4^3 (=12[Sz,S+^3 +S-^3 ]) operator unfreezes odd-k QTM resonances and generates three-fold patterns of BPI minima in all resonances, including k = 0! This behavior cannot be reproduced with operators that possess even rotational symmetry about the quantization axis. We find also that the k = 0 BPI minima shift away from zero longitudinal field. The wider implications of these results will be discussed in terms of the QTM behavior observed in other SMMs.

  12. A Low Spin Manganese(IV) Nitride Single Molecule Magnet.

    Science.gov (United States)

    Ding, Mei; Cutsail, George E; Aravena, Daniel; Amoza, Martín; Rouzières, Mathieu; Dechambenoit, Pierre; Losovyj, Yaroslav; Pink, Maren; Ruiz, Eliseo; Clérac, Rodolphe; Smith, Jeremy M

    2016-09-01

    Structural, spectroscopic and magnetic methods have been used to characterize the tris(carbene)borate compound PhB(MesIm) 3 Mn≡N as a four-coordinate manganese(IV) complex with a low spin ( S = 1/2) configuration. The slow relaxation of the magnetization in this complex, i.e. its single-molecule magnet (SMM) properties, is revealed under an applied dc field. Multireference quantum mechanical calculations indicate that this SMM behavior originates from an anisotropic ground doublet stabilized by spin-orbit coupling. Consistent theoretical and experiment data show that the resulting magnetization dynamics in this system is dominated by ground state quantum tunneling, while its temperature dependence is influenced by Raman relaxation.

  13. Spin thermoelectric effects in organic single-molecule devices

    Energy Technology Data Exchange (ETDEWEB)

    Wang, H.L.; Wang, M.X.; Qian, C.; Hong, X.K.; Zhang, D.B.; Liu, Y.S.; Yang, X.F., E-mail: xfyang@cslg.edu.cn

    2017-05-25

    Highlights: • A stronger spin thermoelectric performance in a polyacetylene device is observed. • For the antiferromagnetic (AFM) ordering, a transport gap is opened. Thus the thermoelectric effects are largely enhanced. - Abstract: The spin thermoelectric performance of a polyacetylene chain bridging two zigzag graphene nanoribbons (ZGNRs) is investigated based on first principles method. Two different edge spin arrangements in ZGNRs are considered. For ferromagnetic (FM) ordering, transmission eigenstates with different spin indices distributed below and above Fermi level are observed, leading directly to a strong spin thermoelectric effect in a wide temperature range. With the edge spins arranged in the antiferromagnetic (AFM) ordering, an obvious transport gap appears in the system, which greatly enhances the thermoelectric effects. The presence of a small spin splitting also induces a spin thermoelectric effect greater than the charge thermoelectric effect in certain temperature range. In general, the single-molecule junction exhibits the potential to be used for the design of perfect thermospin devices.

  14. Force-induced tautomerization in a single molecule.

    Science.gov (United States)

    Ladenthin, Janina N; Frederiksen, Thomas; Persson, Mats; Sharp, John C; Gawinkowski, Sylwester; Waluk, Jacek; Kumagai, Takashi

    2016-10-01

    Heat transfer, electrical potential and light energy are common ways to activate chemical reactions. Applied force is another way, but dedicated studies for such a mechanical activation are limited, and this activation is poorly understood at the single-molecule level. Here, we report force-induced tautomerization in a single porphycene molecule on a Cu(110) surface at 5 K, which is studied by scanning probe microscopy and density functional theory calculations. Force spectroscopy quantifies the force needed to trigger tautomerization with submolecular spatial resolution. The calculations show how the reaction pathway and barrier of tautomerization are modified in the presence of a copper tip and reveal the atomistic origin of the process. Moreover, we demonstrate that a chemically inert tip whose apex is terminated by a xenon atom cannot induce the reaction because of a weak interaction with porphycene and a strong relaxation of xenon on the tip as contact to the molecule is formed.

  15. Low-temperature phonoemissive tunneling rates in single molecule magnets

    Science.gov (United States)

    Liu, Yun; Garg, Anupam

    2016-03-01

    Tunneling between the two lowest energy levels of single molecule magnets with Ising type anisotropy, accompanied by the emission or absorption of phonons, is considered. Quantitatively accurate calculations of the rates for such tunneling are performed for a model Hamiltonian especially relevant to the best studied example, Fe8. Two different methods are used: high-order perturbation theory in the spin-phonon interaction and the non-Ising-symmetric parts of the spin Hamiltonian, and a novel semiclassical approach based on spin-coherent-state-path-integral instantons. The methods are found to be in good quantitative agreement with other, and consistent with previous approaches to the problem. The implications of these results for magnetization of molecular solids of these molecules are discussed briefly.

  16. Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

    CERN Document Server

    Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

    2001-01-01

    Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

  17. Enhancing Single Molecule Imaging in Optofluidics and Microfluidics

    Directory of Open Access Journals (Sweden)

    Andreas E. Vasdekis

    2011-08-01

    Full Text Available Microfluidics and optofluidics have revolutionized high-throughput analysis and chemical synthesis over the past decade. Single molecule imaging has witnessed similar growth, due to its capacity to reveal heterogeneities at high spatial and temporal resolutions. However, both resolution types are dependent on the signal to noise ratio (SNR of the image. In this paper, we review how the SNR can be enhanced in optofluidics and microfluidics. Starting with optofluidics, we outline integrated photonic structures that increase the signal emitted by single chromophores and minimize the excitation volume. Turning then to microfluidics, we review the compatible functionalization strategies that reduce noise stemming from non-specific interactions and architectures that minimize bleaching and blinking.

  18. A thin permeable-membrane device for single-molecule manipulation.

    Science.gov (United States)

    Park, Chang-Young; Jacobson, David R; Nguyen, Dan T; Willardson, Sam; Saleh, Omar A

    2016-01-01

    Single-molecule manipulation instruments have unparalleled abilities to interrogate the structure and elasticity of single biomolecules. Key insights are derived by measuring the system response in varying solution conditions; yet, typical solution control strategies require imposing a direct fluid flow on the measured biomolecule that perturbs the high-sensitivity measurement and/or removes interacting molecules by advection. An alternate approach is to fabricate devices that permit solution changes by diffusion of the introduced species through permeable membranes, rather than by direct solution flow through the sensing region. Prior implementations of permeable-membrane devices are relatively thick, disallowing their use in apparatus that require the simultaneous close approach of external instrumentation from two sides, as occurs in single-molecule manipulation devices like the magnetic tweezer. Here, we describe the construction and use of a thin microfluidic device appropriate for single-molecule studies. We create a flow cell of only ∼500 μm total thickness by sandwiching glass coverslips around a thin plastic gasket and then create permeable walls between laterally separated channels in situ through photo-induced cross-linking of poly(ethylene glycol) diacrylate hydrogels. We show that these membranes permit passage of ions and small molecules (thus permitting solution equilibration in the absence of direct flow), but the membranes block the passage of larger biomolecules (thus retaining precious samples). Finally, we demonstrate the suitability of the device for high-resolution magnetic-tweezer experiments by measuring the salt-dependent folding of a single RNA hairpin under force.

  19. A thin permeable-membrane device for single-molecule manipulation

    Science.gov (United States)

    Park, Chang-Young; Jacobson, David R.; Nguyen, Dan T.; Willardson, Sam; Saleh, Omar A.

    2016-01-01

    Single-molecule manipulation instruments have unparalleled abilities to interrogate the structure and elasticity of single biomolecules. Key insights are derived by measuring the system response in varying solution conditions; yet, typical solution control strategies require imposing a direct fluid flow on the measured biomolecule that perturbs the high-sensitivity measurement and/or removes interacting molecules by advection. An alternate approach is to fabricate devices that permit solution changes by diffusion of the introduced species through permeable membranes, rather than by direct solution flow through the sensing region. Prior implementations of permeable-membrane devices are relatively thick, disallowing their use in apparatus that require the simultaneous close approach of external instrumentation from two sides, as occurs in single-molecule manipulation devices like the magnetic tweezer. Here, we describe the construction and use of a thin microfluidic device appropriate for single-molecule studies. We create a flow cell of only ˜500 μm total thickness by sandwiching glass coverslips around a thin plastic gasket and then create permeable walls between laterally separated channels in situ through photo-induced cross-linking of poly(ethylene glycol) diacrylate hydrogels. We show that these membranes permit passage of ions and small molecules (thus permitting solution equilibration in the absence of direct flow), but the membranes block the passage of larger biomolecules (thus retaining precious samples). Finally, we demonstrate the suitability of the device for high-resolution magnetic-tweezer experiments by measuring the salt-dependent folding of a single RNA hairpin under force.

  20. Fluorescence spectroscopy of single molecules at room temperature and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Taekjip [Univ. of California, Berkeley, CA (United States)

    1996-12-01

    We performed fluorescence spectroscopy of single and pairs of dye molecules on a surface at room temperature. Near field scanning optical microscope (NSOM) and far field scanning optical microscope with multi-color excitation/detection capability were built. The instrument is capable of optical imaging with 100nm resolution and has the sensitivity necessary for single molecule detection. A variety of dynamic events which cannot be observed from an ensemble of molecules is revealed when the molecules are probed one at a time. They include (1) spectral jumps correlated with dark states, (2) individually resolved quantum jumps to and from the meta-stable triplet state, (3) rotational jumps due to desorption/readsorption events of single molecules on the surface. For these studies, a computer controlled optical system which automatically and rapidly locates and performs spectroscopic measurements on single molecules was developed. We also studied the interaction between closely spaced pairs of molecules. In particular, fluorescence resonance energy transfer between a single resonant pair of donor and acceptor molecules was measured. Photodestruction dynamics of the donor or acceptor were used to determine the presence and efficiency of energy transfer Dual molecule spectroscopy was extended to a non-resonant pair of molecules to obtain high resolution differential distance information. By combining NSOM and dual color scheme, we studied the co-localization of parasite proteins and host proteins on a human red blood cell membrane infected with malaria. These dual-molecule techniques can be used to measure distances, relative orientations, and changes in distances/orientations of biological macromolecules with very good spatial, angular and temporal resolutions, hence opening new capabilities in the study of such systems.

  1. Electronic Transport in Single Molecule Junctions: Control of the Molecule-Electrode Coupling Through Intramolecular Tunneling Barriers

    DEFF Research Database (Denmark)

    Danilov, Andrey; Kubatkin, Sergey; Kafanov, Sergey

    2008-01-01

    We report on single molecule electron transport measurements of two oligophenylenevinylene (OPV3) derivatives placed in a nanogap between gold (Au) or lead (Pb) electrodes in a field effect transistor device. Both derivatives contain thiol end groups that allow chemical binding to the electrodes...... to sequential tunneling and Coulomb blockade behavior....

  2. DNA-cisplatin binding mechanism peculiarities studied with single molecule stretching experiments

    Science.gov (United States)

    Crisafuli, F. A. P.; Cesconetto, E. C.; Ramos, E. B.; Rocha, M. S.

    2012-02-01

    We propose a method to determine the DNA-cisplatin binding mechanism peculiarities by monitoring the mechanical properties of these complexes. To accomplish this task, we have performed single molecule stretching experiments by using optical tweezers, from which the persistence and contour lengths of the complexes can be promptly measured. The persistence length of the complexes as a function of the drug total concentration in the sample was used to deduce the binding data, from which we show that cisplatin binds cooperatively to the DNA molecule, a point which so far has not been stressed in binding equilibrium studies of this ligand.

  3. Kondo-correlated transport in single molecule ferromagnetic break junction devices with controllable electrode magnetization alignment

    Science.gov (United States)

    Scott, Gavin; Hu, Ting-Chen

    A quantum dot attached to electrodes with magnetizations that can be switched between parallel and anti-parallel alignment has been proposed as a platform for investigating quantum criticality associated with the destruction of Kondo entanglement. We have fabricated single molecule break junction devices with elliptical ferromagnetic electrodes designed to suit this purpose. Low temperature transport measurements, supported by micromagnetic simulations, were used to investigate the magnetoresistance response on control samples during the magnetization reversal process. We show results of Kondo-correlated transport as the source and drain contacts are switched between parallel and anti-parallel magnetization configurations.

  4. The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

    Science.gov (United States)

    Hua, Boyang; Wang, Yanbo; Park, Seongjin; Han, Kyu Young; Singh, Digvijay; Kim, Jin H; Cheng, Wei; Ha, Taekjip

    2018-03-13

    Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

  5. Single Molecule Screening of Disease DNA Without Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji-Young [Iowa State Univ., Ames, IA (United States)

    2006-01-01

    The potential of single molecule detection as an analysis tool in biological and medical fields is well recognized today. This fast evolving technique will provide fundamental sensitivity to pick up individual pathogen molecules, and therefore contribute to a more accurate diagnosis and a better chance for a complete cure. Many studies are being carried out to successfully apply this technique in real screening fields. In this dissertation, several attempts are shown that have been made to test and refine the application of the single molecule technique as a clinical screening method. A basic applicability was tested with a 100% target content sample, using electrophoretic mobility and multiple colors as identification tools. Both electrophoretic and spectral information of individual molecule were collected within a second, while the molecule travels along the flow in a capillary. Insertion of a transmission grating made the recording of the whole spectrum of a dye-stained molecule possible without adding complicated instrumental components. Collecting two kinds of information simultaneously and combining them allowed more thorough identification, up to 98.8% accuracy. Probing mRNA molecules with fluorescently labeled cDNA via hybridization was also carried out. The spectral differences among target, probe, and hybrid were interpreted in terms of dispersion distances after transmission grating, and used for the identification of each molecule. The probes were designed to have the least background when they are free, but have strong fluorescence after hybridization via fluorescence resonance energy transfer. The mRNA-cDNA hybrids were further imaged in whole blood, plasma, and saliva, to test how far a crude preparation can be tolerated. Imaging was possible with up to 50% of clear bio-matrix contents, suggesting a simple lysis and dilution would be sufficient for imaging for some cells. Real pathogen DNA of human papillomavirus (HPV) type-I6 in human genomic DNA

  6. Compact quantum dots for single-molecule imaging.

    Science.gov (United States)

    Smith, Andrew M; Nie, Shuming

    2012-10-09

    Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology (1-4). To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study. Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation (5). Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large (4,6,7). Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past (8,9). The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an

  7. Examining Sources of Error in PCR by Single-Molecule Sequencing

    Science.gov (United States)

    Potapov, Vladimir

    2017-01-01

    Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification. PMID:28060945

  8. Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

    Science.gov (United States)

    Phelps, Carey; Israels, Brett; Marsh, Morgan C; von Hippel, Peter H; Marcus, Andrew H

    2016-12-29

    Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.

  9. Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53.

    Science.gov (United States)

    Rajagopalan, Sridharan; Huang, Fang; Fersht, Alan R

    2011-03-01

    The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.

  10. Magnetic Quantum Tunneling and Symmetry in Single Molecule Magnets

    Science.gov (United States)

    Kent, Andrew D.

    2003-03-01

    We have studied the symmetry of magnetic quantum tunneling (MQT) in single molecule magnets (SMMs) using a micro-Hall effect magnetometer and high field vector superconducting magnet system. In the most widely studied SMM, Mn12-acetate, an average crystal 4-fold symmetry in the magnetic response is shown to be due to local molecular environments of 2-fold symmetry that are rotated by 90 degrees with respect to one another. We attribute this to ligand disorder that leads to local rhombic distortions, a model first proposed by Cornia et al. based on x-ray diffraction data [1]. We have magnetically distilled a Mn12-acetate crystal to study a subset of these lower (2-fold) site symmetry molecules and present evidence for a spin-parity effect consistent with a local 2-fold symmetry [2]. These results highlight the importance of subtle changes in molecule environment in modulating magnetic anisotropy and MQT. [1] Cornia et al. Phys. Rev. Lett. 89, 257201 (2002) [2] E. del Barco, A. D. Kent, E. Rumberger, D. H. Hendrickson, G. Christou, submitted for publication (2002) and Europhys. Lett. 60, 768 (2002)

  11. Single molecule atomic force microscopy and force spectroscopy of chitosan.

    Science.gov (United States)

    Kocun, Marta; Grandbois, Michel; Cuccia, Louis A

    2011-02-01

    Atomic force microscopy (AFM) and AFM-based force spectroscopy was used to study the desorption of individual chitosan polymer chains from substrates with varying chemical composition. AFM images of chitosan adsorbed onto a flat mica substrate show elongated single strands or aggregated bundles. The aggregated state of the polymer is consistent with the high level of flexibility and mobility expected for a highly positively charged polymer strand. Conversely, the visualization of elongated strands indicated the presence of stabilizing interactions with the substrate. Surfaces with varying chemical composition (glass, self-assembled monolayer of mercaptoundecanoic acid/decanethiol and polytetrafluoroethylene (PTFE)) were probed with chitosan modified AFM tips and the corresponding desorption energies, calculated from plateau-like features, were attributed to the desorption of individual polymer strands. Desorption energies of 2.0±0.3×10(-20)J, 1.8±0.3×10(-20)J and 3.5±0.3×10(-20)J were obtained for glass, SAM of mercaptoundecanoic/dodecanethiol and PTFE, respectively. These single molecule level results can be used as a basis for investigating chitosan and chitosan-based materials for biomaterial applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Experimental techniques for single cell and single molecule biomechanics

    International Nuclear Information System (INIS)

    Lim, C.T.; Zhou, E.H.; Li, A.; Vedula, S.R.K.; Fu, H.X.

    2006-01-01

    Stresses and strains that act on the human body can arise either from external physical forces or internal physiological environmental conditions. These biophysical interactions can occur not only at the musculoskeletal but also cellular and molecular levels and can determine the health and function of the human body. Here, we seek to investigate the structure-property-function relationship of cells and biomolecules so as to understand their important physiological functions as well as establish possible connections to human diseases. With the recent advancements in cell and molecular biology, biophysics and nanotechnology, several innovative and state-of-the-art experimental techniques and equipment have been developed to probe the structural and mechanical properties of biostructures from the micro- down to picoscale. Some of these experimental techniques include the optical or laser trap method, micropipette aspiration, step-pressure technique, atomic force microscopy and molecular force spectroscopy. In this article, we will review the basic principles and usage of these techniques to conduct single cell and single molecule biomechanics research

  13. Photothermal cantilever actuation for fast single-molecule force spectroscopy

    Science.gov (United States)

    Stahl, Stefan W.; Puchner, Elias M.; Gaub, Hermann E.

    2009-07-01

    Photothermal cantilever excitation provides a fast and easy to implement means to control the deflection of standard atomic force microscopy cantilevers. Minute heat pulses yield deflections on the order of several tens of nanometers or when the deflection is kept constant, forces of several hundreds of piconewton can be applied. In our case these pulses resulted in less than 1 K temperature changes at the sample position. Here we present and characterize the implementation of photothermal actuation for single-molecule force-spectroscopy experiments. When molecules are stretched under force-clamp conditions, fast control cycles that re-establish the pulling force after the rupture of molecular domains are essential for detecting the complete unfolding pattern with high precision. By combining the fast response of photothermal cantilever excitation with a conventional piezoactuator, a fast force-clamp with high accuracy and large working distances is reached. Simple feedback mechanisms and standard cantilever geometries lead to step response times of less than 90 μs, which is more than one order of magnitude faster than those of conventional force-clamp systems that are based only on piezo feedback. We demonstrate the fast and accurate performance of the setup by unfolding a protein construct consisting of one green fluorescent protein and eight surrounding immunoglobulin domains at constant force.

  14. A single molecule study of cellulase hydrolysis of crystalline cellulose

    Science.gov (United States)

    Liu, Yu-San; Luo, Yonghua; Baker, John O.; Zeng, Yining; Himmel, Michael E.; Smith, Steve; Ding, Shi-You

    2010-02-01

    Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate β-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

  15. Tetramethylammonium-filled protein nanopore for single-molecule analysis.

    Science.gov (United States)

    Wang, Ying; Yao, Fujun; Kang, Xiao-feng

    2015-10-06

    Nanopore technology, as the simplest and most inexpensive single-molecule tool, is being intensively developed. In nanopore stochastic sensing, KCl and NaCl have traditionally been employed as pore-filled electrolytes for recording the change of ion conductance in nanopores triggered by analyte translocation through the pore. However, some challenges limit its further advance. Here we used tetramethylammonium (TMA) chloride, instead of KCl, as a novel analysis system for nanopores. Some unique nanopore characteristics were observed: (1) The stability of the planar lipid bilayer for embedding the protein pores was elevated at least 6 times. (2) The TMA-Cl system could effectively reduce the noise of single-channel recording. (3) It was easy to control the insertion of protein pores into the lipid bilayer, and the formed single nanopore could last for a sufficiently long time. (4) TMA-Cl could be used as a DNA speed bump in the nanopore to slow DNA translocation speed. (5) The capacity of the nanopore capture of DNA (capture rate) increased significantly and simultaneously increased the translocation time of DNA in the pore. (6) The TMA-filled nanopore could discriminate between various polynucleotides.

  16. Photon-Induced Magnetization Reversal in Single Molecule Magnets

    Science.gov (United States)

    Bal, Mustafa

    2005-03-01

    Single-molecule magnets (SMM) have been the subject of intensive research for more than a decade now because of their unique properties such as macroscopic quantum tunneling. Recent work in this area is focused on whether SMM are potential qubits, as proposed theoretically [1]. We use continuous millimeter wave radiation to manipulate the populations of the energy levels of a single crystal molecular magnet Fe8 [2]. When radiation is in resonance with the transitions between energy levels, the steady state magnetization exhibits dips. As expected, the magnetic field locations of these dips vary linearly with the radiation frequency. We will describe our experimental results, which provide a lower bound of 0.17 ns for transverse relaxation time. Transitions between excited states are found even though these states have negligible population at the experimental temperature. We find evidence that the sample heating is significant when the resonance condition is satisfied. Recent experiments are concentrated on the spin dynamics of Fe8 induced by pulsed radiation and results of these studies will also be presented. [1] Leuenberger, M. N. and Loss, D., Nature 410, 789 (2001). [2] M. Bal et al., Phys. Rev. B 70, 100408(R) (2004).

  17. Coherent interaction of single molecules and plasmonic nanowires

    Science.gov (United States)

    Gerhardt, Ilja; Grotz, Bernhard; Siyushev, Petr; Wrachtrup, Jörg

    2017-09-01

    Quantum plasmonics opens the option to integrate complex quantum optical circuitry onto chip scale devices. In the past, often external light sources were used and nonclassical light was coupled in and out of plasmonic structures, such as hole arrays or waveguide structures. Another option to launch single plasmonic excitations is the coupling of single emitters in the direct proximity of, e.g., a silver or gold nanostructure. Here, we present our attempts to integrate the research of single emitters with wet-chemically grown silver nanowires. The emitters of choice are single organic dye molecules under cryogenic conditions, which are known to act as high-brightness and extremely narrow-band single photon sources. Another advantage is their high optical nonlinearity, such that they might mediate photon-photon interactions on the nanoscale. We report on the coupling of a single molecule fluorescence emission through the wire over the length of several wavelengths. The transmission of coherently emitted photons is proven by an extinction type experiment. As for influencing the spectral properties of a single emitter, we are able to show a remote change of the line-width of a single terrylene molecule, which is in close proximity to the nanowire.

  18. Single molecule approaches for quantifying transcription and degradation rates in intact mammalian tissues.

    Science.gov (United States)

    Bahar Halpern, Keren; Itzkovitz, Shalev

    2016-04-01

    A key challenge in mammalian biology is to understand how rates of transcription and mRNA degradation jointly shape cellular gene expression. Powerful techniques have been developed for measuring these rates either genome-wide or at the single-molecule level, however these techniques are not applicable to assessment of cells within their native tissue microenvironment. Here we describe a technique based on single molecule Fluorescence in-situ Hybridization (smFISH) to measure transcription and degradation rates in intact mammalian tissues. The technique is based on dual-color libraries targeting the introns and exons of the genes of interest, enabling visualization and quantification of both nascent and mature mRNA. We present a software, TransQuant, that facilitates quantifying these rates from smFISH images. Our approach enables assessment of both transcription and degradation rates of any gene of interest while controlling for the inherent heterogeneity of intact tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Fluorescence molecule counting for single-molecule studies in crowded environment of living cells without and with broken ergodicity.

    Science.gov (United States)

    Földes-Papp, Zeno; Baumann, Gerd

    2011-05-01

    We present a new approach to distinguish between non-ergodic and ergodic behavior. Performing ensemble averaging in a subpopulation of individual molecules leads to a mean value that can be similar to the mean value obtained in an ergodic system. The averaging is carried out by minimizing the variation between the sum of the temporal averaged mean square deviation of the simulated data with respect to the logarithmic scaling behavior of the subpopulation. For this reason, we first introduce a kind of Continuous Time Random Walks (CTRW), which we call Limited Continuous Time Random Walks (LCTRW) on fractal support. The random waiting time distributions are sampled at points which fulfill the condition N <1, where N is the Poisson probability of finding a single molecule in the femtoliter-sized observation volume ΔV at the single-molecule level. Given a subpopulation of different single molecules of the same kind, the ratio T/ T(m) between the measurement time T and the meaningful time T(m), which is the time for observing just one and the same single molecule, is the experimentally accessible quantity that allows to compare different molecule numbers in the subpopulation. In addition, the mean square displacement traveled by the molecule during the time t is determined by an upper limit of the geometric dimension of the living cell or its nucleus.

  20. Lab-on-a-chip technologies for single-molecule studies.

    Science.gov (United States)

    Zhao, Yanhui; Chen, Danqi; Yue, Hongjun; French, Jarrod B; Rufo, Joseph; Benkovic, Stephen J; Huang, Tony Jun

    2013-06-21

    Recent developments on various lab-on-a-chip techniques allow miniaturized and integrated devices to perform on-chip single-molecule studies. Fluidic-based platforms that utilize unique microscale fluidic behavior are capable of conducting single-molecule experiments with high sensitivities and throughputs, while biomolecular systems can be studied on-chip using techniques such as DNA curtains, magnetic tweezers, and solid-state nanopores. The advances of these on-chip single-molecule techniques lead to next-generation lab-on-a-chip devices, such as DNA transistors, and single-molecule real-time (SMRT) technology for rapid and low-cost whole genome DNA sequencing. In this Focus article, we will discuss some recent successes in the development of lab-on-a-chip techniques for single-molecule studies and expound our thoughts on the near future of on-chip single-molecule studies.

  1. Modulation of intermolecular interactions in single-molecule magnets

    Science.gov (United States)

    Heroux, Katie Jeanne

    Polynuclear manganese clusters exhibiting interesting magnetic and quantum properties have been an area of intense research since the discovery of the first single-molecule magnet (SMM) in 1993. These molecules, below their blocking temperature, function as single-domain magnetic particles which exhibit classical macroscale magnetic properties as well as quantum mechanical phenomena such as quantum tunnelling of magnetization (QTM) and quantum phase interference. The union of classical and quantum behavior in these nanomaterials makes SMMs ideal candidates for high-density information storage and quantum computing. However, environmental coupling factors (nuclear spins, phonons, neighboring molecules) must be minimized if such applications are ever to be fully realized. The focus of this work is making small structural changes in well-known manganese SMMs in order to drastically enhance the overall magnetic and quantum properties of the system. Well-isolated molecules of high crystalline quality should lead to well-defined energetic and spectral properties as well. An advantage of SMMs over bulk magnetic materials is that they can be chemically altered from a "bottom-up" approach providing a synthetic tool for tuning magnetic properties. This systematic approach is utilized in the work presented herein by incorporating bulky ligands and/or counterions to "isolate" the magnetic core of [Mn4] dicubane SMMs. Reducing intermolecular interactions in the crystal lattice (neighboring molecules, solvate molecules, dipolar interactions) is an important step toward developing viable quantum computing devices. Detailed bulk magnetic studies as well as single crystal magnetization hysteresis and high-frequency EPR studies on these sterically-isolated complexes show enhanced, and sometimes even unexpected, quantum dynamics. The importance of intra- and intermolecular interactions remains a common theme throughout this work, extending to other SMMs of various topology including

  2. Computational and Experimental Insight Into Single-Molecule Piezoelectric Materials

    Science.gov (United States)

    Marvin, Christopher Wayne

    Piezoelectric materials allow for the harvesting of ambient waste energy from the environment. Producing lightweight, highly responsive materials is a challenge for this type of material, requiring polymer, foam, or bio-inspired materials. In this dissertation, I explore the origin of the piezoelectric effect in single molecules through density functional theory (DFT), analyze the piezoresponse of bio-inspired peptidic materials through the use of atomic and piezoresponse force microscopy (AFM and PFM), and develop a novel class of materials combining flexible polyurethane foams and non-piezoelectric, polar dopants. For the DFT calculations, functional group, regiochemical, and heteroatom derivatives of [6]helicene were examined for their influence on the piezoelectric response. An aza[6]helicene derivative was found to have a piezoelectric response (108 pm/V) comparable to ceramics such as lead zirconium titanate (200+ pm/V). These computed materials have the possibility to compete with current field-leading piezomaterials such as lead zirconium titanate (PZT), zinc oxide (ZnO), and polyvinylidene difluoride (PVDF) and its derivatives. The use of AFM/PFM allows for the demonstration of the piezoelectric effect of the selfassembled monolayer (SAM) peptidic systems. Through PFM, the influence that the helicity and sequence of the peptide has on the overall response of the molecule can be analyzed. Finally, development of a novel class of piezoelectrics, the foam-based materials, expands the current understanding of the qualities required for a piezoelectric material from ceramic and rigid materials to more flexible, organic materials. Through the exploration of these novel types of piezoelectric materials, new design rules and figures of merit have been developed.

  3. Probing static disorder in Arrhenius kinetics by single-molecule force spectroscopy.

    Science.gov (United States)

    Kuo, Tzu-Ling; Garcia-Manyes, Sergi; Li, Jingyuan; Barel, Itay; Lu, Hui; Berne, Bruce J; Urbakh, Michael; Klafter, Joseph; Fernández, Julio M

    2010-06-22

    The widely used Arrhenius equation describes the kinetics of simple two-state reactions, with the implicit assumption of a single transition state with a well-defined activation energy barrier DeltaE, as the rate-limiting step. However, it has become increasingly clear that the saddle point of the free-energy surface in most reactions is populated by ensembles of conformations, leading to nonexponential kinetics. Here we present a theory that generalizes the Arrhenius equation to include static disorder of conformational degrees of freedom as a function of an external perturbation to fully account for a diverse set of transition states. The effect of a perturbation on static disorder is best examined at the single-molecule level. Here we use force-clamp spectroscopy to study the nonexponential kinetics of single ubiquitin proteins unfolding under force. We find that the measured variance in DeltaE shows both force-dependent and independent components, where the force-dependent component scales with F(2), in excellent agreement with our theory. Our study illustrates a novel adaptation of the classical Arrhenius equation that accounts for the microscopic origins of nonexponential kinetics, which are essential in understanding the rapidly growing body of single-molecule data.

  4. Single-molecule study on polymer diffusion in a melt state: Effect of chain topology

    KAUST Repository

    Habuchi, Satoshi

    2013-08-06

    We report a new methodology for studying diffusion of individual polymer chains in a melt state, with special emphasis on the effect of chain topology. A perylene diimide fluorophore was incorporated into the linear and cyclic poly(THF)s, and real-time diffusion behavior of individual chains in a melt of linear poly(THF) was measured by means of a single-molecule fluorescence imaging technique. The combination of mean squared displacement (MSD) and cumulative distribution function (CDF) analysis demonstrated the broad distribution of diffusion coefficient of both the linear and cyclic polymer chains in the melt state. This indicates the presence of spatiotemporal heterogeneity of the polymer diffusion which occurs at much larger time and length scales than those expected from the current polymer physics theory. We further demonstrated that the cyclic chains showed marginally slower diffusion in comparison with the linear counterparts, to suggest the effective suppression of the translocation through the threading-entanglement with the linear matrix chains. This coincides with the higher activation energy for the diffusion of the cyclic chains than of the linear chains. These results suggest that the single-molecule imaging technique provides a powerful tool to analyze complicated polymer dynamics and contributes to the molecular level understanding of the chain interaction. © 2013 American Chemical Society.

  5. Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

    Science.gov (United States)

    Jonas, Kim C; Huhtaniemi, Ilpo; Hanyaloglu, Aylin C

    2016-01-01

    The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Probing the conductance superposition law in single-molecule circuits with parallel paths.

    Science.gov (United States)

    Vazquez, H; Skouta, R; Schneebeli, S; Kamenetska, M; Breslow, R; Venkataraman, L; Hybertsen, M S

    2012-10-01

    According to Kirchhoff's circuit laws, the net conductance of two parallel components in an electronic circuit is the sum of the individual conductances. However, when the circuit dimensions are comparable to the electronic phase coherence length, quantum interference effects play a critical role, as exemplified by the Aharonov-Bohm effect in metal rings. At the molecular scale, interference effects dramatically reduce the electron transfer rate through a meta-connected benzene ring when compared with a para-connected benzene ring. For longer conjugated and cross-conjugated molecules, destructive interference effects have been observed in the tunnelling conductance through molecular junctions. Here, we investigate the conductance superposition law for parallel components in single-molecule circuits, particularly the role of interference. We synthesize a series of molecular systems that contain either one backbone or two backbones in parallel, bonded together cofacially by a common linker on each end. Single-molecule conductance measurements and transport calculations based on density functional theory show that the conductance of a double-backbone molecular junction can be more than twice that of a single-backbone junction, providing clear evidence for constructive interference.

  7. Single-Molecule Imaging of DNAs with Sticky Ends at Water/Fused Silica Interface

    Energy Technology Data Exchange (ETDEWEB)

    Isailovic, Slavica [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    Total internal reflection fluorescence microscopy (TIRFM) was used to study intermolecular interactions of DNAs with unpaired (sticky) ends of different lengths at water/fused silica interface at the single-molecule level. Evanescent field residence time, linear velocity and adsorption/desorption frequency were measured in a microchannel for individual DNA molecules from T7, Lambda, and PSP3 phages at various pH values. The longest residence times and the highest adsorption/desorption frequencies at the constant flow at pH 5.5 were found for PSP3 DNA, followed by lower values for Lambda DNA, and the lowest values for T7 DNA. Since T7, Lambda, and PSP3 DNA molecules contain none, twelve and nineteen unpaired bases, respectively, it was concluded that the affinity of DNAs for the surface increases with the length of the sticky ends. This confirms that hydrophobic and hydrogen-bonding interactions between sticky ends and fused-silica surface are driving forces for DNA adsorption at the fused-silica surface. Described single-molecule methodology and results therein can be valuable for investigation of interactions in liquid chromatography, as well as for design of DNA hybridization sensors and drug delivery systems.

  8. Single-molecule transport across an individual biomimetic nuclear pore complex

    Science.gov (United States)

    Kowalczyk, Stefan W.; Kapinos, Larisa; Blosser, Timothy R.; Magalhães, Tomás; van Nies, Pauline; Lim, Roderick Y. H.; Dekker, Cees

    2011-07-01

    Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon. Here, we show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of ~2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to ~25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro.

  9. Nanopore arrays in a silicon membrane for parallel single-molecule detection: DNA translocation.

    Science.gov (United States)

    Zhang, Miao; Schmidt, Torsten; Jemt, Anders; Sahlén, Pelin; Sychugov, Ilya; Lundeberg, Joakim; Linnros, Jan

    2015-08-07

    Optical nanopore sensing offers great potential in single-molecule detection, genotyping, or DNA sequencing for high-throughput applications. However, one of the bottle-necks for fluorophore-based biomolecule sensing is the lack of an optically optimized membrane with a large array of nanopores, which has large pore-to-pore distance, small variation in pore size and low background photoluminescence (PL). Here, we demonstrate parallel detection of single-fluorophore-labeled DNA strands (450 bps) translocating through an array of silicon nanopores that fulfills the above-mentioned requirements for optical sensing. The nanopore array was fabricated using electron beam lithography and anisotropic etching followed by electrochemical etching resulting in pore diameters down to ∼7 nm. The DNA translocation measurements were performed in a conventional wide-field microscope tailored for effective background PL control. The individual nanopore diameter was found to have a substantial effect on the translocation velocity, where smaller openings slow the translocation enough for the event to be clearly detectable in the fluorescence. Our results demonstrate that a uniform silicon nanopore array combined with wide-field optical detection is a promising alternative with which to realize massively-parallel single-molecule detection.

  10. Virtual reality visual feedback for hand-controlled scanning probe microscopy manipulation of single molecules.

    Science.gov (United States)

    Leinen, Philipp; Green, Matthew F B; Esat, Taner; Wagner, Christian; Tautz, F Stefan; Temirov, Ruslan

    2015-01-01

    Controlled manipulation of single molecules is an important step towards the fabrication of single molecule devices and nanoscale molecular machines. Currently, scanning probe microscopy (SPM) is the only technique that facilitates direct imaging and manipulations of nanometer-sized molecular compounds on surfaces. The technique of hand-controlled manipulation (HCM) introduced recently in Beilstein J. Nanotechnol. 2014, 5, 1926-1932 simplifies the identification of successful manipulation protocols in situations when the interaction pattern of the manipulated molecule with its environment is not fully known. Here we present a further technical development that substantially improves the effectiveness of HCM. By adding Oculus Rift virtual reality goggles to our HCM set-up we provide the experimentalist with 3D visual feedback that displays the currently executed trajectory and the position of the SPM tip during manipulation in real time, while simultaneously plotting the experimentally measured frequency shift (Δf) of the non-contact atomic force microscope (NC-AFM) tuning fork sensor as well as the magnitude of the electric current (I) flowing between the tip and the surface. The advantages of the set-up are demonstrated by applying it to the model problem of the extraction of an individual PTCDA molecule from its hydrogen-bonded monolayer grown on Ag(111) surface.

  11. Virtual reality visual feedback for hand-controlled scanning probe microscopy manipulation of single molecules

    Directory of Open Access Journals (Sweden)

    Philipp Leinen

    2015-11-01

    Full Text Available Controlled manipulation of single molecules is an important step towards the fabrication of single molecule devices and nanoscale molecular machines. Currently, scanning probe microscopy (SPM is the only technique that facilitates direct imaging and manipulations of nanometer-sized molecular compounds on surfaces. The technique of hand-controlled manipulation (HCM introduced recently in Beilstein J. Nanotechnol. 2014, 5, 1926–1932 simplifies the identification of successful manipulation protocols in situations when the interaction pattern of the manipulated molecule with its environment is not fully known. Here we present a further technical development that substantially improves the effectiveness of HCM. By adding Oculus Rift virtual reality goggles to our HCM set-up we provide the experimentalist with 3D visual feedback that displays the currently executed trajectory and the position of the SPM tip during manipulation in real time, while simultaneously plotting the experimentally measured frequency shift (Δf of the non-contact atomic force microscope (NC-AFM tuning fork sensor as well as the magnitude of the electric current (I flowing between the tip and the surface. The advantages of the set-up are demonstrated by applying it to the model problem of the extraction of an individual PTCDA molecule from its hydrogen-bonded monolayer grown on Ag(111 surface.

  12. Minimizing pulling geometry errors in atomic force microscope single molecule force spectroscopy.

    Science.gov (United States)

    Rivera, Monica; Lee, Whasil; Ke, Changhong; Marszalek, Piotr E; Cole, Daniel G; Clark, Robert L

    2008-10-01

    In atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS), it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Recent studies, however, have indicated that the pulling geometry errors can drastically alter the measured force-extension relationship of molecules. Here we describe a software-based alignment method that repositions the cantilever such that it is located directly above the molecule's substrate attachment site. By aligning the applied force with the measurement axis, the molecule is no longer undergoing combined loading, and the full force can be measured by the cantilever. Simulations and experimental results verify the ability of the alignment program to minimize pulling geometry errors in AFM-SMFS studies.

  13. Characteristic of fretting damage in metal material

    Energy Technology Data Exchange (ETDEWEB)

    Li, D.; Zhi, F.

    1988-10-01

    The fretting fatigue experiment of LC4 high strength aluminum alloy is described. An SEM examination of the fractology and morphology of fretting damage is carried out as well as an EDAX analysis of the chemical composition of fretting particles. The results show that many loose oxide particles were produced and accumulated in the fretting damage region. 10 references.

  14. Nonlinear and Nonsymmetric Single-Molecule Electronic Properties Towards Molecular Information Processing.

    Science.gov (United States)

    Tamaki, Takashi; Ogawa, Takuji

    2017-09-05

    This review highlights molecular design for nonlinear and nonsymmetric single-molecule electronic properties such as rectification, negative differential resistance, and switching, which are important components of future single-molecule information processing devices. Perspectives on integrated "molecular circuits" are also provided. Nonlinear and nonsymmetric single-molecule electronics can be designed by utilizing (1) asymmetric molecular cores, (2) asymmetric anchoring groups, (3) an asymmetric junction environment, and (4) asymmetric electrode materials. This review mainly focuses on the design of molecular cores.

  15. Using FRET to Measure the Angle at Which a Protein Bends DNA: TBP Binding a TATA Box as a Model System

    Science.gov (United States)

    Kugel, Jennifer F.

    2008-01-01

    An undergraduate biochemistry laboratory experiment that will teach the technique of fluorescence resonance energy transfer (FRET) while analyzing protein-induced DNA bending is described. The experiment uses the protein TATA binding protein (TBP), which is a general transcription factor that recognizes and binds specific DNA sequences known as…

  16. Fluctuation in Interface and Electronic Structure of Single-Molecule Junctions Investigated by Current versus Bias Voltage Characteristics.

    Science.gov (United States)

    Isshiki, Yuji; Fujii, Shintaro; Nishino, Tomoaki; Kiguchi, Manabu

    2018-03-14

    Structural and electronic detail at the metal-molecule interface has a significant impact on the charge transport across the molecular junctions, but its precise understanding and control still remain elusive. On the single-molecule scale, the metal-molecule interface structures and relevant charge transport properties are subject to fluctuation, which contain the fundamental science of single-molecule transport and implication for manipulability of the transport properties in electronic devices. Here, we present a comprehensive approach to investigate the fluctuation in the metal-molecule interface in single-molecule junctions, based on current-voltage ( I- V) measurements in combination with first-principles simulation. Contrary to conventional molecular conductance studies, this I- V approach provides a correlated statistical description of both the degree of electronic coupling across the metal-molecule interface and the molecular orbital energy level. This statistical approach was employed to study fluctuation in single-molecule junctions of 1,4-butanediamine (DAB), pyrazine (PY), 4,4'-bipyridine (BPY), and fullerene (C 60 ). We demonstrate that molecular-dependent fluctuation of σ-, π-, and π-plane-type interfaces can be captured by analyzing the molecular orbital (MO) energy level under mechanical perturbation. While the MO level of DAB with the σ-type interface shows weak distance dependence and fluctuation, the MO level of PY, BPY, and C 60 features unique distance dependence and molecular-dependent fluctuation against the mechanical perturbation. The MO level of PY and BPY with the σ+π-type interface increases with the increase in the stretch distance. In contrast, the MO level of C 60 with the π-plane-type interface decreases with the increase in the stretching perturbation. This study provides an approach to resolve the structural and electronic fluctuation in the single-molecule junctions and insight into the molecular-dependent fluctuation in

  17. Sniffing for gene-silencing efficiency of siRNAs in HeLa cells in comparison with that in HEK293T cells: correlation between knockdown efficiency and sustainability of sirnas revealed by FRET-based probing.

    Science.gov (United States)

    Shin, Seonmi; Kim, Yea Seul; Kim, Jisu; Kwon, Hyun-Mi; Kim, Dong-Eun; Hah, Sang Soo

    2013-04-01

    Investigation of the intracellular fate of small interfering RNAs (siRNAs) following their delivery into cells is of great importance to elucidate their dynamics in cytoplasm. Here we describe the use of an advanced fluorescence-based method to probe the dissociation and/or degradation of double-labeled siRNAs in HeLa cells in comparison with that in human embryonic kidney 293T (HEK293T) cells. This work was performed with three siRNAs labeled with fluorescence resonance energy transfer (FRET) dyes, allowing a non-destructive and non-invasive assessment of the dissociation and degradation state of siRNAs in cultured cells. Our FRET analysis not only shows the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand during the measured time period, underlining the high intrinsic nuclease resistance of duplex siRNAs, but also reveals the longer sustainability of siRNAs in HeLa cells compared with that in HEK293T cells, explaining the gene silencing in HeLa cells is more efficient than that in HEK293T cells. In addition, our single-molecule FRET assays demonstrate the potential of the delineated fluorescence-based technique for future research on biological behavior of siRNAs even at the single-molecule level. The fluorescence-based method is a straightforward technique to gain direct information on siRNA integrity inside living cells, which can provide a detection tool for dynamics of biological molecules.

  18. Single-molecule tracking in living cells using single quantum dot applications.

    Science.gov (United States)

    Baba, Koichi; Nishida, Kohji

    2012-01-01

    Revealing the behavior of single molecules in living cells is very useful for understanding cellular events. Quantum dot probes are particularly promising tools for revealing how biological events occur at the single molecule level both in vitro and in vivo. In this review, we will introduce how single quantum dot applications are used for single molecule tracking. We will discuss how single quantum dot tracking has been used in several examples of complex biological processes, including membrane dynamics, neuronal function, selective transport mechanisms of the nuclear pore complex, and in vivo real-time observation. We also briefly discuss the prospects for single molecule tracking using advanced probes.

  19. Fluorescent proteins as genetically encoded FRET biosensors in life sciences.

    Science.gov (United States)

    Hochreiter, Bernhard; Garcia, Alan Pardo; Schmid, Johannes A

    2015-10-16

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  20. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Directory of Open Access Journals (Sweden)

    Bernhard Hochreiter

    2015-10-01

    Full Text Available Fluorescence- or Förster resonance energy transfer (FRET is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a cleavage; (b conformational-change; (c mechanical force and (d changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  1. QD-Based FRET Probes at a Glance

    Directory of Open Access Journals (Sweden)

    Armen Shamirian

    2015-06-01

    Full Text Available The unique optoelectronic properties of quantum dots (QDs give them significant advantages over traditional organic dyes, not only as fluorescent labels for bioimaging, but also as emissive sensing probes. QD sensors that function via manipulation of fluorescent resonance energy transfer (FRET are of special interest due to the multiple response mechanisms that may be utilized, which in turn imparts enhanced flexibility in their design. They may also function as ratiometric, or “color-changing” probes. In this review, we describe the fundamentals of FRET and provide examples of QD-FRET sensors as grouped by their response mechanisms such as link cleavage and structural rearrangement. An overview of early works, recent advances, and various models of QD-FRET sensors for the measurement of pH and oxygen, as well as the presence of metal ions and proteins such as enzymes, are also provided.

  2. Signal/noise analysis of FRET-based sensors.

    Science.gov (United States)

    Woehler, Andrew; Wlodarczyk, Jakub; Neher, Erwin

    2010-10-06

    Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Single molecule studies of DNA packaging by bacteriophages

    Science.gov (United States)

    Fuller, Derek Nathan

    The DNA packaging dynamics of bacteriophages φ29, gamma, and T4 were studied at the single molecule level using a dual trap optical tweezers. Also, a method for producing long DNA molecules by PCR for optical tweezers studies of protein DNA interactions is presented and thoroughly characterized. This DNA preparation technique provided DNA samples for the φ29 and T4 studies. In the studies of φ29, the role of charge was investigated by varying the ionic conditions of the packaging buffer. Ionic conditions in which the DNA charge was highly screened due to divalent and trivalent cations showed the lowest resistance to packaging of the DNA to high density. This confirmed the importance of counterions in shielding the DNA interstrand repulsion when packaged to high density. While the ionic nature of the packaging buffer had a strong effect on packaging velocities, there was no clear trend between the counterion-screened charge of the DNA and the maximum packaging velocity. The packaging studies of lambda and T4 served as systems for comparative studies with φ29. Each system showed similarities to the φ29 system and unique differences. Both the lambda and T4 packaging motors were capable of generating forces in excess of 50 pN and showed remarkably high processivity, similar to φ29. However, dynamic structural transitions were observed with lambda that are not observed with φ29. The packaging of the lambda genome showed capsid expansion at approximately 30 percent of the genome packaged and capsid rupture at 90 percent of the genome packaged in the absence of capsid stabilizing protein gpD. Unique to the T4 packaging motor, packaging dynamics showed a remarkable amount of variability in velocities. This variability was seen both within individual packaging phages and from one phage to the next. This is possibly due to different conformational states of the packaging machinery. Additionally, lambda and T4 had average packaging velocities under minimal load of 600

  4. Calix[4]arene Based Single-Molecule Magnets

    Energy Technology Data Exchange (ETDEWEB)

    Karotsis, Georgios; Teat, Simon J.; Wernsdorfer, Wolfgang; Piligkos, Stergios; Dalgarno, Scott J.; Brechin, Euan K.

    2009-06-04

    Single-molecule magnets (SMMs) have been the subject of much interest in recent years because their molecular nature and inherent physical properties allow the crossover between classical and quantum physics to be observed. The macroscopic observation of quantum phenomena - tunneling between different spin states, quantum interference between tunnel paths - not only allows scientists to study quantum mechanical laws in great detail, but also provides model systems with which to investigate the possible implementation of spin-based solid state qubits and molecular spintronics. The isolation of small, simple SMMs is therefore an exciting prospect. To date almost all SMMs have been made via the self-assembly of 3d metal ions in the presence of bridging/chelating organic ligands. However, very recently an exciting new class of SMMs, based on 3d metal clusters (or single lanthanide ions) housed within polyoxometalates, has appeared. These types of molecule, in which the SMM is completely encapsulated within (or shrouded by) a 'protective' organic or inorganic sheath have much potential for design and manipulation: for example, for the removal of unwanted dipolar interactions, the introduction of redox activity, or to simply aid functionalization for surface grafting. Calix[4]arenes are cyclic (typically bowl-shaped) polyphenols that have been used extensively in the formation of versatile self-assembled supramolecular structures. Although many have been reported, p-{sup t}But-calix[4]arene and calix[4]arene (TBC4 and C4 respectively, Figure 1A) are frequently encountered due to (a) synthetic accessibility, and (b) vast potential for alteration at either the upper or lower rim of the macrocyclic framework. Within the field of supramolecular chemistry, TBC4 is well known for interesting polymorphic behavior and phase transformations within anti-parallel bi-layer arrays, while C4 often forms self-included trimers. The polyphenolic nature of calix[n]arenes (where

  5. Functionalization of gold and nanocrystalline diamond atomic force microscope tips for single molecule force spectroscopy

    Science.gov (United States)

    Drew, Michael E.

    The atomic force microscope (AFM) has fueled interest in nanotechnology because of its ability to image surfaces at the nanometer level and act as a molecular force sensor. Functionalization of the surface of an AFM tip surface in a stable, controlled manner expands the capabilities of the AFM and enables additional applications in the fields of single molecule force spectroscopy and nanolithography. Two AFM tip functionalizations are described: the assembly of tripodal molecular tips onto gold AFM tips and the photochemical attachment of terminal alkenes to nanocrystalline diamond (NCD) AFM tips. Two separate tripodal molecules with different linker lengths and a monopodal molecule terminated with biotin were synthesized to attach to a gold AFM tip for single molecule force spectroscopy. The immobilization of these molecules was examined by contact angle measurements, spectroscopic ellipsometry, infrared, and near edge x-ray absorption fine structure (NEXAFS) spectroscopy. All three molecules displayed rupture forces that agreed with previously reported values for the biotin--avidin rupture. The tripodal molecular tip displayed narrower distribution in their force histograms than the monopodal molecular tip. The performance of the tripodal molecular tip was compared to the monopodal molecular tip in single molecule force spectroscopy studies. Over repeated measurements, the distribution of forces for the monopodal molecular tip shifted to lower forces, whereas the distribution for the tripodal molecular tip remained constant throughout. Loading rate dependence and control experiments further indicated that the rupture forces of the tripod molecular tips were specific to the biotin--NeutrAvidin interaction. The second functionalization method used the photochemical attachment of undecylenic acid to NCD AFM tips. The photochemical attachment of undecylenic acid to hydrogen-terminated NCD wafer surfaces was investigated by contact angle measurements, x

  6. An integrated system for optical and electrical detection of single molecules/particles inside a solid-state nanopore.

    Science.gov (United States)

    Shi, Xin; Gao, Rui; Ying, Yi-Lun; Si, Wei; Chen, Yunfei; Long, Yi-Tao

    2015-01-01

    Nanopore techniques have proven to be useful tools for single-molecule detection. The combination of optical detection and ionic current measurements enables a new possibility for the parallel readout of multiple nanopores without complex nanofluidics and embedded electrodes. In this study, we developed a new integrated system for the label-free optical and electrical detection of single molecules based on a metal-coated nanopore. The entire system, containing a dark-field microscopy system and an ultralow current detection system with high temporal resolution, was designed and fabricated. An Au-coated nanopore was used to generate the optical signal. Light scattering from a single Au-coated nanopore was measured under a dark-field microscope. A lab-built ultralow current detection system was designed for the correlated optical and electrical readout. This integrated system might provide more direct and detailed information on single analytes inside the nanopore compared with classical ionic current measurements.

  7. Tetrairon(III) Single Molecule Magnet Studied by Scanning Tunneling Microscopy and Spectroscopy

    Science.gov (United States)

    Oh, Youngtek; Jeong, Hogyun; Lee, Minjun; Kwon, Jeonghoon; Yu, Jaejun; Mamun, Shariful Islam; Gupta, Gajendra; Kim, Jinkwon; Kuk, Young

    2011-03-01

    Tetrairon(III) single-molecule magnet (SMM) on a clean Au(111) has studied using scanning tunneling microscopy (STM) and spectroscopy (STS) to understand quantum mechanical tunneling of magnetization and hysteresis of pure molecular origin. Before the STM studies, elemental analysis, proton nuclear magnetic resonance (NMR) measurement and Energy Dispersive X- ray Spectroscopy (EDS) were carried out to check the robustness of the sample. The STM image of this molecule shows a hexagonal shape, with a phenyl ring at the center and surrounding six dipivaloylmethane ligands. Two peaks are observed at 0.5 eV, 1.5 eV in the STS results, agreeing well with the first principles calculations. Spin-polarized scanning tunneling microscopy (SPSTM) measurements have been performed with a magnetic tip to get the magnetization image of the SMM. We could observe the antiferromagnetic coupling and a centered- triangular topology with six alkoxo bridges inside the molecule while applying external magnetic fields.

  8. Spin quantum tunneling via entangled states in a dimer of exchange coupled single-molecule magnets

    Science.gov (United States)

    Tiron, R.; Wernsdorfer, W.; Aliaga-Alcalde, N.; Foguet-Albiol, D.; Christou, G.

    2004-03-01

    A new family of supramolecular, antiferromagnetically exchange-coupled dimers of single-molecule magnets (SMMs) has recently been reported [W. Wernsdorfer, N. Aliaga-Alcalde, D.N. Hendrickson, and G. Christou, Nature 416, 406 (2002)]. Each SMM acts as a bias on its neighbor, shifting the quantum tunneling resonances of the individual SMMs. Hysteresis loop measurements on a single crystal of SMM-dimers have now established quantum tunneling of the magnetization via entangled states of the dimer. This shows that the dimer really does behave as a quantum-mechanically coupled dimer. The transitions are well separated, suggesting long coherence times compared to the time scale of the energy splitting. This result is of great importance if such systems are to be used for quantum computing. It also allows the measurement of the longitudinal and transverse superexchange coupling constants [Phys. Rev. Lett. 91, 227203 (2003)].

  9. Topoisomerase I as a Biomarker: Detection of Activity at the Single Molecule Level

    DEFF Research Database (Denmark)

    Proszek, Joanna; Roy, Amit; Jakobsen, Ann-Katrine

    2014-01-01

    of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring......Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations...... in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement...

  10. Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

    Energy Technology Data Exchange (ETDEWEB)

    Dynan, William S.; Li, Shuyi; Mernaugh, Raymond; Wragg, Stephanie; Takeda, Yoshihiko

    2003-03-27

    Exposure to low doses of ionizing radiation is universal. The signature injury from ionizing radiation exposure is induction of DNA double-strand breaks (DSBs). The first line of defense against DSBs is direct ligation of broken DNA ends via the nonhomologous end-joining pathway. Because even a relatively high environmental exposure induces only a few DSBs per cell, our current understanding of the response to this exposure is limited by the ability to measure DSB repair events reliably in situ at a single-molecule level. To address this need, we have taken advantage of biological amplification, measuring relocalization of proteins and detection of protein phosphorylation as a surrogate for detection of broken ends themselves. We describe the use of specific antibodies to investigate the kinetics and mechanism of repair of very small numbers of DSBs in human cells by the nonhomologous end-joining pathway.

  11. Single Molecule Spectroelectrochemistry of Interfacial Charge Transfer Dynamics In Hybrid Organic Solar Cell

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Shanlin [Univ. of Alabama, Tuscaloosa, AL (United States)

    2014-11-16

    Our research under support of this DOE grant is focused on applied and fundamental aspects of model organic solar cell systems. Major accomplishments are: 1) we developed a spectroelectorchemistry technique of single molecule single nanoparticle method to study charge transfer between conjugated polymers and semiconductor at the single molecule level. The fluorescence of individual fluorescent polymers at semiconductor surfaces was shown to exhibit blinking behavior compared to molecules on glass substrates. Single molecule fluorescence excitation anisotropy measurements showed the conformation of the polymer molecules did not differ appreciably between glass and semiconductor substrates. The similarities in molecular conformation suggest that the observed differences in blinking activity are due to charge transfer between fluorescent polymer and semiconductor, which provides additional pathways between states of high and low fluorescence quantum efficiency. Similar spectroelectrochemistry work has been done for small organic dyes for understand their charge transfer dynamics on various substrates and electrochemical environments; 2) We developed a method of transferring semiconductor nanoparticles (NPs) and graphene oxide (GO) nanosheets into organic solvent for a potential electron acceptor in bulk heterojunction organic solar cells which employed polymer semiconductor as the electron donor. Electron transfer from the polymer semiconductor to semiconductor and GO in solutions and thin films was established through fluorescence spectroscopy and electroluminescence measurements. Solar cells containing these materials were constructed and evaluated using transient absorption spectroscopy and dynamic fluorescence techniques to understand the charge carrier generation and recombination events; 3) We invented a spectroelectorchemistry technique using light scattering and electroluminescence for rapid size determination and studying electrochemistry of single NPs in an

  12. Engineering Genetically Encoded FRET Sensors

    Science.gov (United States)

    Lindenburg, Laurens; Merkx, Maarten

    2014-01-01

    Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940

  13. Injection molded nanofluidic chips: Fabrication method and functional tests using single-molecule DNA experiments

    DEFF Research Database (Denmark)

    Utko, Pawel; Persson, Karl Fredrik; Kristensen, Anders

    2011-01-01

    We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels.......We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels....

  14. Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

    NARCIS (Netherlands)

    Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; van der Zant, Herre S. J.

    It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the

  15. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Energy Technology Data Exchange (ETDEWEB)

    Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia; Hoffbauer, Mark A.

    2017-09-12

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  16. Single-molecule analysis of DNA replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Yardimci, Hasan; Loveland, Anna B.; van Oijen, Antoine M.; Walter, Johannes C.; Mechali, Marcel

    The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the

  17. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Science.gov (United States)

    Sansinena, Jose-Maria [Los Alamos, NM; Redondo, Antonio [Los Alamos, NM; Olazabal, Virginia [Los Alamos, NM; Hoffbauer, Mark A [Los Alamos, NM; Akhadov, Elshan A [Los Alamos, NM

    2009-12-29

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  18. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Energy Technology Data Exchange (ETDEWEB)

    Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia; Hoffbauer, Mark A.; Akhadov, Elshan A.

    2017-10-31

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  19. Rational design of single-molecule magnets: a supramolecular approach.

    Science.gov (United States)

    Glaser, Thorsten

    2011-01-07

    Since the discovery that Mn(12)OAc acts as a single-molecule magnet (SMM), an increasing number of transition metal complexes have been demonstrated to behave as SMMs. The signature of a SMM is a slow relaxation of the magnetization at low temperatures accompanied by a magnetic hysteresis. The origin of SMM behaviour is the existence of an appreciable thermal barrier U for spin-reversal called magnetic anisotropy barrier which is related to the combination of a large total spin ground state (S(t)) and an easy-axis magnetic anisotropy. The extensive research on Mn(12)OAc and other SMMs has established more prerequisites for a rational development of new SMMs besides the high-spin ground state and the magnetic anisotropy: the symmetry should be at least C(3) to minimize the quantum tunneling of the magnetization through the anisotropy barrier but lower than cubic to avoid the cancellation of the local anisotropies upon projection onto the spin ground state. Based on these prerequisites, we have designed the ligand triplesalen which combines the phloroglucinol bridging unit for high spin ground states by the spin-polarization mechanism with a salen-like ligand environment for single-site magnetic anisotropies by a strong tetragonal ligand field. The C(3) symmetric, trinuclear complexes of the triplesalen ligand (talen(t-Bu(2)))(6-) exhibit a strong ligand folding resulting in an overall bowl-shaped molecular structure. This ligand folding preorganizes the axial coordination sites of the metal salen subunits for the complementary binding of three facial nitrogen atoms of a hexacyanometallate unit. This leads to a high driving force for the formation of heptanuclear complexes [M(t)(6)M(c)](n+) by the assembly of three molecular building blocks. Attractive van der Waals interactions of the tert-butyl phenyl units of two triplesalen trinuclear building blocks increase the driving force. In this respect, we have been able to synthesize the isostructural series [Mn(III)(6

  20. Monitoring and Manipulating Motions of Single Molecules/Nanoparticles

    Science.gov (United States)

    Chen, Fang

    -nanometer pores in theory. We then experimentally studied nanoparticles diffusing on membrane filters containing 200 nm polyethyleneglycol- or C18-modified pores. Using STED microscopy, we resolved for the first time how small particles are retained by the pores. Trapping by the pore entrances rather than adsorption is responsible for the retention. Further studies on C18-modified pores showed consistency in Gibbs free energy about the retention process. In addition, in order to understand how nanoparticles interact with the surface when they are forced to be on, or very close to, the surface, we studied nanosecond rotation dynamics of gold nanorods with one end attached on the surface. We found that the nanorod motion is dominated by van der Waals interaction-induced immobilization rather Brownian rotational diffusion as previously thought. The actual rotation, during which the nanorod transits from one immobilized state to the other, slows down by 50 times. The second part of the research is the collaboration with Tour's group in Rice University. The ultimate goal is to use light to drive a motorized nanocar at ambient conditions. To fulfill this goal, we first studied the moving kinetics of adamantane-wheeled nanocars on hydroxylated and PEG-modified surfaces using single molecule fluorescence microscopy. We found that nanocars' diffusion slows down on solid surface over time, which is possibly caused by the increased hydrophobicity of the substrate surface due to the adsorbates from the air. A sticky-spots model was proposed to explain the observed slowing down. To find out whether a light-activatable motor works when it is incorporated into a nanocar, we carefully designed a series of molecules containing a regular motor, a slow motor, a nonunidirectional motor, and no motor. We found that a fast unidirectional rotating motor enhanced the diffusion of the molecule in solution upon UV-illumination. Detailed analysis suggested that the unimolecular submersible nanomachine (USN

  1. Experimental and computational characterization of biological liquid crystals: a review of single-molecule bioassays.

    Science.gov (United States)

    Eom, Kilho; Yang, Jaemoon; Park, Jinsung; Yoon, Gwonchan; Soo Sohn, Young; Park, Shinsuk; Yoon, Dae Sung; Na, Sungsoo; Kwon, Taeyun

    2009-09-10

    Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM) have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.

  2. Frequency-domain terahertz transmission spectra of Mn3 and Mn12 single-molecule magnets

    Science.gov (United States)

    Liu, RuiYuan; Zuo, JunWei; Li, YanRong; Zhou, YuRong; Wang, YunPing

    2012-07-01

    Frequency-domain terahertz transmission spectra of Mn3 and Mn12 single molecule magnets (SMMs) have been measured at different temperatures, and hence the anisotropic parameters D 2 and D 4 of the spin Hamiltonian hat H = D_2 hat S_z^2 + D_4 hat S_z^4 have been calculated. For Mn12 SMM, D 2=-10.9 GHz and D 4=-2.59×10-2 GHz, while for Mn3 SMM, D 2=-22.0 GHz and D 4 can be considered negligible. This suggests Mn3 SMM can be considered as a simpler and more suitable candidate for magnetic quantum tunneling research.

  3. A binuclear Fe(III)Dy(III) single molecule magnet. Quantum effects and models.

    Science.gov (United States)

    Ferbinteanu, Marilena; Kajiwara, Takashi; Choi, Kwang-Yong; Nojiri, Hiroyuki; Nakamoto, Akio; Kojima, Norimichi; Cimpoesu, Fanica; Fujimura, Yuichi; Takaishi, Shinya; Yamashita, Masahiro

    2006-07-19

    The binuclear [FeIII(bpca)(mu-bpca)Dy(NO3)4], having Single Molecule Magnet (SMM) properties, belonging to a series of isostructural FeIIILnIII complexes (Ln = Eu, Gd, Tb, Dy, Ho) and closely related FeIILnIII chain structures, was characterized in concise experimental and theoretical respects. The low temperature magnetization data showed hysteresis and tunneling. The anomalous temperature dependence of Mössbauer spectra is related to the onset of magnetic order, consistent with the magnetization relaxation time scale resulting from AC susceptibility measurements. The advanced ab initio calculations (CASSCF and spin-orbit) revealed the interplay of ligand field, spin-orbit, and exchange effects and probed the effective Ising nature of the lowest states, involved in the SMM and tunneling effects.

  4. Exploration of the Berry phase interference in a single-molecule magnets of trigonal symmetry

    Science.gov (United States)

    Quddusi, H. M.; Liu, J.; Feng, P. L.; Del Barco, E.; Hill, S.; Hendrickson, D. N.

    2012-02-01

    The quantum behavior of single-molecule magnets (SMM) is mainly governed by their molecular composition and crystallographic symmetries, thus playing an essential role in the tunneling dynamics. We present low temperature magnetometry measurements on a trigonal symmetric, low nuclearity Mn3 SMM. The experiments are designed to explore the behavior of the tunnel splittings within the transverse field magnitude/direction phase space, by applying a transverse field (0-1 T) along different directions within the hard anisotropy plane of the molecules. The expected quantum interference pattern can be understood as an outcome of a competition between different intramolecular magnetic interactions. A multi-spin description using non-collinear zero-field splitting tensors and intra molecular dipolar interactions between the manganese ions is employed to explain the symmetry patterns.

  5. Detection of single molecules with a scanning near-field optical microscope: Absorption and luminescence

    International Nuclear Information System (INIS)

    Plakhotnik, T.V.

    1995-01-01

    An analysis is given of the absorption and emission of single molecules located in the waist of a Gaussian beam with diameter smaller than the wavelength of light or near a conducting flat screen with a round aperture. The intensity of light in the far field was determined from the self-consistent solution of Maxwell's equations with appropriate boundary conditions. It is shown that the intensity, shape, and position of absorption and luminescence spectral lines measured in a broad beam significantly change if a molecule is located near the waist or the aperture. If their diameter is far smaller than the wavelength of light, then the presence of a molecule that is resonant with the exciting field results in an increase in the intensity of light in the far field, i.e., in negative absorption. 19 refs., 3 figs

  6. Surface single-molecule dynamics controlled by entropy at low temperatures

    Science.gov (United States)

    Gehrig, J. C.; Penedo, M.; Parschau, M.; Schwenk, J.; Marioni, M. A.; Hudson, E. W.; Hug, H. J.

    2017-02-01

    Configuration transitions of individual molecules and atoms on surfaces are traditionally described using an Arrhenius equation with energy barrier and pre-exponential factor (attempt rate) parameters. Characteristic parameters can vary even for identical systems, and pre-exponential factors sometimes differ by orders of magnitude. Using low-temperature scanning tunnelling microscopy (STM) to measure an individual dibutyl sulfide molecule on Au(111), we show that the differences arise when the relative position of tip apex and molecule changes by a fraction of the molecule size. Altering the tip position on that scale modifies the transition's barrier and attempt rate in a highly correlated fashion, which results in a single-molecular enthalpy-entropy compensation. Conversely, appropriately positioning the STM tip allows selecting the operating point on the compensation line and modifying the transition rates. The results highlight the need to consider entropy in transition rates of single molecules, even at low temperatures.

  7. A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy.

    Science.gov (United States)

    Li, Hao; Yang, Haw

    2018-03-28

    This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

  8. Determining molecular orientation via single molecule SERS in a plasmonic nano-gap.

    Science.gov (United States)

    Marshall, Addison R L; Stokes, Jamie; Viscomi, Francesco N; Proctor, John E; Gierschner, Johannes; Bouillard, Jean-Sebastien G; Adawi, Ali M

    2017-11-16

    In this work, plasmonic nano-gaps consisting of a silver nanoparticle coupled to an extended silver film have been fully optimized for single molecule Surface-Enhanced Raman Scattering (SERS) spectroscopy. The SERS signal was found to be strongly dependent on the particle size and the molecule orientation with respect to the field inside the nano-gap. Using Finite Difference Time Domain (FDTD) simulations to complement the experimental measurements, the complex interplay between the excitation enhancement and the emission enhancement of the system as a function of particle size were highlighted. Additionally, in conjunction with Density Functional Theory (DFT), the well-defined field direction in the nano-gap enables to recover the orientation of individual molecules.

  9. A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy

    Science.gov (United States)

    Li, Hao; Yang, Haw

    2018-03-01

    This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

  10. Variable contact gap single-molecule conductance determination for a series of conjugated molecular bridges

    DEFF Research Database (Denmark)

    Haiss, W.; Wang, Christian; Jitchati, R.

    2008-01-01

    It is now becoming clear that the characteristics of the whole junction are important in determining the conductance of single molecules bound between two metal contacts. This paper shows through measurements on a series of seven conjugated molecular bridges that contact separation is an important...... that conductance increases rather dramatically at higher tilt angle away from the normal for conformationally rigid molecular wires and that this increase in conductance arises from increased electronic coupling between the molecular bridge and the gold contacts.......-distance curves and knowledge of the terminal to terminal length of the molecular wire. The contact gap separation dependence is interpreted as arising from tilting of these molecules in the junction and this model is underpinned by ab initio transport computations. In this respect we make the general observation...

  11. In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

    International Nuclear Information System (INIS)

    Hsu, Y.-Y.; Liu, Y.-N.; Wang Wenyen; Kao, Fu-Jen; Kung, S.-H.

    2007-01-01

    An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP 2 )-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A pro ) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A pro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research

  12. Generic Schemes for Single-Molecule Kinetics. 3: Self-Consistent Pathway Solutions for Nonrenewal Processes.

    Science.gov (United States)

    Piephoff, D Evan; Cao, Jianshu

    2018-04-23

    We recently developed a pathway analysis framework (paper 1) for describing single-molecule kinetics for renewal (i.e., memoryless) processes based on the decomposition of a kinetic scheme into generic structures. In our approach, waiting time distribution functions corresponding to such structures are expressed in terms of self-consistent pathway solutions and concatenated to form measurable probability distribution functions (PDFs), affording a simple way to decompose and recombine a network. Here, we extend this framework to nonrenewal processes, which involve correlations between events, and employ it to formulate waiting time PDFs, including the first-passage time PDF, for a general kinetic network model. Our technique does not require the assumption of Poissonian kinetics, permitting a more general kinetic description than the usual rate approach, with minimal topological restrictiveness. To demonstrate the usefulness of this technique, we provide explicit calculations for our general model, which we adapt to two generic schemes for single-enzyme turnover with conformational interconversion. For each generic scheme, wherein the intermediate state(s) need not undergo Poissonian decay, the functional dependence of the mean first-passage time on the concentration of an external substrate is analyzed. When conformational detailed balance is satisfied, the enzyme turnover rate (related to the mean first-passage time) reduces to the celebrated Michaelis-Menten functional form, consistent with our previous work involving a similar scheme with all rate processes, thereby establishing further generality to this intriguing result. Our framework affords a general and intuitive approach for evaluating measurable waiting time PDFs and their moments, making it a potentially useful kinetic tool for a wide variety of single-molecule processes.

  13. A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

    Science.gov (United States)

    Jo, Kyubong; Schramm, Timothy M.; Schwartz, David C.

    Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels

  14. Possibility of 1-nm level localization of a single molecule with gap-mode surface-enhanced Raman scattering

    International Nuclear Information System (INIS)

    Choi, Han Kyu; Kim, Zee Hwan

    2015-01-01

    The electromagnetic (EM) enhancement mechanism of surface-enhanced Raman scattering (SERS) has been well established through 30 years of extensive investigation: molecules adsorbed on resonantly driven silver or gold nanoparticles (NPs) experience strongly enhanced field and thus show enhanced Raman scattering. Even stronger SERS enhancement is possible with a gap structure in which two or more NPs form assemblies with gap sizes of 1 nm or less. We have theoretically shown that the measurement of SERS angular distribution can reveal the position of a single molecule near the gap with 1-nm accuracy, even though the spatial extent of the enhanced field is ~10 nm. Real implementation of such experiment requires extremely well-defined (preferably a single crystal) dimeric junctions. Nevertheless, the experiment will provide spatial as well as frequency domain information on single-molecule dynamics at metallic surfaces

  15. Precision and accuracy in smFRET based structural studies—A benchmark study of the Fast-Nano-Positioning System

    Science.gov (United States)

    Nagy, Julia; Eilert, Tobias; Michaelis, Jens

    2018-03-01

    Modern hybrid structural analysis methods have opened new possibilities to analyze and resolve flexible protein complexes where conventional crystallographic methods have reached their limits. Here, the Fast-Nano-Positioning System (Fast-NPS), a Bayesian parameter estimation-based analysis method and software, is an interesting method since it allows for the localization of unknown fluorescent dye molecules attached to macromolecular complexes based on single-molecule Förster resonance energy transfer (smFRET) measurements. However, the precision, accuracy, and reliability of structural models derived from results based on such complex calculation schemes are oftentimes difficult to evaluate. Therefore, we present two proof-of-principle benchmark studies where we use smFRET data to localize supposedly unknown positions on a DNA as well as on a protein-nucleic acid complex. Since we use complexes where structural information is available, we can compare Fast-NPS localization to the existing structural data. In particular, we compare different dye models and discuss how both accuracy and precision can be optimized.

  16. Vesicle Encapsulation Studies Reveal that Single Molecule Ribozyme Heterogeneities Are Intrinsic

    Science.gov (United States)

    Okumus, Burak; Wilson, Timothy J.; Lilley, David M. J.; Ha, Taekjip

    2004-01-01

    Single-molecule measurements have revealed that what were assumed to be identical molecules can differ significantly in their static and dynamic properties. One of the most striking examples is the hairpin ribozyme, which was shown to exhibit two to three orders of magnitude variation in folding kinetics between molecules. Although averaged behavior of single molecules matched the bulk solution data, it was not possible to exclude rigorously the possibility that the variations around the mean values arose from different ways of interacting with the surface environment. To test this, we minimized the molecules' interaction with the surface by encapsulating DNA or RNA molecules inside 100- to 200-nm diameter unilamellar vesicles, following the procedures described by Haran and coworkers. Vesicles were immobilized on a supported lipid bilayer via biotin-streptavidin linkages. We observed no direct binding of DNA or RNA on the supported bilayer even at concentrations exceeding 100 nM, indicating that these molecules do not bind stably on the membrane. Since the vesicle diameter is smaller than the resolution of optical microscopy, the lateral mobility of the molecules is severely constrained, allowing long observation periods. We used fluorescence correlation spectroscopy, nuclease digestion, and external buffer exchange to show that the molecules were indeed encapsulated within the vesicles. When contained within vesicles, the natural form of the hairpin ribozyme exhibited 50-fold variation in both folding and unfolding rates in 0.5 mM Mg2+, which is identical to what was observed from the molecules tethered directly on the surface. This strongly indicates that the observed heterogeneity in dynamic properties does not arise as an artifact of surface attachment, but is intrinsic to the nature of the molecules. PMID:15454471

  17. Roughness Influence on Initiation of Fretting Fatigue Scar of Ti-6Al-4V Alloy

    Science.gov (United States)

    Capitanu, L.; Badita, L. L.; Florescu, V.; Tiganesteanu, C.

    2018-01-01

    This paper reports on the experimental studies undertaken to detect the early stage when appears the fretting wear of the Ti-6Al-4V alloy used for the hip prostheses. Wear is a critical aspect for estimating the fretting fatigue. Studies were performed on samples of special shape, in order to be able to study the influence of in contact surfaces roughness on the durability to fretting. Fretting buffers, with roughnesses Ra of the contact surface of 0.015 and 0.045 μm, and Ti-6Al-4V samples with roughnesses Ra = 0.045 μm, Ra = 0.075 μm and Ra = 0.19 μm, were used. Testing periods of 3 seconds, 1 minute and 5 minutes were selected to capture the moment of the fretting scar appearance, long before these initiate the eventual fretting cracking. Simultaneously with fretting wear of the surface, the friction coefficient was also measured. From the in time evolution determinations of the fretting wear, it resulted that, under the experimental conditions used, the minimum wear occurs at a certain value of the roughness and not at the minimum roughness. Surprisingly, the minimum friction coefficient does not coincide with the minimum fretting wear.

  18. Single-molecule fluorescence resonance energy transfer shows uniformity in TATA binding protein-induced DNA bending and heterogeneity in bending kinetics.

    Science.gov (United States)

    Blair, Rebecca H; Goodrich, James A; Kugel, Jennifer F

    2012-09-25

    TATA binding protein (TBP) is a key component of the eukaryotic RNA polymerase II transcription machinery that binds to TATA boxes located in the core promoter regions of many genes. Structural and biochemical studies have shown that when TBP binds DNA, it sharply bends the DNA. We used single-molecule fluorescence resonance energy transfer (smFRET) to study DNA bending by human TBP on consensus and mutant TATA boxes in the absence and presence of TFIIA. We found that the state of the bent DNA within populations of TBP-DNA complexes is homogeneous; partially bent intermediates were not observed. In contrast to the results of previous ensemble studies, TBP was found to bend a mutant TATA box to the same extent as the consensus TATA box. Moreover, in the presence of TFIIA, the extent of DNA bending was not significantly changed, although TFIIA did increase the fraction of DNA molecules bound by TBP. Analysis of the kinetics of DNA bending and unbending revealed that on the consensus TATA box two kinetically distinct populations of TBP-DNA complexes exist; however, the bent state of the DNA is the same in the two populations. Our smFRET studies reveal that human TBP bends DNA in a largely uniform manner under a variety of different conditions, which was unexpected given previous ensemble biochemical studies. Our new observations led to us to revise the model for the mechanism of DNA binding by TBP and for how DNA bending is affected by TATA sequence and TFIIA.

  19. Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Chi, Qijin; Albrecht, Tim

    2005-01-01

    Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class...... tunnelling spectroscopic (STS) features. Mapping of redox metalloproteins from the three major classes, i.e. blue copper proteins, heme proteins, and iron-sulfur proteins, at the monolayer and single-molecule levels have also been achieved. In situ STM and spectroscopy of redox molecules and biomolecules...

  20. Central dogma at the single-molecule level in living cells.

    Science.gov (United States)

    Li, Gene-Wei; Xie, X Sunney

    2011-07-20

    Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

  1. Smart SERS Hot Spots: Single Molecules Can Be Positioned in a Plasmonic Nanojunction Using Host-Guest Chemistry.

    Science.gov (United States)

    Kim, Nam Hoon; Hwang, Wooseup; Baek, Kangkyun; Rohman, Md Rumum; Kim, Jeehong; Kim, Hyun Woo; Mun, Jungho; Lee, So Young; Yun, Gyeongwon; Murray, James; Ha, Ji Won; Rho, Junsuk; Moskovits, Martin; Kim, Kimoon

    2018-03-01

    Single-molecule surface-enhanced Raman spectroscopy (SERS) offers new opportunities for exploring the complex chemical and biological processes that cannot be easily probed using ensemble techniques. However, the ability to place the single molecule of interest reliably within a hot spot, to enable its analysis at the single-molecule level, remains challenging. Here we describe a novel strategy for locating and securing a single target analyte in a SERS hot spot at a plasmonic nanojunction. The "smart" hot spot was generated by employing a thiol-functionalized cucurbit[6]uril (CB[6]) as a molecular spacer linking a silver nanoparticle to a metal substrate. This approach also permits one to study molecules chemically reluctant to enter the hot spot, by conjugating them to a moiety, such as spermine, that has a high affinity for CB[6]. The hot spot can accommodate at most a few, and often only a single, analyte molecule. Bianalyte experiments revealed that one can reproducibly treat the SERS substrate such that 96% of the hot spots contain a single analyte molecule. Furthermore, by utilizing a series of molecules each consisting of spermine bound to perylene bisimide, a bright SERS molecule, with polymethylene linkers of varying lengths, the SERS intensity as a function of distance from the center of the hot spot could be measured. The SERS enhancement was found to decrease as 1 over the square of the distance from the center of the hot spot, and the single-molecule SERS cross sections were found to increase with AgNP diameter.

  2. Yoctoliter thermometry for single-molecule investigations: a generic bead-on-a-tip temperature-control module.

    Science.gov (United States)

    Koirala, Deepak; Punnoose, Jibin Abraham; Shrestha, Prakash; Mao, Hanbin

    2014-03-24

    A new temperature-jump (T-jump) strategy avoids photo-damage of individual molecules by focusing a low-intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T-jump of 65 °C within milliseconds. To measure the temperature in situ in single-molecule experiments, the temperature-dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead-on-a-tip module and the robust single-molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T-jump and single-molecule techniques. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase.

    Science.gov (United States)

    Volkán-Kacsó, Sándor; Marcus, Rudolph A

    2016-10-25

    A recently proposed chemomechanical group transfer theory of rotary biomolecular motors is applied to treat single-molecule controlled rotation experiments. In these experiments, single-molecule fluorescence is used to measure the binding and release rate constants of nucleotides by monitoring the occupancy of binding sites. It is shown how missed events of nucleotide binding and release in these experiments can be corrected using theory, with F 1 -ATP synthase as an example. The missed events are significant when the reverse rate is very fast. Using the theory the actual rate constants in the controlled rotation experiments and the corrections are predicted from independent data, including other single-molecule rotation and ensemble biochemical experiments. The effective torsional elastic constant is found to depend on the binding/releasing nucleotide, and it is smaller for ADP than for ATP. There is a good agreement, with no adjustable parameters, between the theoretical and experimental results of controlled rotation experiments and stalling experiments, for the range of angles where the data overlap. This agreement is perhaps all the more surprising because it occurs even though the binding and release of fluorescent nucleotides is monitored at single-site occupancy concentrations, whereas the stalling and free rotation experiments have multiple-site occupancy.

  4. Simulation of FRET dyes allows quantitative comparison against experimental data

    Science.gov (United States)

    Reinartz, Ines; Sinner, Claude; Nettels, Daniel; Stucki-Buchli, Brigitte; Stockmar, Florian; Panek, Pawel T.; Jacob, Christoph R.; Nienhaus, Gerd Ulrich; Schuler, Benjamin; Schug, Alexander

    2018-03-01

    Fully understanding biomolecular function requires detailed insight into the systems' structural dynamics. Powerful experimental techniques such as single molecule Förster Resonance Energy Transfer (FRET) provide access to such dynamic information yet have to be carefully interpreted. Molecular simulations can complement these experiments but typically face limits in accessing slow time scales and large or unstructured systems. Here, we introduce a coarse-grained simulation technique that tackles these challenges. While requiring only few parameters, we maintain full protein flexibility and include all heavy atoms of proteins, linkers, and dyes. We are able to sufficiently reduce computational demands to simulate large or heterogeneous structural dynamics and ensembles on slow time scales found in, e.g., protein folding. The simulations allow for calculating FRET efficiencies which quantitatively agree with experimentally determined values. By providing atomically resolved trajectories, this work supports the planning and microscopic interpretation of experiments. Overall, these results highlight how simulations and experiments can complement each other leading to new insights into biomolecular dynamics and function.

  5. Fretting-wear damage: a summary of recent findings

    Energy Technology Data Exchange (ETDEWEB)

    Guerout, F.M.; Fisher, N.J. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    1998-07-01

    Flow-induced vibration of steam generator (SG) tubes may sometimes result in fretting-wear damage at the tube-to-support locations. Fretting-wear damage predictions are largely experimental data obtained at representative test conditions. Fretting-wear of steam generator materials has been studied at the Chalk River Laboratories for two decades. Tests are conducted in fretting-wear test machines that simulate steam generator environmental conditions and tube-to-support dynamic interactions. A new high-temperature force and displacement measuring system was developed to monitor tube-to-support interaction (i.e., work-rate) at operating conditions. This improvement in experimental fretting-wear technology was used to perform a comprehensive study of the effect of various environment and design parameters on steam generator tube wear damage. This paper summarizes the results of tests performed over the past four years to study the effect of temperature, water chemistry, support geometry, and tube material on fretting-wear. The results show a significant effect of temperature on tube wear damage. Therefore fretting-wear tests must be performed at operating temperatures in order to be relevant. No significant effect of the type of water treatment on tube wear damage was observed. For predominantly impacting motion, the wear of steam generator tubes in contact with 410 stainless steel is similar regardless of whether Alloy 690 or Alloy 800 is used as tubing material or whether lattice bars or broached hole supports are used. Based on results presented in this paper, an average wear coefficient value is recommended that is used for the prediction of SG tube wear depth versus time. (author)

  6. Fretting-wear damage: a summary of recent findings

    International Nuclear Information System (INIS)

    Guerout, F.M.; Fisher, N.J.

    1998-01-01

    Flow-induced vibration of steam generator (SG) tubes may sometimes result in fretting-wear damage at the tube-to-support locations. Fretting-wear damage predictions are largely experimental data obtained at representative test conditions. Fretting-wear of steam generator materials has been studied at the Chalk River Laboratories for two decades. Tests are conducted in fretting-wear test machines that simulate steam generator environmental conditions and tube-to-support dynamic interactions. A new high-temperature force and displacement measuring system was developed to monitor tube-to-support interaction (i.e., work-rate) at operating conditions. This improvement in experimental fretting-wear technology was used to perform a comprehensive study of the effect of various environment and design parameters on steam generator tube wear damage. This paper summarizes the results of tests performed over the past four years to study the effect of temperature, water chemistry, support geometry, and tube material on fretting-wear. The results show a significant effect of temperature on tube wear damage. Therefore fretting-wear tests must be performed at operating temperatures in order to be relevant. No significant effect of the type of water treatment on tube wear damage was observed. For predominantly impacting motion, the wear of steam generator tubes in contact with 410 stainless steel is similar regardless of whether Alloy 690 or Alloy 800 is used as tubing material or whether lattice bars or broached hole supports are used. Based on results presented in this paper, an average wear coefficient value is recommended that is used for the prediction of SG tube wear depth versus time. (author)

  7. Fretting fatigue in AISI 1015 steel

    Indian Academy of Sciences (India)

    A strength reduction factor of about 1.30 was obtained due to fretting for the test material under the present experimental conditions. Influence of contact load on fretting was also studied. Increasing fretting contact load decreased the fatigue life in the range investigated. Failure analysis showed typical stage I oblique crack ...

  8. Ordered array of CoPc-vacancies filled with single-molecule rotors

    Science.gov (United States)

    Xie, Zheng-Bo; Wang, Ya-Li; Tao, Min-Long; Sun, Kai; Tu, Yu-Bing; Yuan, Hong-Kuan; Wang, Jun-Zhong

    2018-05-01

    We report the highly ordered array of CoPc-vacancies and the single-molecule rotors inside the vacancies. When CoPc molecules are deposited on Cd(0001) at low-temperature, three types of molecular vacancies appeared randomly in the CoPc monolayer. Annealing the sample to higher temperature leads to the spontaneous phase separation and self-organized arrangement of the vacancies. Highly ordered arrays of two-molecule vacancies and single-molecule vacancies have been obtained. In particular, there is a rotating CoPc molecule inside each single-molecule vacancy, which constitutes the array of single-molecule rotors. These results provide a new routine to fabricate the nano-machines on a large scale.

  9. Single vesicle biochips for ultra-miniaturized nanoscale fluidics and single molecule bioscience

    DEFF Research Database (Denmark)

    Christensen, Andreas Lauge; Lohr, Christina; Christensen, Sune M.

    2013-01-01

    , their fabrication via controlled self-assembly, and their characterization using fluorescence microscopy. We also highlight their applications in selected fields such as nanofluidics and single molecule bioscience. Despite their great potential for improved biocompatibility, extreme miniaturization and high...

  10. Targeting neurotransmitter receptors with nanoparticles in vivo allows single-molecule tracking in acute brain slices

    Science.gov (United States)

    Varela, Juan A.; Dupuis, Julien P.; Etchepare, Laetitia; Espana, Agnès; Cognet, Laurent; Groc, Laurent

    2016-03-01

    Single-molecule imaging has changed the way we understand many biological mechanisms, particularly in neurobiology, by shedding light on intricate molecular events down to the nanoscale. However, current single-molecule studies in neuroscience have been limited to cultured neurons or organotypic slices, leaving as an open question the existence of fast receptor diffusion in intact brain tissue. Here, for the first time, we targeted dopamine receptors in vivo with functionalized quantum dots and were able to perform single-molecule tracking in acute rat brain slices. We propose a novel delocalized and non-inflammatory way of delivering nanoparticles (NPs) in vivo to the brain, which allowed us to label and track genetically engineered surface dopamine receptors in neocortical neurons, revealing inherent behaviour and receptor activity regulations. We thus propose a NP-based platform for single-molecule studies in the living brain, opening new avenues of research in physiological and pathological animal models.

  11. Stereoelectronic Effect-Induced Conductance Switching in Aromatic Chain Single-Molecule Junctions.

    Science.gov (United States)

    Xin, Na; Wang, Jinying; Jia, Chuancheng; Liu, Zitong; Zhang, Xisha; Yu, Chenmin; Li, Mingliang; Wang, Shuopei; Gong, Yao; Sun, Hantao; Zhang, Guanxin; Liu, Zhirong; Zhang, Guangyu; Liao, Jianhui; Zhang, Deqing; Guo, Xuefeng

    2017-02-08

    Biphenyl, as the elementary unit of organic functional materials, has been widely used in electronic and optoelectronic devices. However, over decades little has been fundamentally understood regarding how the intramolecular conformation of biphenyl dynamically affects its transport properties at the single-molecule level. Here, we establish the stereoelectronic effect of biphenyl on its electrical conductance based on the platform of graphene-molecule single-molecule junctions, where a specifically designed hexaphenyl aromatic chain molecule is covalently sandwiched between nanogapped graphene point contacts to create stable single-molecule junctions. Both theoretical and temperature-dependent experimental results consistently demonstrate that phenyl twisting in the aromatic chain molecule produces different microstates with different degrees of conjugation, thus leading to stochastic switching between high- and low-conductance states. These investigations offer new molecular design insights into building functional single-molecule electrical devices.

  12. Electrochemical Control of Single-Molecule Conductance by Fermi- Level Tuning and Conjugation Switching

    DEFF Research Database (Denmark)

    Baghernejad, Masoud; Zhao, Xiaotao; Ørnsø, Kristian Baruël

    2014-01-01

    Controlling charge transport through a single molecule connected to metallic electrodes remains one of the most fundamental challenges of nanoelectronics. Here we use electrochemical gating to reversibly tune the conductance of two different organic molecules, both containing anthraquinone (AQ...

  13. Break junction under electrochemical gating: testbed for single-molecule electronics.

    Science.gov (United States)

    Huang, Cancan; Rudnev, Alexander V; Hong, Wenjing; Wandlowski, Thomas

    2015-02-21

    Molecular electronics aims to construct functional molecular devices at the single-molecule scale. One of the major challenges is to construct a single-molecule junction and to further manipulate the charge transport through the molecular junction. Break junction techniques, including STM break junctions and mechanically controllable break junctions are considered as testbed to investigate and control the charge transport on a single-molecule scale. Moreover, additional electrochemical gating provides a unique opportunity to manipulate the energy alignment and molecular redox processes for a single-molecule junction. In this review, we start from the technical aspects of the break junction technique, then discuss the molecular structure-conductance correlation derived from break junction studies, and, finally, emphasize electrochemical gating as a promising method for the functional molecular devices.

  14. Single Molecule Arrays for ultra-sensitive detection of rat cytokines in serum.

    Science.gov (United States)

    Cohen, Limor; Xie, Liangxia; Xylas, Mark E; Walt, David R

    2018-01-01

    Rats are used as animal models for many human diseases. Cytokines can serve as biomarkers indicative of these diseases or disease states. Techniques for measuring cytokine expression levels often do not provide the sensitivity needed to measure these biomarkers in biological fluids because the concentrations of many cytokines are below the detection limits of conventional methods. In this paper, we present ultra-sensitive digital immunoassays using Single Molecule Arrays (Simoa) for seven rat cytokines: TNF-α, IL-10, IL-17F, GM-CSF, IFN-γ, IL-4, and IL-1α. These ultra-sensitive immunoassays have limits of detection (LODs) in the femtomolar range and provide the ability to measure rat cytokines in serum below the LODs of conventional immunoassays. We also measured these cytokines in healthy rat serum to obtain baseline levels. The ability to measure cytokines present at low concentrations in rat serum will facilitate future studies of disease using rats as animal models. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    Science.gov (United States)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  16. Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

    International Nuclear Information System (INIS)

    Zhang Jingdong; Chi Qijin; Albrecht, Tim; Kuznetsov, Alexander M.; Grubb, Mikala; Hansen, Allan G.; Wackerbarth, Hainer; Welinder, Anne C.; Ulstrup, Jens

    2005-01-01

    Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class of metallic and semiconductor-based nanoparticles, nano-arrays, nanotubes, and nanopits. Others are based on self-assembled molecular monolayers. The latter extend to bioelectrochemical systems with redox metalloproteins and DNA-based molecules as targets. We overview here some recent achievements in areas of interfacial electrochemical ET systems, mapped to the nanoscale and single-molecule levels. Focus is on both experimental and theoretical studies in our group. Systems addressed are organized monolayers of redox active transition metal complexes, and metalloproteins and metalloenzymes on single-crystal Au(1 1 1)-electrode surfaces. These systems have been investigated by voltammetry, spectroscopy, microcantilever technology, and scanning probe microscopy. A class of Os-complexes has shown suitable as targets for electrochemical in situ scanning tunnelling microscopy (STM), with close to single-molecule scanning tunnelling spectroscopic (STS) features. Mapping of redox metalloproteins from the three major classes, i.e. blue copper proteins, heme proteins, and iron-sulfur proteins, at the monolayer and single-molecule levels have also been achieved. In situ STM and spectroscopy of redox molecules and biomolecules have been supported by new theoretical frames, which extend established theory of interfacial electrochemical ET. The electrochemical nanoscale and single-molecule systems discussed are compared with other recent nanoscale and single-molecule systems with conspicuous device-like properties, particularly unimolecular rectifiers and single-molecule transistors. Both of these show analogies to electrochemical in situ STM features of redox molecules and

  17. Forces and conductances in a single-molecule bipyridine junction

    DEFF Research Database (Denmark)

    Stadler, Robert; Thygesen, Kristian Sommer; Jacobsen, Karsten Wedel

    2005-01-01

    Inspired by recent measurements of forces and conductances of bipyridine nanojunctions, we have performed density functional theory calculations of structure and electron transport in a bipyridine molecule attached between gold electrodes for seven different contact geometries. The calculations...

  18. Detection of kinetic change points in piece-wise linear single molecule motion

    Science.gov (United States)

    Hill, Flynn R.; van Oijen, Antoine M.; Duderstadt, Karl E.

    2018-03-01

    Single-molecule approaches present a powerful way to obtain detailed kinetic information at the molecular level. However, the identification of small rate changes is often hindered by the considerable noise present in such single-molecule kinetic data. We present a general method to detect such kinetic change points in trajectories of motion of processive single molecules having Gaussian noise, with a minimum number of parameters and without the need of an assumed kinetic model beyond piece-wise linearity of motion. Kinetic change points are detected using a likelihood ratio test in which the probability of no change is compared to the probability of a change occurring, given the experimental noise. A predetermined confidence interval minimizes the occurrence of false detections. Applying the method recursively to all sub-regions of a single molecule trajectory ensures that all kinetic change points are located. The algorithm presented allows rigorous and quantitative determination of kinetic change points in noisy single molecule observations without the need for filtering or binning, which reduce temporal resolution and obscure dynamics. The statistical framework for the approach and implementation details are discussed. The detection power of the algorithm is assessed using simulations with both single kinetic changes and multiple kinetic changes that typically arise in observations of single-molecule DNA-replication reactions. Implementations of the algorithm are provided in ImageJ plugin format written in Java and in the Julia language for numeric computing, with accompanying Jupyter Notebooks to allow reproduction of the analysis presented here.

  19. Extracting physics of life at the molecular level: A review of single-molecule data analyses.

    Science.gov (United States)

    Colomb, Warren; Sarkar, Susanta K

    2015-06-01

    Studying individual biomolecules at the single-molecule level has proved very insightful recently. Single-molecule experiments allow us to probe both the equilibrium and nonequilibrium properties as well as make quantitative connections with ensemble experiments and equilibrium thermodynamics. However, it is important to be careful about the analysis of single-molecule data because of the noise present and the lack of theoretical framework for processes far away from equilibrium. Biomolecular motion, whether it is free in solution, on a substrate, or under force, involves thermal fluctuations in varying degrees, which makes the motion noisy. In addition, the noise from the experimental setup makes it even more complex. The details of biologically relevant interactions, conformational dynamics, and activities are hidden in the noisy single-molecule data. As such, extracting biological insights from noisy data is still an active area of research. In this review, we will focus on analyzing both fluorescence-based and force-based single-molecule experiments and gaining biological insights at the single-molecule level. Inherently nonequilibrium nature of biological processes will be highlighted. Simulated trajectories of biomolecular diffusion will be used to compare and validate various analysis techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Modeling single molecule junction mechanics as a probe of interface bonding

    Science.gov (United States)

    Hybertsen, Mark S.

    2017-03-01

    Using the atomic force microscope based break junction approach, applicable to metal point contacts and single molecule junctions, measurements can be repeated thousands of times resulting in rich data sets characterizing the properties of an ensemble of nanoscale junction structures. This paper focuses on the relationship between the measured force extension characteristics including bond rupture and the properties of the interface bonds in the junction. A set of exemplary model junction structures has been analyzed using density functional theory based calculations to simulate the adiabatic potential surface that governs the junction elongation. The junction structures include representative molecules that bond to the electrodes through amine, methylsulfide, and pyridine links. The force extension characteristics are shown to be most effectively analyzed in a scaled form with maximum sustainable force and the distance between the force zero and force maximum as scale factors. Widely used, two parameter models for chemical bond potential energy versus bond length are found to be nearly identical in scaled form. Furthermore, they fit well to the present calculations of N-Au and S-Au donor-acceptor bonds, provided no other degrees of freedom are allowed to relax. Examination of the reduced problem of a single interface, but including relaxation of atoms proximal to the interface bond, shows that a single-bond potential form renormalized by an effective harmonic potential in series fits well to the calculated results. This allows relatively accurate extraction of the interface bond energy. Analysis of full junction models shows cooperative effects that go beyond the mechanical series inclusion of the second bond in the junction, the spectator bond that does not rupture. Calculations for a series of diaminoalkanes as a function of molecule length indicate that the most important cooperative effect is due to the interactions between the dipoles induced by the donor

  1. A trinuclear radical-bridged lanthanide single-molecule magnet

    Energy Technology Data Exchange (ETDEWEB)

    Gould, Colin A.; Darago, Lucy E.; Gonzalez, Miguel I. [Department of Chemistry, University of California, Berkeley, CA (United States); Demir, Selvan [Institut fuer Anorganische Chemie, Georg-August-Universitaet, Goettingen (Germany); Long, Jeffrey R. [Department of Chemistry, University of California, Berkeley, CA (United States); Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA (United States); Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA (United States)

    2017-08-14

    Assembly of the triangular, organic radical-bridged complexes Cp*{sub 6}Ln{sub 3}(μ{sub 3}-HAN) (Cp*=pentamethylcyclopentadienyl; Ln=Gd, Tb, Dy; HAN=hexaazatrinaphthylene) proceeds through the reaction of Cp*{sub 2}Ln(BPh{sub 4}) with HAN under strongly reducing conditions. Significantly, magnetic susceptibility measurements of these complexes support effective magnetic coupling of all three Ln{sup III} centers through the HAN{sup 3-.} radical ligand. Thorough investigation of the Dy{sup III} congener through both ac susceptibility and dc magnetic relaxation measurements reveals slow relaxation of the magnetization, with an effective thermal relaxation barrier of U{sub eff}=51 cm{sup -1}. Magnetic coupling in the Dy{sup III} complex enables a large remnant magnetization at temperatures up to 3.0 K in the magnetic hysteresis measurements and hysteresis loops that are open at zero-field up to 3.5 K. (copyright 2017 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim)

  2. Spin Quantum Tunneling via Entangled States in a Dimer of Exchange-Coupled Single-Molecule Magnets

    Science.gov (United States)

    Tiron, R.; Wernsdorfer, W.; Foguet-Albiol, D.; Aliaga-Alcalde, N.; Christou, G.

    2003-11-01

    A new family of supramolecular, antiferromagnetically exchange-coupled dimers of single-molecule magnets (SMMs) has recently been reported. Each SMM acts as a bias on its neighbor, shifting the quantum tunneling resonances of the individual SMMs. Hysteresis loop measurements on a single crystal of SMM dimers have now established quantum tunneling of the magnetization via entangled states of the dimer. This shows that the dimer really does behave as a quantum mechanically coupled dimer, and also allows the measurement of the longitudinal and transverse superexchange coupling constants.

  3. A Development and Application of the Fretting Fatigue Test Apparatus

    Directory of Open Access Journals (Sweden)

    Wang Jun

    2016-01-01

    Full Text Available Fretting fatigue occurs in contacting parts which are subjected to fluctuating loads and very small sliding movements at the same time. Fretting fatigue can significantly reduce the fatigue life of materials. In this investigation, a new device is introduced, which performed using a combination of a servo-hydraulic push-pull fatigue testing machine and a specially designed jig for appropriate positioning of pads. The axial and contact loads are measured by load cells. The functionality of the device is examined by making a comparison between fretting fatigue lives and plain fatigue lives of a number of ZL702A aluminum alloy specimens. The main advantages of the device are its simplicity and cheapness. It can be developed further for high and low temperature tests in future.

  4. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

    Science.gov (United States)

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, Jongone; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic `fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

  5. Studying Chemical Reactions, One Bond at a Time, with Single Molecule AFM Techniques

    Science.gov (United States)

    Fernandez, Julio M.

    2008-03-01

    The mechanisms by which mechanical forces regulate the kinetics of a chemical reaction are unknown. In my lecture I will demonstrate how we use single molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bond via the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is of common occurrence in mechanically stressed proteins. While reduction is thought to proceed through a substitution nucleophilic bimolecular (SN2) reaction, the role of a mechanical force in modulating this chemical reaction is unknown. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by dithiothreitol (DTT). We find that while the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300 pN range. This result predicts that the disulfide bond lengthens by 0.34 å at the transition state of the thiol/disulfide exchange reaction. In addition to DTT, we also study the reduction of the engineered disulfide bond by the E. coli enzyme thioredoxin (Trx). Thioredoxins are enzymes that catalyze disulfide bond reduction in all organisms. As before, we apply a mechanical force in the range of 25-450 pN to the engineered disulfide bond substrate and monitor the reduction of these bonds by individual enzymes. In sharp contrast with the data obtained with DTT, we now observe two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulfide bond, causing a shortening of the substrate polypeptide by 0.76±0.07 å, and the second elongating the substrate disulfide bond by 0.21±0.01 å. These results support the view that the Trx active site regulates the geometry of the participating sulfur atoms, with sub-ångström precision, in order to achieve efficient catalysis. Single molecule

  6. Single Molecule Kinetics of ENTH Binding to Lipid Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Rozovsky, Sharon [Univ. of Delaware, Newark, DE (United States); Forstner, Martin B. [Syracuse Univ., NY (United States); Sondermann, Holger [Cornell Univ., Ithaca, NY (United States); Groves, Jay T. [Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2012-04-03

    Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins’ localization and interactions. Thus, the specificity and the kinetics of peripheral proteins’ membrane residence are an attribute of their function. In this article, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P2 despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. In addition, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH’s diffusive transport appears to be the result of at least three different diffusion processes.

  7. Spin dynamics in the single molecule magnet Ni4 under microwave irradiation

    Science.gov (United States)

    de Loubens, Gregoire

    2009-03-01

    Quantum mechanical effects such as quantum tunneling of magnetization (QTM) and quantum phase interference have been intensively studied in single molecule magnets (SMMs). These materials have also been suggested as candidates for qubits and are promising for molecular spintronics. Understanding decoherence and energy relaxation mechanisms in SMMs is then both of fundamental interest and important for the use of SMMs in applications. Interestingly, the single-spin relaxation rate due to direct process of a SMM embedded in an elastic medium can be derived without any unknown coupling constant [1]. Moreover, nontrivial relaxation mechanisms are expected from collective effects in SMM single crystals, such as phonon superradiance or phonon bottleneck. In order to investigate the spin relaxation between the two lowest lying spin-states of the S=4 single molecule magnet Ni4, we have developed an integrated sensor that combines a microstrip resonator and micro-Hall effect magnetometer on a chip [2]. This sensor enables both real time studies of magnetization dynamics under pulse irradiation as well as simultaneous measurements of the absorbed power and magnetization changes under continuous microwave irradiation. The latter technique permits the study of small deviations from equilibrium under steady state conditions, i.e. small amplitude cw microwave irradiation. This has been used to determine the energy relaxation rate of a Ni4 single crystal as a function of temperature at two frequencies, 10 and 27.8 GHz. A strong temperature dependence is observed below 1.5 K, which is not consistent with a direct spin-phonon relaxation process. The data instead suggest that the spin relaxation is dominated by a phonon bottleneck at low temperatures and occurs by an Orbach process involving excited spin-levels at higher temperatures [3]. Experimental results will be compared with detailed calculations of the relaxation rate using the density matrix equation with the relaxation

  8. A Single Molecule Study of Two Bacteriophage Epigenetic Switches

    Science.gov (United States)

    Wang, Haowei

    Epigenetic switches allow organisms to evolve into different states by activating/repressing different sets of genes without mutations of the underlying DNA sequence. The study of epigenetic switches is very important to understand the mechanism of human development, the origin of cancer, mental illness and fundamental processes such as gene regulation. The coliphage lambda epigenetic switch, which allows switching from lysogeny to lysis, has been studied for more than 50 years as a paradigm, and has recently received renewed attention. Atomic force microscopy (AFM) was used here to show that the lambda repressor oligomerizes on DNA, primarily as a dodecamer, to secure a DNA loop, which is the basis of the lambda switch. This study also provides support for the idea that specifically bound repressor stabilizes adjacent, non-specifically bound repressor molecules, which confers robustness to the switch. 186 is a member of a different coliphage family. One of the major differences between the two coliphage families is that lambda phages can be induced to switch from the lysogenic to the lytic state by UV radiation, but most coliphages of P2 family, to which 186 belongs, cannot. Interaction between coliphage 186 repressor and DNA is characterized by AFM and tethered particle motion (TPM). To expedite analysis of the AFM data, MatLab codes were written to automate the laborious, manual tracing procedures. The programs automatically recognize DNA segments and protein particles in an image, in order to measure the DNA length and position of bound particles as well as their height, diameter and volume. Application of these algorithms greatly improved the efficiency of AFM analysis. It was showed that 186 CI dimers form heptameric wheels, which induce DNA wrapping and different kinds of DNA looping producing various conformations of nucleoprotein complexes. Information about the dynamics of DNA wrapping and looping on 186 CI particles was also obtained by TPM.

  9. Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra.

    Science.gov (United States)

    Du, Mengyan; Zhang, Lili; Xie, Shusen; Chen, Tongsheng

    2016-07-11

    Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has the inherent ability to resolve donor emission, fluorescence resonance energy transfer (FRET)-sensitized acceptor emission and directly excited acceptor emission. We here develop an ExEm unmixing-based quantitative FRET measurement method (EES-FRET) independent of excitation intensity and detector parameter setting. The ratio factor (rK), predetermined using a donor-acceptor tandem construct, of total acceptor absorption to total donor absorption in excitation wavelengths used is introduced for determining the concentration ratio of acceptor to donor. We implemented EES-FRET method on a wide-field microscope to image living cells expressing tandem FRET constructs with different donor-acceptor stoichiometry.

  10. Photon-HDF5: Open Data Format and Computational Tools for Timestamp-based Single-Molecule Experiments.

    Science.gov (United States)

    Ingargiola, Antonino; Laurence, Ted; Boutelle, Robert; Weiss, Shimon; Michalet, Xavier

    2016-02-13

    Archival of experimental data in public databases has increasingly become a requirement for most funding agencies and journals. These data-sharing policies have the potential to maximize data reuse, and to enable confirmatory as well as novel studies. However, the lack of standard data formats can severely hinder data reuse. In photon-counting-based single-molecule fluorescence experiments, data is stored in a variety of vendor-specific or even setup-specific (custom) file formats, making data interchange prohibitively laborious, unless the same hardware-software combination is used. Moreover, the number of available techniques and setup configurations make it difficult to find a common standard. To address this problem, we developed Photon-HDF5 (www.photon-hdf5.org), an open data format for timestamp-based single-molecule fluorescence experiments. Building on the solid foundation of HDF5, Photon-HDF5 provides a platform- and language-independent, easy-to-use file format that is self-describing and supports rich metadata. Photon-HDF5 supports different types of measurements by separating raw data (e.g. photon-timestamps, detectors, etc) from measurement metadata. This approach allows representing several measurement types and setup configurations within the same core structure and makes possible extending the format in backward-compatible way. Complementing the format specifications, we provide open source software to create and convert Photon-HDF5 files, together with code examples in multiple languages showing how to read Photon-HDF5 files. Photon-HDF5 allows sharing data in a format suitable for long term archival, avoiding the effort to document custom binary formats and increasing interoperability with different analysis software. We encourage participation of the single-molecule community to extend interoperability and to help defining future versions of Photon-HDF5.

  11. Blinking effect and the use of quantum dots in single molecule spectroscopy

    International Nuclear Information System (INIS)

    Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland; Petersen, Jan; Domingo, M.P.; Pardo, Julian; Gräber, P.; Galvez, E.M.

    2013-01-01

    Highlights: ► It is possible to eliminate the blinking effect of a water-soluble QD. ► We provide a direct method to study protein function and dynamics at the single level. ► QD, potent tool for single molecule studies of biochemical and biological processes. -- Abstract: Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the “on”/“off” states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein–protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.

  12. Experimental and Computational Characterization of Biological Liquid Crystals: A Review of Single-Molecule Bioassays

    Directory of Open Access Journals (Sweden)

    Sungsoo Na

    2009-09-01

    Full Text Available Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.

  13. Blinking effect and the use of quantum dots in single molecule spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland; Petersen, Jan [Albert-Ludwigs-Universitaet Freiburg, Institut fuer Physikalische Chemie, Albertstrasse 23a, 79104 Freiburg (Germany); Domingo, M.P. [Instituto de Carboquimica (CSIC), Miguel Luesma 4, 50018 Zaragoza (Spain); Pardo, Julian [Grupo Apoptosis, Inmunidad y Cancer, Departamento Bioquimica y Biologia Molecular y Celular, Fac. Ciencias, Universidad de Zaragoza, Zaragoza (Spain); Fundacion Aragon I-D (ARAID), Gobierno de Aragon, Zaragoza (Spain); Immune Effector Cells Group, Aragon Health Research Institute (IIS Aragon), Biomedical Research Centre of Aragon (CIBA) Fundacion Aragon I-D - ARAID, Gobierno de Aragon, Zaragoza (Spain); Graeber, P. [Albert-Ludwigs-Universitaet Freiburg, Institut fuer Physikalische Chemie, Albertstrasse 23a, 79104 Freiburg (Germany); Galvez, E.M., E-mail: eva@icb.csic.es [Instituto de Carboquimica (CSIC), Miguel Luesma 4, 50018 Zaragoza (Spain); Immune Effector Cells Group, Aragon Health Research Institute (IIS Aragon), Biomedical Research Centre of Aragon (CIBA) Fundacion Aragon I-D - ARAID, Gobierno de Aragon, Zaragoza (Spain)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer It is possible to eliminate the blinking effect of a water-soluble QD. Black-Right-Pointing-Pointer We provide a direct method to study protein function and dynamics at the single level. Black-Right-Pointing-Pointer QD, potent tool for single molecule studies of biochemical and biological processes. -- Abstract: Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the 'on'/'off' states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein-protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.

  14. Electronic Transport in Single Molecule Junctions: Control of the Molecule-Electrode Coupling Through Intramolecular Tunneling Barriers

    DEFF Research Database (Denmark)

    Danilov, Andrey; Kubatkin, Sergey; Kafanov, Sergey

    2008-01-01

    We report on single molecule electron transport measurements of two oligophenylenevinylene (OPV3) derivatives placed in a nanogap between gold (Au) or lead (Pb) electrodes in a field effect transistor device. Both derivatives contain thiol end groups that allow chemical binding to the electrodes....... One derivative has additional methylene groups separating the thiols from the delocalized -electron system. The insertion of methylene groups changes the open state conductance by 3-4 orders of magnitude and changes the transport mechanism from a coherent regime with finite zero-bias conductance...

  15. How to determine the handedness of single molecules using Coulomb explosion imaging

    International Nuclear Information System (INIS)

    Pitzer, Martin

    2017-01-01

    This tutorial is based on a doctoral thesis that was shortlisted for the 2016 AMOP dissertation prize of the German Physical Society (DPG). The principal achievement of the thesis was to use Coulomb explosion imaging (CEI) to determine the microscopic handedness (‘chirality’) of molecular structures on a single-molecule level. It thus shows how a technique developed in atomic physics can address a long-standing problem in chemistry. Owing to these disparate backgrounds, the tutorial has two facets: on the one hand, the history of molecular chirality and recent developments are very briefly reviewed. On the other hand, an account is given of different experimental approaches to CEI, on the physical processes in light-induced Coulomb explosion and—most importantly—on the aspects that are relevant when designing and performing such an experiment. As structural chirality occurs only in polyatomic molecules, special attention will be given to multiple ionization and multi-coincidence measurements. A short discussion of the results presented in earlier papers is given, followed by an outlook on experiments that are under way or can realistically be performed within the next years. (phd tutorial)

  16. Comprehensive high frequency electron paramagnetic resonance studies of single molecule magnets

    Science.gov (United States)

    Lawrence, Jonathan D.

    This dissertation presents research on a number of single molecule magnet (SMM) compounds conducted using high frequency, low temperature magnetic resonance spectroscopy of single crystals. By developing a new technique that incorporated other devices such as a piezoelectric transducer or Hall magnetometer with our high frequency microwaves, we were able to collect unique measurements on SMMs. This class of materials, which possess a negative, axial anisotropy barrier, exhibit unique magnetic properties such as quantum tunneling of a large magnetic moment vector. There are a number of spin Hamiltonians used to model these systems, the most common one being the giant spin approximation. Work done on two nickel systems with identical symmetry and microenvironments indicates that this model can contain terms that lack any physical significance. In this case, one must turn to a coupled single ion approach to model the system. This provides information on the nature of the exchange interactions between the constituent ions of the molecule. Additional studies on two similar cobalt systems show that, for these compounds, one must use a coupled single ion approach since the assumptions of the giant spin model are no longer valid. Finally, we conducted a collection of studies on the most famous SMM, Mn12Ac. Three different techniques were used to study magnetization dynamics in this system: stand-alone HFEPR in two different magnetization relaxation regimes, HFEPR combined with magnetometry, and HFEPR combined with surface acoustic waves. All of this research gives insight into the relaxation mechanisms in Mn12Ac.

  17. How to probe transverse magnetic anisotropy of a single-molecule magnet by electronic transport?

    Science.gov (United States)

    Misiorny, M.; Burzuri, E.; Gaudenzi, R.; Park, K.; Leijnse, M.; Wegewijs, M.; Paaske, J.; Cornia, A.; van der Zant, H.

    We propose an approach for in-situ determination of the transverse magnetic anisotropy (TMA) of an individual molecule by electronic transport measurements, see Phys. Rev. B 91, 035442 (2015). We study a Fe4 single-molecule magnet (SMM) captured in a gateable junction, a unique tool for addressing the spin in different redox states of a molecule. We show that, due to mixing of the spin eigenstates of the SMM, the TMA significantly manifests itself in transport. We predict and experimentally observe the pronounced intensity modulation of the Coulomb peak amplitude with the magnetic field in the linear-response transport regime, from which the TMA parameter E can be estimated. Importantly, the method proposed here does not rely on the small induced tunnelling effects and, hence, works well at temperatures and electron tunnel broadenings by far exceeding the tunnel splittings and even E itself. We deduce that the TMA for a single Fe4 molecule captured in a junction is substantially larger than the bulk value. Work supported by the Polish Ministry of Science and Education as `Iuventus Plus' project (IP2014 030973) in years 2015-2016.

  18. How to determine the handedness of single molecules using Coulomb explosion imaging

    Science.gov (United States)

    Pitzer, Martin

    2017-08-01

    This tutorial is based on a doctoral thesis that was shortlisted for the 2016 AMOP dissertation prize of the German Physical Society (DPG). The principal achievement of the thesis was to use Coulomb explosion imaging (CEI) to determine the microscopic handedness (‘chirality’) of molecular structures on a single-molecule level. It thus shows how a technique developed in atomic physics can address a long-standing problem in chemistry. Owing to these disparate backgrounds, the tutorial has two facets: on the one hand, the history of molecular chirality and recent developments are very briefly reviewed. On the other hand, an account is given of different experimental approaches to CEI, on the physical processes in light-induced Coulomb explosion and—most importantly—on the aspects that are relevant when designing and performing such an experiment. As structural chirality occurs only in polyatomic molecules, special attention will be given to multiple ionization and multi-coincidence measurements. A short discussion of the results presented in earlier papers is given, followed by an outlook on experiments that are under way or can realistically be performed within the next years.

  19. MCD spectroscopy of hexanuclear Mn(III) salicylaldoxime single-molecule magnets

    DEFF Research Database (Denmark)

    Bradley, Justin M; Thomson, Andrew J; Inglis, Ross

    2010-01-01

    The hexanuclear cages [Mn(6)O(2)(R-sao)(6)L(2)(EtOH)(x)(H(2)O)(y)] "Mn(6)" behave as single-molecule magnets (SMMs) below a characteristic blocking temperature. As with [Mn(12)O(12)(O(2)CR)(16)(H(2)O)(4)] "Mn(12)" the electronic absorption spectra are rather featureless, yielding little information...... structural perturbations of a generic Mn(6)O(2) core. Absorbance and MCD spectra are reported for [Mn(6)O(2)(Et-sao)(6){O(2)CPh(Me)(2)}(2)(EtOH)(6)] (1), [Mn(6)O(2)(Et-sao)(6){O(2)CPh}(2)(EtOH)(4)(H(2)O)(2)] (2), [Mn(6)O(2)(sao)(6){O(2)CPh}(2)(EtOH)(4)]·EtOH (3) and the trinuclear precursor [Mn(3)O(Et-sao)(3......)(MeOH)(3)](ClO(4)) (4) cast into polymer film. SMM behaviour has previously been observed using magnetic susceptibility measurements on powder and single-crystal samples. The ligand field environment of the magnetic ions is assumed to be similar in (1) and (2) and their different blocking temperatures...

  20. From isolated light-harvesting complexes to the thylakoid membrane: a single-molecule perspective

    Science.gov (United States)

    Gruber, J. Michael; Malý, Pavel; Krüger, Tjaart P. J.; Grondelle, Rienk van

    2018-01-01

    The conversion of solar radiation to chemical energy in plants and green algae takes place in the thylakoid membrane. This amphiphilic environment hosts a complex arrangement of light-harvesting pigment-protein complexes that absorb light and transfer the excitation energy to photochemically active reaction centers. This efficient light-harvesting capacity is moreover tightly regulated by a photoprotective mechanism called non-photochemical quenching to avoid the stress-induced destruction of the catalytic reaction center. In this review we provide an overview of single-molecule fluorescence measurements on plant light-harvesting complexes (LHCs) of varying sizes with the aim of bridging the gap between the smallest isolated complexes, which have been well-characterized, and the native photosystem. The smallest complexes contain only a small number (10-20) of interacting chlorophylls, while the native photosystem contains dozens of protein subunits and many hundreds of connected pigments. We discuss the functional significance of conformational dynamics, the lipid environment, and the structural arrangement of this fascinating nano-machinery. The described experimental results can be utilized to build mathematical-physical models in a bottom-up approach, which can then be tested on larger in vivo systems. The results also clearly showcase the general property of biological systems to utilize the same system properties for different purposes. In this case it is the regulated conformational flexibility that allows LHCs to switch between efficient light-harvesting and a photoprotective function.

  1. A versatile low-temperature setup for the electrical characterization of single-molecule junctions

    Science.gov (United States)

    Martin, Christian A.; Smit, Roel H. M.; Egmond, Ruud van; van der Zant, Herre S. J.; van Ruitenbeek, Jan M.

    2011-05-01

    We present a modular high-vacuum setup for the electrical characterization of single molecules down to liquid helium temperatures. The experimental design is based on microfabricated mechanically controllable break junctions, which offer control over the distance of two electrodes via the bending of a flexible substrate. The actuator part of the setup is divided into two stages. The slow stage is based on a differential screw drive with a large bending range. An amplified piezoceramic actuator forms the fast stage of the setup, which can operate at bending speeds of up to 800 μm/s. In our microfabricated break junctions this is translated into breaking speeds of several 10 nm/s, sufficient for the fast acquisition of large statistical datasets. The bandwidth of the measurement electronics has been optimized to enable fast dI/dV spectroscopy on molecular junctions with resistances up to 100 MΩ. The performance of the setup is demonstrated for a π-conjugated oligo(phenylene-ethynylene)-dithiol molecule.

  2. Single molecule localization imaging of exosomes using blinking silicon quantum dots

    Science.gov (United States)

    Zong, Shenfei; Zong, Junzhu; Chen, Chen; Jiang, Xiaoyue; Zhang, Yizhi; Wang, Zhuyuan; Cui, Yiping

    2018-02-01

    Discovering new fluorophores, which are suitable for single molecule localization microscopy (SMLM) is important for promoting the applications of SMLM in biological or material sciences. Here, we found that silicon quantum dots (Si QDs) possess a fluorescence blinking behavior, making them an excellent candidate for SMLM. The Si QDs are fabricated using a facile microwave-assisted method. Blinking of Si QDs is confirmed by single particle fluorescence measurement and the spatial resolution achieved is about 30 nm. To explore the potential application of Si QDs as the nanoprobes for SMLM imaging, cell derived exosomes are chosen as the object owing to their small size (50-100 nm in diameter). Since CD63 is commonly presented on the membrane of exosomes, CD63 aptamers are attached to the surface of Si QDs to form nanoprobes which can specifically recognize exosomes. SMLM imaging shows that Si QDs based nanoprobes can indeed realize super resolved optical imaging of exosomes. More importantly, blinking of Si QDs is observed in water or PBS buffer with no need for special imaging buffers. Besides, considering that silicon is highly biocompatible, Si QDs should have minimal cytotoxicity. These features make Si QDs quite suitable for SMLM applications especially for live cell imaging.

  3. A mononuclear uranium(IV) single-molecule magnet with an azobenzene radical ligand

    Energy Technology Data Exchange (ETDEWEB)

    Antunes, Maria A.; Coutinho, Joana T.; Santos, Isabel C.; Marcalo, Joaquim; Almeida, Manuel; Pereira, Laura C.J. [C" 2TN, Instituto Superior Tecnico, Universidade de Lisboa, Bobadela (Portugal); Baldovi, Jose J.; Gaita-Arino, Alejandro; Coronado, Eugenio [Instituto de Ciencia Molecular, Universitat de Valencia, Paterna (Spain)

    2015-12-01

    A tetravalent uranium compound with a radical azobenzene ligand, namely, [{(SiMe_2NPh)_3-tacn}U{sup IV}(η{sup 2}-N{sub 2}Ph{sub 2{sup .}})] (2), was obtained by one-electron reduction of azobenzene by the trivalent uranium compound [U{sup III}{(SiMe_2NPh)_3-tacn}] (1). Compound 2 was characterized by single-crystal X-ray diffraction and {sup 1}H NMR, IR, and UV/Vis/NIR spectroscopy. The magnetic properties of 2 and precursor 1 were studied by static magnetization and ac susceptibility measurements, which for the former revealed single-molecule magnet behaviour for the first time in a mononuclear U{sup IV} compound, whereas trivalent uranium compound 1 does not exhibit slow relaxation of the magnetization at low temperatures. A first approximation to the magnetic behaviour of these compounds was attempted by combining an effective electrostatic model with a phenomenological approach using the full single-ion Hamiltonian. (copyright 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  4. Single-molecule magnet behavior in 2,2’-bipyrimidine-bridged dilanthanide complexes

    Directory of Open Access Journals (Sweden)

    Wen Yu

    2016-01-01

    Full Text Available A series of 2,2’-bipyrimidine-bridged dinuclear lanthanide complexes with the general formula [Ln(tmhd3]2bpm (tmhd = 2,2,6,6-tetramethyl-3,5-heptanedionate, bpm = 2,2’-bipyrimidine, Ln = Gd(III, 1; Tb(III, 2; Dy(III, 3; Ho(III, 4 and Er(III, 5 has been synthesized and characterized. Sublimation of [Tb(tmhd3]2bpm onto a Au(111 surface leads to the formation of a homogeneous film with hexagonal pattern, which was studied by scanning tunneling microscopy (STM. The bulk magnetic properties of all complexes have been studied comprehensively. The dynamic magnetic behavior of the Dy(III and Er(III compounds clearly exhibits single molecule magnet (SMM characteristics with an energy barrier of 97 and 25 K, respectively. Moreover, micro-SQUID measurements on single crystals confirm their SMM behavior with the presence of hysteresis loops.

  5. Single-molecule analysis of the major glycopolymers of pathogenic and non-pathogenic yeast cells

    Science.gov (United States)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Sarazin, Aurore; Jouault, Thierry; Dufrêne, Yves F.

    2013-05-01

    Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous β-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.

  6. Single-molecule detection of dihydroazulene photo-thermal reaction using break junction technique

    Science.gov (United States)

    Huang, Cancan; Jevric, Martyn; Borges, Anders; Olsen, Stine T.; Hamill, Joseph M.; Zheng, Jue-Ting; Yang, Yang; Rudnev, Alexander; Baghernejad, Masoud; Broekmann, Peter; Petersen, Anne Ugleholdt; Wandlowski, Thomas; Mikkelsen, Kurt V.; Solomon, Gemma C.; Brøndsted Nielsen, Mogens; Hong, Wenjing

    2017-05-01

    Charge transport by tunnelling is one of the most ubiquitous elementary processes in nature. Small structural changes in a molecular junction can lead to significant difference in the single-molecule electronic properties, offering a tremendous opportunity to examine a reaction on the single-molecule scale by monitoring the conductance changes. Here, we explore the potential of the single-molecule break junction technique in the detection of photo-thermal reaction processes of a photochromic dihydroazulene/vinylheptafulvene system. Statistical analysis of the break junction experiments provides a quantitative approach for probing the reaction kinetics and reversibility, including the occurrence of isomerization during the reaction. The product ratios observed when switching the system in the junction does not follow those observed in solution studies (both experiment and theory), suggesting that the junction environment was perturbing the process significantly. This study opens the possibility of using nano-structured environments like molecular junctions to tailor product ratios in chemical reactions.

  7. Surface-enhanced Raman spectroscopy of dyes: from single molecules to the artists' canvas.

    Science.gov (United States)

    Wustholz, Kristin L; Brosseau, Christa L; Casadio, Francesca; Van Duyne, Richard P

    2009-09-14

    This perspective presents recent surface-enhanced Raman spectroscopy (SERS) studies of dyes, with applications to the fields of single-molecule spectroscopy and art conservation. First we describe the development and outlook of single-molecule SERS (SMSERS). Rather than providing an exhaustive review of the literature, SMSERS experiments that we consider essential for its future development are emphasized. Shifting from single-molecule to ensemble-averaged experiments, we describe recent efforts toward SERS analysis of colorants in precious artworks. Our intention is to illustrate through these examples that the forward development of SERS is dependent upon both fundamental (e.g., SMSERS) and applied (e.g., on-the-specimen SERS of historical art objects) investigations and that the future of SERS is very bright indeed.

  8. Estimating single molecule conductance from spontaneous evolution of a molecular contact

    Science.gov (United States)

    Gil, M.; Malinowski, T.; Iazykov, M.; Klein, H. R.

    2018-03-01

    We present an original method to estimate the conductivity of a single molecule anchored to nanometric-sized metallic electrodes, using a Mechanically Controlled Break Junction operated at room temperature in the liquid. We record the conductance through the metal/molecules/metal nanocontact while keeping the metallic electrodes at a fixed distance. Taking advantage of thermal diffusion and electromigration, we let the contact naturally explore the more stable configurations around a chosen conductance value. The conductance of a single molecule is estimated from a statistical analysis of raw conductance and conductance standard deviation data for molecular contacts containing up to 14 molecules. The single molecule conductance values are interpreted as time-averaged conductance of an ensemble of conformers at thermal equilibrium.

  9. Assembly and diploid architecture of an individual human genome via single-molecule technologies.

    Science.gov (United States)

    Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

    2015-08-01

    We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

  10. Single-molecule detection of dihydroazulene photo-thermal reaction using break junction technique

    DEFF Research Database (Denmark)

    Huang, Cancan; Jevric, Martyn; Borges, Anders Christian

    2017-01-01

    a quantitative approach for probing the reaction kinetics and reversibility, including the occurrence of isomerization during the reaction. The product ratios observed when switching the system in the junction does not follow those observed in solution studies (both experiment and theory), suggesting......Charge transport by tunnelling is one of the most ubiquitous elementary processes in nature. Small structural changes in a molecular junction can lead to significant difference in the single-molecule electronic properties, offering a tremendous opportunity to examine a reaction on the single......-molecule scale by monitoring the conductance changes. Here, we explore the potential of the single-molecule break junction technique in the detection of photo-thermal reaction processes of a photochromic dihydroazulene/vinylheptafulvene system. Statistical analysis of the break junction experiments provides...

  11. Single-molecule electronics: Cooling individual vibrational modes by the tunneling current.

    Science.gov (United States)

    Lykkebo, Jacob; Romano, Giuseppe; Gagliardi, Alessio; Pecchia, Alessandro; Solomon, Gemma C

    2016-03-21

    Electronic devices composed of single molecules constitute the ultimate limit in the continued downscaling of electronic components. A key challenge for single-molecule electronics is to control the temperature of these junctions. Controlling heating and cooling effects in individual vibrational modes can, in principle, be utilized to increase stability of single-molecule junctions under bias, to pump energy into particular vibrational modes to perform current-induced reactions, or to increase the resolution in inelastic electron tunneling spectroscopy by controlling the life-times of phonons in a molecule by suppressing absorption and external dissipation processes. Under bias the current and the molecule exchange energy, which typically results in heating of the molecule. However, the opposite process is also possible, where energy is extracted from the molecule by the tunneling current. Designing a molecular "heat sink" where a particular vibrational mode funnels heat out of the molecule and into the leads would be very desirable. It is even possible to imagine how the vibrational energy of the other vibrational modes could be funneled into the "cooling mode," given the right molecular design. Previous efforts to understand heating and cooling mechanisms in single molecule junctions have primarily been concerned with small models, where it is unclear which molecular systems they correspond to. In this paper, our focus is on suppressing heating and obtaining current-induced cooling in certain vibrational modes. Strategies for cooling vibrational modes in single-molecule junctions are presented, together with atomistic calculations based on those strategies. Cooling and reduced heating are observed for two different cooling schemes in calculations of atomistic single-molecule junctions.

  12. Crystal lattice desolvation effects on the magnetic quantum tunneling of single-molecule magnets

    Science.gov (United States)

    Redler, G.; Lampropoulos, C.; Datta, S.; Koo, C.; Stamatatos, T. C.; Chakov, N. E.; Christou, G.; Hill, S.

    2009-09-01

    High-frequency electron paramagnetic resonance (HFEPR) and alternating current (ac) susceptibility measurements are reported for a new high-symmetry Mn12 complex, [Mn12O12(O2CCH3)16(CH3OH)4]ṡCH3OH . The results are compared to those of other high-symmetry spin S=10Mn12 single-molecule magnets (SMMs), including the original acetate, [Mn12(O2CCH3)16(H2O)4]ṡ2CH3CO2Hṡ4H2O , and the [Mn12O12(O2CCH2Br)16(H2O)4]ṡ4CH2Cl2 and [Mn12O12(O2CCH2But)16(CH3OH)4]ṡCH3OH complexes. These comparisons reveal important insights into the factors that influence the values of the effective barrier to magnetization reversal, Ueff , deduced on the basis of ac susceptibility measurements. In particular, we find that variations in Ueff can be correlated with the degree of disorder in a crystal which can be controlled by desolvating (drying) samples. This highlights the importance of careful sample handling when making measurements on SMM crystals containing volatile lattice solvents. The HFEPR data additionally provide spectroscopic evidence suggesting that the relatively weak disorder induced by desolvation influences the quantum tunneling interactions and that it is under-barrier tunneling that is responsible for a consistent reduction in Ueff that is found upon drying samples. Meanwhile, the axial anisotropy deduced from HFEPR is found to be virtually identical for all four Mn12 complexes, with no measurable reduction upon desolvation.

  13. Fast recognition of single molecules based on single-event photon statistics

    International Nuclear Information System (INIS)

    Dong Shuangli; Huang Tao; Liu Yuan; Wang Jun; Zhang Guofeng; Xiao Liantuan; Jia Suotang

    2007-01-01

    Mandel's Q parameter, which is determined from single-event photon statistics, provides an alternative way to recognize single molecules with fluorescence detection, other than the second-order correlation function. It is shown that the Q parameter of an assumed ideal double-molecule fluorescence with the same average photon number as that of the sample fluorescence can act as the criterion for single-molecule recognition. The influence of signal-to-background ratio and the error estimates for photon statistics are also presented. We have applied this method to ascertain single Cy5 dye molecules within hundreds of milliseconds

  14. Chemical Principles and Interference in the Electrical Conductance of Single Molecules

    DEFF Research Database (Denmark)

    Borges, Anders Christian

    The electrical conductance of single molecules are routinely reported in the scientific literature and off-resonant coherent tunneling is believed to be the mechanism for transport in some of these experiments. In these experiments it is observed that, in spite of similar molecular structures......-Tunneling Microscope Break-Junction experiments (STM-BJ). It is demonstrated that these links can be used to design molecules exhibiting surprising interference effects and to interpret and predict the trends in the characteristic conductance of single molecules without resorting to numerical computational methods...

  15. Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

    OpenAIRE

    Oiwa, K; Eccleston, J F; Anson, M; Kikumoto, M; Davis, C T; Reid, G P; Ferenczi, M A; Corrie, J E; Yamada, A; Nakayama, H; Trentham, D R

    2000-01-01

    Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-A...

  16. Manipulation and Motion of Organelles and Single Molecules in Living Cells

    DEFF Research Database (Denmark)

    Norregaard, Kamilla; Metzler, Ralf; Ritter, Christine M.

    2017-01-01

    driving many cellular processes. The forces on a molecular scale are exactly in the range that can be manipulated and probed with single molecule force spectroscopy. The natural environment of a biomolecule is inside a living cell, hence, this is the most relevant environment for probing their function....... In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theoretical tools for analyzing single molecule data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly...

  17. Site-specific labeling of Saccharomyces cerevisiae ribosomes for single-molecule manipulations

    Science.gov (United States)

    Petrov, Alexey; Puglisi, Joseph D.

    2010-01-01

    Site-specific labeling of Escherichia coli ribosomes has allowed application of single-molecule fluorescence spectroscopy and force methods to probe the mechanism of translation. To apply these approaches to eukaryotic translation, eukaryotic ribosomes must be specifically labeled with fluorescent labels and molecular handles. Here, we describe preparation and labeling of the small and large yeast ribosomal subunits. Phylogenetically variable hairpin loops in ribosomal RNA are mutated to allow hybridization of oligonucleotides to mutant ribosomes. We demonstrate specific labeling of the ribosomal subunits, and their use in single-molecule fluorescence and force experiments. PMID:20501598

  18. Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

    Science.gov (United States)

    Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; Zant, Herre S. J. van der

    2015-08-01

    It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the molecule is reduced resulting into a ten-fold increase in the off-resonant differential conductance. Theoretical calculations link the change in differential conductance to a reduction-induced change in conjugation, thereby lifting destructive interference of transport pathways.

  19. Single molecule experiments challenge the strict wave-particle dualism of light.

    Science.gov (United States)

    Greulich, Karl Otto

    2010-01-21

    Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the "single photon limit" of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source. "Single photon detectors" do not meet their promise-only "photon number resolving single photon detectors" do so. Particularly, the accumulation time argument, the only safe basis for the postulate of a strictly particle like photon, has so far not yet been verified.

  20. Controlled switching of single-molecule junctions by mechanical motion of a phenyl ring

    Directory of Open Access Journals (Sweden)

    Yuya Kitaguchi

    2015-10-01

    Full Text Available Mechanical methods for single-molecule control have potential for wide application in nanodevices and machines. Here we demonstrate the operation of a single-molecule switch made functional by the motion of a phenyl ring, analogous to the lever in a conventional toggle switch. The switch can be actuated by dual triggers, either by a voltage pulse or by displacement of the electrode, and electronic manipulation of the ring by chemical substitution enables rational control of the on-state conductance. Owing to its simple mechanics, structural robustness, and chemical accessibility, we propose that phenyl rings are promising components in mechanical molecular devices.

  1. Single Molecule Experiments Challenge the Strict Wave-Particle Dualism of Light

    Directory of Open Access Journals (Sweden)

    Karl Otto Greulich

    2010-01-01

    Full Text Available Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the “single photon limit” of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source. “Single photon detectors” do not meet their promise―only “photon number resolving single photon detectors” do so. Particularly, the accumulation time argument, the only safe basis for the postulate of a strictly particle like photon, has so far not yet been verified.

  2. EXPERIMENTAL INVESTIGTION OF THE FRETTING PHENOMENON

    Directory of Open Access Journals (Sweden)

    Ştefan GHIMISI

    2015-12-01

    Full Text Available Fretting is now fully identified as a small amplitude oscilatory motion which induces a harmonic tangential force between two surfaces in contact.It is related to three main loadings, i.e. fretting-wear, fretting-fatigue and fretting corrosion.Fretting regimes were first mapped by Vingsbo. In a similar way, three fretting regimes will be considered: stick regime,slip regime and mixed regime. The mixed regime was made up of initial gross slip followed by partial slip condition after a few hundred cycles. Obviously the partial slip transition develops the highest stress levels which can induce fatigue crack nucleation depending on the fatigue properties of the two contacting first bodies. Therefore prediction of the frontier between partial slip and gross slip is required.

  3. Fast, Background-Free DNA-PAINT Imaging Using FRET-Based Probes.

    Science.gov (United States)

    Auer, Alexander; Strauss, Maximilian T; Schlichthaerle, Thomas; Jungmann, Ralf

    2017-10-11

    DNA point accumulation in nanoscale topography (DNA-PAINT) enables super-resolution microscopy by harnessing the predictable, transient hybridization between short dye-labeled "imager" and complementary target-bound "docking" strands. DNA-PAINT microscopy allows sub-5 nm spatial resolution, spectrally unlimited multiplexing, and quantitative image analysis. However, these abilities come at the cost of nonfluorogenic imager strands, also emitting fluorescence when not bound to their docking strands. This has thus far prevented rapid image acquisition with DNA-PAINT, as the blinking rate of probes is limited by an upper-bound of imager strand concentrations, which in turn is dictated by the necessity to facilitate the detection of single-molecule binding events over the background of unbound, freely diffusing probes. To overcome this limitation and enable fast, background-free DNA-PAINT microscopy, we here introduce FRET-based imaging probes, alleviating the concentration-limit of imager strands and speeding up image acquisition by several orders of magnitude. We assay two approaches for FRET-based DNA-PAINT (or FRET-PAINT) using either fixed or transient acceptor dyes in combination with transiently binding donor-labeled DNA strands and achieve high-quality super-resolution imaging on DNA origami structures in a few tens of seconds. Finally, we also demonstrate the applicability of FRET-PAINT in a cellular environment by performing super-resolution imaging of microtubules in under 30 s. FRET-PAINT combines the advantages of conventional DNA-PAINT with fast image acquisition times, facilitating the potential study of dynamic processes.

  4. Nanofluidic single-molecule sorting of DNA: a new concept in separation and analysis of biomolecules towards ultimate level performance

    International Nuclear Information System (INIS)

    Yamamoto, Takatoki; Fujii, Teruo

    2010-01-01

    Separation and separation-based analysis of biomolecules are fundamentally important techniques in the field of biotechnology. These techniques, however, depend on stochastic processes that intrinsically involve uncertainty, and thus it is not possible to achieve 100% separation accuracy. Theoretically, the ultimate resolution and sensitivity should be realized in a single-molecule system because of the deterministic nature of single-molecule manipulation. Here, we have proposed and experimentally demonstrated the concept of a 'single-molecule sorter' that detects and correctly identifies individual single molecules, realizing the ultimate level of resolution and sensitivity for any separation-based technology. The single-molecule sorter was created using a nanofluidic network consisting of a single inlet channel that branches off into multiple outlet channels. It includes two major functional elements, namely a single-molecule detection and identification element and a flow path switching element to accurately separate single molecules. With this system we have successfully demonstrated the world's first single-molecule sorting using DNA as a sample molecule. In the future, we hope to expand the application of such devices to comprehensive sorting of single-proteins from a single cell. We also believe that in addition to the single-molecule sorting method reported here, other types of single-molecule based processes will emerge and find use in a wide variety of applications.

  5. Electronic and magnetic properties of Mn{sub 12} single-molecule magnets on the Au(111) surface

    Energy Technology Data Exchange (ETDEWEB)

    Voss, Soenke; Burgert, Michael; Fonin, Mikhail; Groth, Ulrich; Ruediger, Ulrich [Universitaet Konstanz (Germany); Michaelis, Christian; Brihuega, Ivan; Kern, Klaus [Max-Planck-Institut fuer Festkoerperforschung, Stuttgart (Germany); Dedkov, Yury S. [Institut fuer Festkoerperphysik, Technische Universitaet Dresden (Germany)

    2008-07-01

    The paramount interest in single-molecule magnets (SMMs) like Mn{sub 12}-acetate and its derivatives was inspired by numerous experimental and theoretical insights indicating the feasibility of addressing quantum effects of magnetism on a molecular scale. Due to its relatively high blocking temperature ({proportional_to}3 K) combined with the ability to identify well-defined spin states, Mn{sub 12} still remains the most favoured SMM possibly allowing the detection of magnetic fingerprints in transport properties of a single molecule. In this work, the electronic properties of Mn{sub 12} molecules chemically grafted on Au(111) surfaces have been studied by means of low temperature as well as room temperature scanning tunneling microscopy and spectroscopy (STS), X-ray absorption spectroscopy and photoelectron spectroscopy. The results revealed signatures from most probably intact Mn{sub 12} molecules while STS measurements in magnetic fields indicate the possibility to identify magnetic fingerprints in scanning tunneling spectra. The results will be discussed with respect to previous attempts to perform transport measurements on Mn{sub 12} SMMs.

  6. A novel aptasensor based on single-molecule force spectroscopy for highly sensitive detection of mercury ions.

    Science.gov (United States)

    Li, Qing; Michaelis, Monika; Wei, Gang; Colombi Ciacchi, Lucio

    2015-08-07

    We have developed a novel aptasensor based on single-molecule force spectroscopy (SMFS) capable of detecting mercury ions (Hg(2+)) with sub-nM sensitivity. The single-strand (ss) DNA aptamer used in this work is rich in thymine (T) and readily forms T-Hg(2+)-T complexes in the presence of Hg(2+). The aptamer was conjugated to an atomic force microscope (AFM) probe, and the adhesion force between the probe and a flat graphite surface was measured by single-molecule force spectroscopy (SMFS). The presence of Hg(2+) ions above a concentration threshold corresponding to the affinity constant of the ions for the aptamer (about 5 × 10(9) M(-1)) could be easily detected by a change of the measured adhesion force. With our chosen aptamer, we could reach an Hg(2+) detection limit of 100 pM, which is well below the maximum allowable level of Hg(2+) in drinking water. In addition, this aptasensor presents a very high selectivity for Hg(2+) over other metal cations, such as K(+), Ca(2+), Zn(2+), Fe(2+), and Cd(2+). Furthermore, the effects of the ionic strength and loading rate on the Hg(2+) detection were evaluated. Its simplicity, reproducibility, high selectivity and sensitivity make our SMFS-based aptasensor advantageous with respect to other current Hg(2+) sensing methods. It is expected that our strategy can be exploited for monitoring the pollution of water environments and the safety of potentially contaminated food.

  7. Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements.

    Science.gov (United States)

    Kudryavtsev, Volodymyr; Sikor, Martin; Kalinin, Stanislav; Mokranjac, Dejana; Seidel, Claus A M; Lamb, Don C

    2012-03-01

    Single-pair Förster resonance energy transfer (spFRET) experiments using single-molecule burst analysis on a confocal microscope are an ideal tool to measure inter- and intramolecular distances and dynamics on the nanoscale. Different techniques have been developed to maximize the amount of information available in spFRET burst analysis experiments. Multiparameter fluorescence detection (MFD) is used to monitor a variety of fluorescence parameters simultaneously and pulsed interleaved excitation (PIE) employs direct excitation of the acceptor to probe its presence and photoactivity. To calculate accurate FRET efficiencies from spFRET experiments with MFD or PIE, several calibration measurements are usually required. Herein, we demonstrate that by combining MFD with PIE information regarding all calibration factors as well as an accurate determination of spFRET histograms can be performed in a single measurement. In addition, the quality of overlap of the different detection volumes as well as the detection of acceptor photophysics can be investigated with MFD-PIE. Bursts containing acceptor photobleaching can be identified and excluded from further investigation while bursts that contain FRET dynamics are unaffected by this analysis. We have employed MFD-PIE to accurately analyze the effects of nucleotides and substrate on the interdomain separation in DnaK, the major bacterial heat shock protein 70 (Hsp70). The interdomain distance increases from 47 Å in the ATP-bound state to 84 Å in the ADP-bound state and slightly contracts to 77 Å when a substrate is bound. This is in contrast to what was observed for the mitochondrial member of the Hsp70s, Ssc1, supporting the notion of evolutionary specialization of Hsp70s for different cellular functions in different organisms and cell organelles. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Analysis of Point Based Image Registration Errors With Applications in Single Molecule Microscopy.

    Science.gov (United States)

    Cohen, E A K; Ober, R J

    2013-12-15

    We present an asymptotic treatment of errors involved in point-based image registration where control point (CP) localization is subject to heteroscedastic noise; a suitable model for image registration in fluorescence microscopy. Assuming an affine transform, CPs are used to solve a multivariate regression problem. With measurement errors existing for both sets of CPs this is an errors-in-variable problem and linear least squares is inappropriate; the correct method being generalized least squares. To allow for point dependent errors the equivalence of a generalized maximum likelihood and heteroscedastic generalized least squares model is achieved allowing previously published asymptotic results to be extended to image registration. For a particularly useful model of heteroscedastic noise where covariance matrices are scalar multiples of a known matrix (including the case where covariance matrices are multiples of the identity) we provide closed form solutions to estimators and derive their distribution. We consider the target registration error (TRE) and define a new measure called the localization registration error (LRE) believed to be useful, especially in microscopy registration experiments. Assuming Gaussianity of the CP localization errors, it is shown that the asymptotic distribution for the TRE and LRE are themselves Gaussian and the parameterized distributions are derived. Results are successfully applied to registration in single molecule microscopy to derive the key dependence of the TRE and LRE variance on the number of CPs and their associated photon counts. Simulations show asymptotic results are robust for low CP numbers and non-Gaussianity. The method presented here is shown to outperform GLS on real imaging data.

  9. Fast magnetization tunneling in tetranickel(II) single-molecule magnets.

    Science.gov (United States)

    Yang, En-Che; Wernsdorfer, Wolfgang; Zakharov, Lev N; Karaki, Yoshitomo; Yamaguchi, Akira; Isidro, Rose M; Lu, Guo-Di; Wilson, Samuel A; Rheingold, Arnold L; Ishimoto, Hidehiko; Hendrickson, David N

    2006-01-23

    A series of Ni(4) cubane complexes with the composition [Ni(hmp)(ROH)Cl](4) complexes 1-4 where R= -CH(3) (complex 1), -CH(2)CH(3) (complex 2), -CH(2)CH(2)(C(4)H(9)) (complex 3), -CH(2)CH(2)CH(2)(C(6)H(11)) (complex 4), hmp(-) is the anion of 2-hydroxymethylpyridine, t-Buhmp(-) is the anion of 4-tert-butyl-2-hydroxymethylpyridine, and dmb is 3,3-dimethyl-1-butanol] and [Ni(hmp)(dmb)Br](4) (complex 5) and [Ni(t-Buhmp)(dmb)Cl](4) (complex 6) were prepared. All six complexes were characterized by dc magnetic susceptibility data to be ferromagnetically coupled to give an S = 4 ground state with significant magnetoanisotropy (D approximately equal to -0.6 cm(-1)). Magnetization hysteresis measurements carried out on single crystals of complexes 1-6 establish the single-molecule magnet (SMM) behavior of these complexes. The exchange bias observed in the magnetization hysteresis loops of complexes 1 and 2 is dramatically decreased to zero in complex 3, where the bulky dmb ligand is employed. Fast tunneling of magnetization is observed for the high-symmetry (S(4) site symmetry) Ni(4) complexes in the crystal of complex 3, and the tunneling rate can even be enhanced by destroying the S(4) site symmetry, as is the case for complex 4, where there are two crystallographically different Ni(4) molecules, one with C(2) and the other with C(1) site symmetry. Magnetic ordering temperatures due to intermolecular dipolar and magnetic exchange interactions were determined by means of very low-temperature ac susceptibility measurements; complex 1 orders at 1100 mK, complex 3 at 290 mK, complex 4 at approximately 80 mK, and complex 6 at lower temperatures for those complexes with the bulkiest ligands.

  10. Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Fluorescence resonance energy transfer (FRET between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.

  11. Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection.

    Science.gov (United States)

    Yao, B C; Wu, Y; Yu, C B; He, J R; Rao, Y J; Gong, Y; Fu, F; Chen, Y F; Li, Y R

    2016-03-24

    Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel 'FRET on Fiber' concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based 'FRET on fiber' configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated 'FRET on Fiber' sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response.

  12. Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy.

    Science.gov (United States)

    Boehm, E M; Subramanyam, S; Ghoneim, M; Washington, M Todd; Spies, M

    2016-01-01

    Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary) complexes using multicolor setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches have become today. © 2016 Elsevier Inc. All rights reserved.

  13. Single-Molecule Transport at a Rectifying GaAs Contact.

    Science.gov (United States)

    Vezzoli, Andrea; Brooke, Richard J; Ferri, Nicolò; Higgins, Simon J; Schwarzacher, Walther; Nichols, Richard J

    2017-02-08

    In most single- or few-molecule devices, the contact electrodes are simple ohmic resistors. Here we describe a new type of single-molecule device in which metal and semiconductor contact electrodes impart a function, namely, current rectification, which is then modified by a molecule bridging the gap. We study junctions with the structure Au STM tip/X/n-GaAs substrate, where "X" is either a simple alkanedithiol or a conjugated unit bearing thiol/methylthiol contacts, and we detect current jumps corresponding to the attachment and detachment of single molecules. From the magnitudes of the current jumps we can deduce values for the conductance decay constant with molecule length that agree well with values determined from Au/molecule/Au junctions. The ability to impart functionality to a single-molecule device through the properties of the contacts as well as through the properties of the molecule represents a significant extension of the single-molecule electronics "tool-box".

  14. Single-molecule fluorescence microscopy review: shedding new light on old problems.

    Science.gov (United States)

    Shashkova, Sviatlana; Leake, Mark C

    2017-08-31

    Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called 'green revolution', has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called 'super-resolution' fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. © 2017 The Author(s).

  15. Single molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update

    Science.gov (United States)

    Widom, Julia R.; Dhakal, Soma; Heinicke, Laurie A.; Walter, Nils G.

    2015-01-01

    Toxicology is the highly interdisciplinary field studying the adverse effects of chemicals on living organisms. It requires sensitive tools to detect such effects. After their initial implementation during the 1990s, single-molecule fluorescence detection tools were quickly recognized for their potential to contribute greatly to many different areas of scientific inquiry. In the intervening time, technical advances in the field have generated ever-improving spatial and temporal resolution, and have enabled the application of single-molecule fluorescence to increasingly complex systems, such as live cells. In this review, we give an overview of the optical components necessary to implement the most common versions of single-molecule fluorescence detection. We then discuss current applications to enzymology and structural studies, systems biology, and nanotechnology, presenting the technical considerations that are unique to each area of study, along with noteworthy recent results. We also highlight future directions that have the potential to revolutionize these areas of study by further exploiting the capabilities of single-molecule fluorescence microscopy. PMID:25212907

  16. Electron-vibron coupling effects on electron transport via a single-molecule magnet

    NARCIS (Netherlands)

    McCaskey, A.; Yamamoto, Y.; Warnock, M.; Burzuri, E.; Van der Zant, H.S.J.; Park, K.

    2015-01-01

    We investigate how the electron-vibron coupling influences electron transport via an anisotropic magnetic molecule, such as a single-molecule magnet (SMM) Fe4, by using a model Hamiltonian with parameter values obtained from density-functional theory (DFT). The magnetic anisotropy parameters,

  17. Ninth international conference on hole burning, single molecule and related spectroscopies: science and applications (HBSM 2006)

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2006-07-01

    This conference was organized around 9 sessions: -) single molecule, -) quantum optics, -) hole-burning materials and mechanisms, -) single nano-particle spectroscopy, -) dephasing and spectral diffusion, -) microwave photonics, -) biological systems, -) rare earth doped materials, -) novel laser sources. This document gathers only the slides of the presentations.

  18. Ninth international conference on hole burning, single molecule and related spectroscopies: science and applications (HBSM 2006)

    International Nuclear Information System (INIS)

    2006-01-01

    This conference was organized around 9 sessions: -) single molecule, -) quantum optics, -) hole-burning materials and mechanisms, -) single nano-particle spectroscopy, -) dephasing and spectral diffusion, -) microwave photonics, -) biological systems, -) rare earth doped materials, -) novel laser sources. This document gathers only the slides of the presentations

  19. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

    Science.gov (United States)

    Neuman, Keir C.; Nagy, Attila

    2012-01-01

    Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. These techniques are described and illustrated with examples highlighting current capabilities and limitations. PMID:18511917

  20. Single-molecule force-conductance spectroscopy of hydrogen-bonded complexes

    DEFF Research Database (Denmark)

    Pirrotta, Alessandro; De Vico, Luca; Solomon, Gemma C.

    2017-01-01

    to inform about molecular recognition events at the single-molecule limit. For this, we consider the force-conductance characteristics of a prototypical class of hydrogen bonded bimolecular complexes sandwiched between gold electrodes. The complexes consist of derivatives of a barbituric acid and a Hamilton...

  1. Single molecule charge transport : From a quantum mechanical to a classical description

    NARCIS (Netherlands)

    Kocherzhenko, A.A.; Grozema, F.C.; Siebbeles, L.D.A.

    2010-01-01

    This paper explores charge transport at the single molecule level. The conductive properties of both small organic molecules and conjugated polymers (molecular wires) are considered. In particular, the reasons for the transition from fully coherent to incoherent charge transport and the approaches

  2. The Relation between Structure and Quantum Interference in Single Molecule Junctions

    DEFF Research Database (Denmark)

    Markussen, Troels; Stadler, Robert; Thygesen, Kristian Sommer

    2010-01-01

    Quantum interference (QI) of electron pathways has recently attracted increased interest as an enabling tool for single-molecule electronic devices. Although various molecular systems have been shown to exhibit QI effects and a number of methods have been proposed for its analysis, simple...

  3. Nonequilibrium Chemical Effects in Single-Molecule SERS Revealed by Ab Initio Molecular Dynamics Simulations

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Sean A.; Apra, Edoardo; Govind, Niranjan; Hess, Wayne P.; El-Khoury, Patrick Z.

    2017-02-03

    Recent developments in nanophotonics have paved the way for achieving significant advances in the realm of single molecule chemical detection, imaging, and dynamics. In particular, surface-enhanced Raman scattering (SERS) is a powerful analytical technique that is now routinely used to identify the chemical identity of single molecules. Understanding how nanoscale physical and chemical processes affect single molecule SERS spectra and selection rules is a challenging task, and is still actively debated. Herein, we explore underappreciated chemical phenomena in ultrasensitive SERS. We observe a fluctuating excited electronic state manifold, governed by the conformational dynamics of a molecule (4,4’-dimercaptostilbene, DMS) interacting with a metallic cluster (Ag20). This affects our simulated single molecule SERS spectra; the time trajectories of a molecule interacting with its unique local environment dictates the relative intensities of the observable Raman-active vibrational states. Ab initio molecular dynamics of a model Ag20-DMS system are used to illustrate both concepts in light of recent experimental results.

  4. 2012 Gordon Research Conference, Single molecule approaches to biology, July 15-20 2012

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez, Julio M. [Columbia Univ., New York, NY (United States)

    2012-04-20

    Single molecule techniques are rapidly occupying a central role in biological research at all levels. This transition was made possible by the availability and dissemination of robust techniques that use fluorescence and force probes to track the conformation of molecules one at a time, in vitro as well as in live cells. Single-molecule approaches have changed the way many biological problems are studied. These novel techniques provide previously unobtainable data on fundamental biochemical processes that are essential for all forms of life. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of the molecular systems that underpin the functioning of living cells. Hence, our conference seeks to disseminate the implementation and use of single molecule techniques in the pursuit of new biological knowledge. Topics covered include: Molecular Motors on the Move; Origin And Fate Of Proteins; Physical Principles Of Life; Molecules and Super-resolution Microscopy; Nanoswitches In Action; Active Motion Or Random Diffusion?; Building Blocks Of Living Cells; From Molecular Mechanics To Physiology; Tug-of-war: Force Spectroscopy Of Single Proteins.

  5. A Nanofluidic Mixing Device for High-throughput Fluorescence Sensing of Single Molecules

    NARCIS (Netherlands)

    Mathwig, Klaus; Fijen, C.; Fontana, M.; Lemay, S.G.; Hohlbein, J.C.

    2017-01-01

    We introduce a nanofluidic mixing device entirely fabricated in glass for the fluorescence detection of single molecules. The design consists of a nanochannel T-junction and allows the continuous monitoring of chemical or enzymatic reactions of analytes as they arrive from two independent inlets.

  6. Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification

    Science.gov (United States)

    Vernick, Sefi; Trocchia, Scott M.; Warren, Steven B.; Young, Erik F.; Bouilly, Delphine; Gonzalez, Ruben L.; Nuckolls, Colin; Shepard, Kenneth L.

    2017-05-01

    The study of biomolecular interactions at the single-molecule level holds great potential for both basic science and biotechnology applications. Single-molecule studies often rely on fluorescence-based reporting, with signal levels limited by photon emission from single optical reporters. The point-functionalized carbon nanotube transistor, known as the single-molecule field-effect transistor, is a bioelectronics alternative based on intrinsic molecular charge that offers significantly higher signal levels for detection. Such devices are effective for characterizing DNA hybridization kinetics and thermodynamics and enabling emerging applications in genomic identification. In this work, we show that hybridization kinetics can be directly controlled by electrostatic bias applied between the device and the surrounding electrolyte. We perform the first single-molecule experiments demonstrating the use of electrostatics to control molecular binding. Using bias as a proxy for temperature, we demonstrate the feasibility of detecting various concentrations of 20-nt target sequences from the Ebolavirus nucleoprotein gene in a constant-temperature environment.

  7. Single-molecule studies of fork dynamics in Escherichia coli DNA replication

    NARCIS (Netherlands)

    Tanner, Nathan A.; Hamdan, Samir M.; Jergic, Slobodan; Loscha, Karin V.; Schaeffer, Patrick M.; Dixon, Nicholas E.; Oijen, Antoine M. van

    2008-01-01

    We present single-molecule studies of the Escherichia coli replication machinery. We visualize individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand

  8. Quantum interference effects at room temperature in OPV-based single-molecule junctions

    NARCIS (Netherlands)

    Arroyo Rodriguez, C.; Frisenda, R.; Moth-Poulsen, K.; Seldenthuis, J.S.; Bjornholm, T.; Van der Zant, H.S.

    2013-01-01

    Interference effects on charge transport through an individual molecule can lead to a notable modulation and suppression on its conductance. In this letter, we report the observation of quantum interference effects occurring at room temperature in single-molecule junctions based on

  9. Electrochemistry of single molecules and biomolecules, molecular scale nanostructures, and low-dimensional systems

    DEFF Research Database (Denmark)

    Nazmutdinov, Renat R.; Zinkicheva, Tamara T.; Zinkicheva, Tamara T.

    2018-01-01

    Electrochemistry at ultra-small scales, where even the single molecule or biomolecule can be characterized and manipulated, is on the way to a consolidated status. At the same time molecular electrochemistry is expanding into other areas of sophisticated nano- and molecular scale systems including...

  10. Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.

    NARCIS (Netherlands)

    van Mameren, J.; Gross, P.; Farge, G.; Hooijman, P.; Modesti, M.; Falkenberg, M.; Wuite, G.J.L.; Peterman, E.J.G.

    2009-01-01

    Single-molecule manipulation studies have revealed that double-stranded DNA undergoes a structural transition when subjected to tension. At forces that depend on the attachment geometry of the DNA (65 pN or 110 pN), it elongates ≈1.7-fold and its elastic properties change dramatically. The nature of

  11. Single-molecule studies of the twisted, knotted, and broken genome

    NARCIS (Netherlands)

    Van Loenhout, M.T.J.

    2012-01-01

    This thesis describes a series of single-molecule experiments aimed at understanding the physical properties of DNA itself and the proteins that interact with it. We developed and applied sensitive techniques that allowed us to directly probe the conformation and interactions of individual DNA

  12. Spectral multitude and spectral dynamics reflect changing conjugation length in single molecules of oligophenylenevinylenes

    KAUST Repository

    Kobayashi, Hiroyuki

    2012-01-01

    Single-molecule study of phenylenevinylene oligomers revealed distinct spectral forms due to different conjugation lengths which are determined by torsional defects. Large spectral jumps between different spectral forms were ascribed to torsional flips of a single phenylene ring. These spectral changes reflect the dynamic nature of electron delocalization in oligophenylenevinylenes and enable estimation of the phenylene torsional barriers. © 2012 The Owner Societies.

  13. Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

    Science.gov (United States)

    Oiwa, K; Eccleston, J F; Anson, M; Kikumoto, M; Davis, C T; Reid, G P; Ferenczi, M A; Corrie, J E; Yamada, A; Nakayama, H; Trentham, D R

    2000-06-01

    Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.

  14. Challenges for single molecule electronic devices with nanographene and organic molecules. Do single molecules offer potential as elements of electronic devices in the next generation?

    Science.gov (United States)

    Enoki, Toshiaki; Kiguchi, Manabu

    2018-03-01

    Interest in utilizing organic molecules to fabricate electronic materials has existed ever since organic (molecular) semiconductors were first discovered in the 1950s. Since then, scientists have devoted serious effort to the creation of various molecule-based electronic systems, such as molecular metals and molecular superconductors. Single-molecule electronics and the associated basic science have emerged over the past two decades and provided hope for the development of highly integrated molecule-based electronic devices in the future (after the Si-based technology era has ended). Here, nanographenes (nano-sized graphene) with atomically precise structures are among the most promising molecules that can be utilized for electronic/spintronic devices. To manipulate single small molecules for an electronic device, a single molecular junction has been developed. It is a powerful tool that allows even small molecules to be utilized. External electric, magnetic, chemical, and mechanical perturbations can change the physical and chemical properties of molecules in a way that is different from bulk materials. Therefore, the various functionalities of molecules, along with changes induced by external perturbations, allows us to create electronic devices that we cannot create using current top-down Si-based technology. Future challenges that involve the incorporation of condensed matter physics, quantum chemistry calculations, organic synthetic chemistry, and electronic device engineering are expected to open a new era in single-molecule device electronic technology.

  15. Magnetic Quantum Tunneling in Single Molecule Magnets: Mn-12 and Others

    Science.gov (United States)

    del Barco, Enrique

    2004-03-01

    Magnetic quantum tunneling (MQT) has been studied in single molecule magnets (SMMs) using a micro-Hall effect magnetometer in a superconducting high field vector magnet system that incorporates the possibility of applying pulsed microwave fields. Mn_12-acetate has been studied extensively over the years. However, only recently the symmetry of MQT and the nature of the transverse interactions important to MQT have been determined [1,2]. Magnetic measurements in the pure quantum tunneling regime (0.6 K) illustrate that an average crystal fourfold MQT symmetry is due to local molecular environments of twofold symmetry that are rotated by 90 degrees with respect to one another, confirming that disorder which lowers the molecule symmetry is important to MQT. We have studied a subset of these lower site symmetry molecules and present evidence for a Berry phase that results from a combination of second and forth order contributions to the transverse magnetic anisotropy. These observations are consistent with high frequency EPR studies of the transverse interactions in Mn_12-acetate [3]. Finally, we discuss recent experiments in which microwave radiation is applied to modulate MQT and characterize the lifetimes and coherence times of states that are superpositions of "up" and "down" high spin-projections. [1] E. del Barco, et al., Phys. Rev. Lett. 91, 047203 (2003) [2] S. Hill, et al., Phys. Rev. Lett. 90, 217204 (2003). [3] E. del Barco, A, D. Kent, R. S. Edwards, S. I. Jones, S. Hill, J. M. North, N. S. Dalal, E. M. Rumnberger, D. N. Hendrickson and G. Christou, to be published.

  16. Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy.

    Science.gov (United States)

    Pozzi, Nicola; Bystranowska, Dominika; Zuo, Xiaobing; Di Cera, Enrico

    2016-08-26

    The coagulation factor prothrombin has a complex spatial organization of its modular assembly that comprises the N-terminal Gla domain, kringle-1, kringle-2, and the C-terminal protease domain connected by three intervening linkers. Here we use single molecule Förster resonance energy transfer to access the conformational landscape of prothrombin in solution and uncover structural features of functional significance that extend recent x-ray crystallographic analysis. Prothrombin exists in equilibrium between two alternative conformations, open and closed. The closed conformation predominates (70%) and features an unanticipated intramolecular collapse of Tyr(93) in kringle-1 onto Trp(547) in the protease domain that obliterates access to the active site and protects the zymogen from autoproteolytic conversion to thrombin. The open conformation (30%) is more susceptible to chymotrypsin digestion and autoactivation, and features a shape consistent with recent x-ray crystal structures. Small angle x-ray scattering measurements of prothrombin wild type stabilized 70% in the closed conformation and of the mutant Y93A stabilized 80% in the open conformation directly document two envelopes that differ 50 Å in length. These findings reveal important new details on the conformational plasticity of prothrombin in solution and the drastic structural difference between its alternative conformations. Prothrombin uses the intramolecular collapse of kringle-1 onto the active site in the closed form to prevent autoactivation. The open-closed equilibrium also defines a new structural framework for the mechanism of activation of prothrombin by prothrombinase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Assaying the binding strength of G-quadruplex ligands using single-molecule TPM experiments.

    Science.gov (United States)

    Liu, Shih-Wei; Chu, Jen-Fei; Tsai, Cheng-Ting; Fang, Hung-Chih; Chang, Ta-Chau; Li, Hung-Wen

    2013-05-15

    G-quadruplexes are stable secondary structures formed by Hoogsteen base pairing of guanine-rich single-stranded DNA sequences in the presence of monovalent cations (Na(+) or K(+)). Folded G-quadruplex (G4) structures in human telomeres have been proposed as a potential target for cancer therapy. In this study, we used single-molecule tethered particle motion (TPM) experiments to assay the binding strength of possible G4 ligands. We found that individual single-stranded DNA molecules containing the human telomeric sequence d[AGGG(TTAGGG)3] fluctuated between the folded and the unfolded states in a 10 mM Na(+) solution at 37 °C. The durations of folded and unfolded states were single-exponentially distributed, and in return the folding and unfolding rate constants were 1.68 ± 0.01 and 1.63 ± 0.03 (s(-1)), respectively. In the presence of G4 ligands, such as TMPyP4, DODCI, BMVC, and BMVPA, the unfolding rate constant decreased appreciably. In addition, combining the Cu(2+)-induced G4 unfolding and TPM assay, we showed that BMVC and TMPyP4 are better G4 stabilizers than DODCI. The capability of monitoring the fluctuation between the folded and the unfolded state of G4 DNA in real time allows the determination of both kinetic and thermodynamic parameters in a single measurement and offers a simple way to assay binding strength under various conditions. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  18. Exploring the Interaction of Ruthenium(II) Polypyridyl Complexes with DNA Using Single-Molecule Techniques†

    Science.gov (United States)

    Mihailovic, Aleksandra; Vladescu, Ioana; McCauley, Micah; Ly, Elaine; Williams, Mark C.; Spain, Eileen M.; Nuñez, Megan E.

    2008-01-01

    Here we explore DNA binding by a family of ruthenium(II) polypyridyl complexes using an atomic force microscope (AFM) and optical tweezers. We demonstrate using AFM that Ru(bpy)2dppz2+ intercalates into DNA (Kb= 1.5 × 105 M−1), as does its close relative Ru(bpy)2dppx2+ (Kb= 1.5 × 105 M−1). However, intercalation by Ru(phen)32+ and other Ru(II) complexes with Kb's lower than Ru(bpy)2dppz2+ are difficult to determine using AFM because of competing aggregation and surface-binding phenomena. At the high Ru(II) concentrations required to evaluate intercalation, most of the DNA strands acquire a twisted, curled conformation that is impossible to measure accurately. The condensation of DNA on mica in the presence of polycations is well known, but it clearly precludes the accurate assessment by AFM of DNA intercalation by most Ru(II) complexes, though not by ethidium bromide and other monovalent intercalators. When stretching individual DNA molecules using optical tweezers the same limitation on high metal concentration does not exist. Using optical tweezers we show that Ru(phen)2dppz2+ intercalates avidly (Kb = 3.2 × 106 M−1) while Ru(bpy)32+ does not intercalate, even at micromolar ruthenium concentrations. Ru(phen)32+ is shown to intercalate weakly, i.e. at micromolar concentrations (Kb= 8.8 × 103 M−1). The distinct differences in DNA stretching behavior between Ru(phen)32+ and Ru(bpy)32+ clearly illustrate that intercalation can be distinguished from groove binding by pulling the DNA with optical tweezers. Our results demonstrate both the benefits and challenges of two single-molecule methods in exploring DNA binding, and help to elucidate the mode of binding of Ru(phen)32+. PMID:16649785

  19. Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase

    Science.gov (United States)

    Wang, Quan; Serban, Andrew J.; Wachter, Rebekka M.; Moerner, W. E.

    2018-03-01

    Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ˜0.1 s-1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.

  20. A Bayesian method for inferring quantitative information from FRET data

    Directory of Open Access Journals (Sweden)

    Lichten Catherine A

    2011-05-01

    Full Text Available Abstract Background Understanding biological networks requires identifying their elementary protein interactions and establishing the timing and strength of those interactions. Fluorescence microscopy and Förster resonance energy transfer (FRET have the potential to reveal such information because they allow molecular interactions to be monitored in living cells, but it is unclear how best to analyze FRET data. Existing techniques differ in assumptions, manipulations of data and the quantities they derive. To address this variation, we have developed a versatile Bayesian analysis based on clear assumptions and systematic statistics. Results Our algorithm infers values of the FRET efficiency and dissociation constant, Kd, between a pair of fluorescently tagged proteins. It gives a posterior probability distribution for these parameters, conveying more extensive information than single-value estimates can. The width and shape of the distribution reflects the reliability of the estimate and we used simulated data to determine how measurement noise, data quantity and fluorophore concentrations affect the inference. We are able to show why varying concentrations of donors and acceptors is necessary for estimating Kd. We further demonstrate that the inference improves if additional knowledge is available, for example of the FRET efficiency, which could be obtained from separate fluorescence lifetime measurements. Conclusions We present a general, systematic approach for extracting quantitative information on molecular interactions from FRET data. Our method yields both an estimate of the dissociation constant and the uncertainty associated with that estimate. The information produced by our algorithm can help design optimal experiments and is fundamental for developing mathematical models of biochemical networks.

  1. Standard guide for fretting fatigue testing

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 This guide defines terminology and covers general requirements for conducting fretting fatigue tests and reporting the results. It describes the general types of fretting fatigue tests and provides some suggestions on developing and conducting fretting fatigue test programs. 1.2 Fretting fatigue tests are designed to determine the effects of mechanical and environmental parameters on the fretting fatigue behavior of metallic materials. This guide is not intended to establish preference of one apparatus or specimen design over others, but will establish guidelines for adherence in the design, calibration, and use of fretting fatigue apparatus and recommend the means to collect, record, and reporting of the data. 1.3 The number of cycles to form a fretting fatigue crack is dependent on both the material of the fatigue specimen and fretting pad, the geometry of contact between the two, and the method by which the loading and displacement are imposed. Similar to wear behavior of materials, it is important t...

  2. Axisymmetric fretting analysis in coated cylinder

    Indian Academy of Sciences (India)

    stress, which in turn controls the severity of fretting. There are cases where the ... shear stress and the stress normal to the interface (particularly when it is tensile) control the fatigue life expectancy of ...... detonation gun sprayed Cu-Ni-In coatings on Ti-6Al-4V under plain fatigue and fretting fatigue loading. Mater. Sci. and ...

  3. FRET pair printing of fluorescent proteins

    NARCIS (Netherlands)

    Escalante, Maryana; Blum, Christian; Cesa, Yanina; Otto, Cees; Subramaniam, Vinod

    2009-01-01

    We report for the first time the directed assembly and characterization of FRET pairs on micrometer patterned surfaces. We used visible fluorescent proteins expressing a hexahistidine affinity tag as component molecules for the construction of the FRET constructs, where His(6)-EGFP served as donor

  4. Color control through FRET efficiency modulation using CDI (Conference Presentation)

    Science.gov (United States)

    Wolowelsky, Karni; Guyes, Eric; Rubin, Shimon; Suss, Matthew; Bercovici, Moran; Rotschild, Carmel

    2017-02-01

    Although much progress was made in light emitting devices, the ability to electrically control their spectral emission remains limited. We will present a novel approach and experimental results for dynamic color control, by electrically modulating the non-radiative Forster resonance energy transfer (FRET) efficiency between donor and acceptor dyes in a solution. FRET efficiency depends on the 6th power of the distance between donor and acceptor dye molecules, and thus, it is sensitive to variations in acceptor's concentration. Controlled acceptor concentrations could be achieved by attracting or repelling ionic dyes from the electrodes using a capacitive deionization (CDI) cell, with high surface area porous electrodes. This approach to dynamic color control may open new directions in 100% fill-factor displays, and can be expanded to energy saving applications such as controlling building's external wall emissivity. We studied the modulation of a single dye emission using a CDI cell with negatively charged Fluorescein Sodium Salt in aquatic solution. Photoluminescence was measured along few charging-discharging CDI cycles and showed the ability to control extensive optical response through CDI. We experimented with two types of FRET-pair dyes: a) anion-cation, where the acceptor and the donor ions are oppositely charged, and b) zwitterion and ion, where the donor is neutral. We found that electrical control on FRET in aquatic solution is weak, due to hydrophobic attractive interaction between the acceptor and the donor. In order to avoid this effect, we are experimenting FRET control in organic solvents. These results will be presented in the talk.

  5. rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

    Science.gov (United States)

    Nagy, Peter; Szabó, Ágnes; Váradi, Tímea; Kovács, Tamás; Batta, Gyula; Szöllősi, János

    2016-04-01

    Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

  6. Optical and Electrical Detection of Single Molecule Translocation through Carbon Nanotubes

    Science.gov (United States)

    Song, Weisi; Pang, Pei; He, Jin; Lindsay, Stuart

    2013-01-01

    Ion current through a single-walled carbon nanotube (SWCNT) was monitored at the same time as fluorescence was recorded from charged dye molecules translocating through the SWCNT. Fluorescence bursts generally follow ion-current peaks with a delay time consistent with diffusion from the end of the SWCNT to the fluorescence collection point. The fluorescence amplitude distribution of the bursts is consistent with single molecule signals. Thus each peak in the ion current flowing through the SWCNT is associated with the translocation of a single molecule. Ion current peaks (as opposed to blockades) were produced by both positively (Rhodamine 6G) and negatively (Alexa 546) charged molecules, showing that the charge filtering responsible for the current bursts is caused by the molecules themselves. PMID:23248975

  7. DNA-Based Single-Molecule Electronics: From Concept to Function

    Science.gov (United States)

    2018-01-01

    Beyond being the repository of genetic information, DNA is playing an increasingly important role as a building block for molecular electronics. Its inherent structural and molecular recognition properties render it a leading candidate for molecular electronics applications. The structural stability, diversity and programmability of DNA provide overwhelming freedom for the design and fabrication of molecular-scale devices. In the past two decades DNA has therefore attracted inordinate amounts of attention in molecular electronics. This review gives a brief survey of recent experimental progress in DNA-based single-molecule electronics with special focus on single-molecule conductance and I–V characteristics of individual DNA molecules. Existing challenges and exciting future opportunities are also discussed. PMID:29342091

  8. madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy

    Science.gov (United States)

    Yi, Jason; Manna, Asit; Barr, Valarie A.; Hong, Jennifer; Neuman, Keir C.; Samelson, Lawrence E.

    2016-01-01

    Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM. PMID:27708141

  9. Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy.

    Science.gov (United States)

    Goudsmits, Joris M H; van Oijen, Antoine M; Slotboom, Dirk J

    2017-01-01

    Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data. © 2017 Elsevier Inc. All rights reserved.

  10. Radio frequency scanning tunneling spectroscopy for single-molecule spin resonance.

    Science.gov (United States)

    Müllegger, Stefan; Tebi, Stefano; Das, Amal K; Schöfberger, Wolfgang; Faschinger, Felix; Koch, Reinhold

    2014-09-26

    We probe nuclear and electron spins in a single molecule even beyond the electromagnetic dipole selection rules, at readily accessible magnetic fields (few mT) and temperatures (5 K) by resonant radio-frequency current from a scanning tunneling microscope. We achieve subnanometer spatial resolution combined with single-spin sensitivity, representing a 10 orders of magnitude improvement compared to existing magnetic resonance techniques. We demonstrate the successful resonant spectroscopy of the complete manifold of nuclear and electronic magnetic transitions of up to ΔI(z)=±3 and ΔJ(z)=±12 of single quantum spins in a single molecule. Our method of resonant radio-frequency scanning tunneling spectroscopy offers, atom-by-atom, unprecedented analytical power and spin control with an impact on diverse fields of nanoscience and nanotechnology.

  11. Single-Molecule Electrochemical Transistor Utilizing a Nickel-Pyridyl Spinterface

    DEFF Research Database (Denmark)

    Brooke, Richard J.; Jin, Chengjun; Szumski, Doug S.

    2015-01-01

    Using a scanning tunnelling microscope break-junction technique, we produce 4,4′-bipyridine (44BP) single-molecule junctions with Ni and Au contacts. Electrochemical control is used to prevent Ni oxidation and to modulate the conductance of the devices via nonredox gating - the first time this has...... been shown using non-Au contacts. Remarkably the conductance and gain of the resulting Ni-44BP-Ni electrochemical transistors is significantly higher than analogous Au-based devices. Ab-initio calculations reveal that this behavior arises because charge transport is mediated by spin-polarized Ni d......-electrons, which hybridize strongly with molecular orbitals to form a "spinterface". Our results highlight the important role of the contact material for single-molecule devices and show that it can be varied to provide control of charge and spin transport....

  12. Inelastic transport and low-bias rectification in a single-molecule diode.

    Science.gov (United States)

    Hihath, Joshua; Bruot, Christopher; Nakamura, Hisao; Asai, Yoshihiro; Díez-Pérez, Ismael; Lee, Youngu; Yu, Luping; Tao, Nongjian

    2011-10-25

    Designing, controlling, and understanding rectification behavior in molecular-scale devices has been a goal of the molecular electronics community for many years. Here we study the transport behavior of a single molecule diode, and its nonrectifying, symmetric counterpart at low temperatures, and at both low and high biases to help elucidate the electron-phonon interactions and transport mechanisms in the rectifying system. We find that the onset of current rectification occurs at low biases, indicating a significant change in the elastic transport pathway. However, the peaks in the inelastic electron tunneling (IET) spectrum are antisymmetric about zero bias and show no significant changes in energy or intensity in the forward or reverse bias directions, indicating that despite the change in the elastic transmission probability there is little impact on the inelastic pathway. These results agree with first principles calculations performed to evaluate the IETS, which also allow us to identify which modes are active in the single molecule junction.

  13. Single-molecule junction of an overcrowded ethylene with binary conductance states

    Science.gov (United States)

    Koike, Masato; Fujii, Shintaro; Cho, Haruna; Shoji, Yoshiaki; Nishino, Tomoaki; Fukushima, Takanori; Kiguchi, Manabu

    2018-03-01

    Overcrowded ethylene 1 shows mechanochromic behavior with contrasting color change between yellow and violet arising from its conformational isomerization, which should also be accompanied by the change in the electronic structure. Here, we report a single-molecule electronic study of 1 using scanning tunneling microcopy (STM) and STM-based break junction techniques. The single-molecule junction of 1 sandwiched by Au electrodes showed two distinct high- and low-conductance states with the conductance values of 0.003 and 0.0002 G 0. The high-conductance state is one order of magnitude more conductive than the low-conductance state. The two states can be ascribed to two conformational isomers of 1 in the junction.

  14. A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell

    Science.gov (United States)

    Xie, Sunney; Choi, Paul; Cai, Long

    2009-03-01

    By monitoring fluorescently labeled lactose permease with single-molecule sensitivity, we investigated the molecular mechanism of how an Escherichia coli cell with the lac operon switches from one phenotype to another. At intermediate inducer concentrations, a population of genetically identical cells exhibits two phenotypes: induced cells with highly fluorescent membranes and uninduced cells with a small number of membrane-bound permeases. We found that this basal-level expression results from partial dissociation of the tetrameric lactose repressor from one of its operators on looped DNA. In contrast, infrequent events of complete dissociation of the repressor from DNA result in large bursts of permease expression that trigger induction of the lac operon. Hence, a stochastic single-molecule event determines a cell's phenotype.

  15. PhotoGate microscopy: tracking single molecules in a cytoplasm (Conference Presentation)

    Science.gov (United States)

    Yildiz, Ahmet

    2016-02-01

    Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density in the cellular environment. We developed a photobleaching gate assay, which controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. Using this method, we tracked single particles at surface densities two orders of magnitude higher than the single-molecule detection limit. We observed ligand-induced dimerization of epidermal growth factor receptors (EGFR) on a live cell membrane. In addition, we tracked individual intraflagellar transport (IFT) trains along the length of a cilium and observed their remodeling at the ciliary tip.

  16. Single-Molecule Imaging with X-Ray Free-Electron Lasers: Dream or Reality?

    KAUST Repository

    Fratalocchi, Andrea

    2011-03-09

    X-ray free-electron lasers (XFEL) are revolutionary photon sources, whose ultrashort, brilliant pulses are expected to allow single-molecule diffraction experiments providing structural information on the atomic length scale of nonperiodic objects. This ultimate goal, however, is currently hampered by several challenging questions basically concerning sample damage, Coulomb explosion, and the role of nonlinearity. By employing an original ab initio approach, we address these issues showing that XFEL-based single-molecule imaging will be only possible with a few-hundred long attosecond pulses, due to significant radiation damage and the formation of preferred multisoliton clusters which reshape the overall electronic density of the molecular system at the femtosecond scale.

  17. Single-Molecule Imaging with X-Ray Free-Electron Lasers: Dream or Reality?

    International Nuclear Information System (INIS)

    Fratalocchi, A.; Ruocco, G.

    2011-01-01

    X-ray free-electron lasers (XFEL) are revolutionary photon sources, whose ultrashort, brilliant pulses are expected to allow single-molecule diffraction experiments providing structural information on the atomic length scale of nonperiodic objects. This ultimate goal, however, is currently hampered by several challenging questions basically concerning sample damage, Coulomb explosion, and the role of nonlinearity. By employing an original ab initio approach, we address these issues showing that XFEL-based single-molecule imaging will be only possible with a few-hundred long attosecond pulses, due to significant radiation damage and the formation of preferred multisoliton clusters which reshape the overall electronic density of the molecular system at the femtosecond scale.

  18. Influence of quantum dot labels on single molecule movement in the plasma membrane

    DEFF Research Database (Denmark)

    Clausen, Mathias P.; Lagerholm, B. Christoffer

    2011-01-01

    Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues...... in labeling single molecules with QDs (and other particles e.g. gold particles) are induction of cross-linking of the target molecules, which can cause activation of signaling pathways or reduced mobility, and steric hindrance as a result of the probe size. Cross-linking can be a result of the multivalent...... functionalization tag (e.g. streptavidin (sAv)) or the presence of multiple mono- or multivalent functionalization tags per QD. In this work, we have compared commercially available sAv-QDs of different sizes with custom prepared Co enzyme A (CoA)-QDs both targeting a GPI-anchored protein modified with either...

  19. Flicker Noise as a Probe of Electronic Interaction at Metal-Single Molecule Interfaces.

    Science.gov (United States)

    Adak, Olgun; Rosenthal, Ethan; Meisner, Jeffery; Andrade, Erick F; Pasupathy, Abhay N; Nuckolls, Colin; Hybertsen, Mark S; Venkataraman, Latha

    2015-06-10

    Charge transport properties of metal-molecule interfaces depend strongly on the character of molecule-electrode interactions. Although through-bond coupled systems have attracted the most attention, through-space coupling is important in molecular systems when, for example, through-bond coupling is suppressed due to quantum interference effects. To date, a probe that clearly distinguishes these two types of coupling has not yet been demonstrated. Here, we investigate the origin of flicker noise in single molecule junctions and demonstrate how the character of the molecule-electrode coupling influences the flicker noise behavior of single molecule junctions. Importantly, we find that flicker noise shows a power law dependence on conductance in all junctions studied with an exponent that can distinguish through-space and through-bond coupling. Our results provide a new and powerful tool for probing and understanding coupling at the metal-molecule interface.

  20. Supramolecular Chemistry and Mechanochemistry of Macromolecules: Recent Advances by Single-Molecule Force Spectroscopy.

    Science.gov (United States)

    Cheng, Bo; Cui, Shuxun

    2015-01-01

    Atomic force spectroscopy (AFM)-based single-molecule force spectroscopy (SMFS) was invented in the 1990s. Since then, SMFS has been developed into a powerful tool to study the inter- and intra-molecular interactions of macromolecules. Using SMFS, a number of problems in the field of supramolecular chemistry and mechanochemistry have been studied at the single-molecule level, which are not accessible by traditional ensemble characterization methods. In this review, the principles of SMFS are introduced, followed by the discussion of several problems of contemporary interest at the interface of supramolecular chemistry and mechanochemistry of macromolecules, including single-chain elasticity of macromolecules, interactions between water and macromolecules, interactions between macromolecules and solid surface, and the interactions in supramolecular polymers.