DNA-psoralen interaction: a single molecule experiment.

PubMed

Rocha, M S; Viana, N B; Mesquita, O N

2004-11-15

By attaching one end of a single lambda-DNA molecule to a microscope coverslip and the other end to a polystyrene microsphere trapped by an optical tweezers, we can study the entropic elasticity of the lambda-DNA by measuring force versus extension as we stretch the molecule. This powerful method permits single molecule studies. We are particularly interested in the effects of the photosensitive drug psoralen on the elasticity of the DNA molecule. We have illuminated the sample with different light sources, studying how the different wavelengths affect the psoralen-DNA linkage. To do this, we measure the persistence length of individual DNA-psoralen complexes.

Kondo effect in single cobalt phthalocyanine molecules adsorbed on Au(111) monoatomic steps

NASA Astrophysics Data System (ADS)

Zhao, Aidi; Hu, Zhenpeng; Wang, Bing; Xiao, Xudong; Yang, Jinlong; Hou, J. G.

2008-06-01

The Kondo effect in single dehydrogenated cobalt phthalocyanine (CoPc) molecules adsorbed on Au(111) monoatomic steps was studied with a low temperature scanning tunneling microscope. The CoPc molecules adsorbed on Au(111) monoatomic steps show two typical configurations, which can be dehydrogenated to reveal Kondo effect. Moreover, the Kondo temperatures (TK) measured for different molecules vary in a large range from ~150 to ~550 K, increasing monotonically with decreasing Co-Au distance. A simple model consisting of a single Co 3dz2 orbital and a Au 6s orbital is considered and gives a qualitative explanation to the dependence. The large variation of TK is attributed to the variation of the interaction between the magnetic-active cobalt ion and the Au substrate resulted from different Co-Au distances.

Single-molecule electronics: Cooling individual vibrational modes by the tunneling current.

PubMed

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.

Giant light-harvesting nanoantenna for single-molecule detection in ambient light

PubMed Central

Trofymchuk, Kateryna; Reisch, Andreas; Didier, Pascal; Fras, François; Gilliot, Pierre; Mely, Yves; Klymchenko, Andrey S.

2017-01-01

Here, we explore the enhancement of single molecule emission by polymeric nano-antenna that can harvest energy from thousands of donor dyes to a single acceptor. In this nano-antenna, the cationic dyes are brought together in very close proximity using bulky counterions, thus enabling ultrafast diffusion of excitation energy (≤30 fs) with minimal losses. Our 60-nm nanoparticles containing >10,000 rhodamine-based donor dyes can efficiently transfer energy to 1-2 acceptors resulting in an antenna effect of ~1,000. Therefore, single Cy5-based acceptors become 25-fold brighter than quantum dots QD655. This unprecedented amplification of the acceptor dye emission enables observation of single molecules at illumination powers (1-10 mW cm-2) that are >10,000-fold lower than typically required in single-molecule measurements. Finally, using a basic setup, which includes a 20X air objective and a sCMOS camera, we could detect single Cy5 molecules by simply shining divergent light on the sample at powers equivalent to sunlight. PMID:28983324

DNA molecule stretching through thermo-electrophoresis and thermal convection in a heated converging-diverging microchannel.

PubMed

Hsieh, Shou-Shing; Chen, Jyun-Hong; Tsai, Cheng-Fung

2013-02-18

A novel DNA molecule stretching technique is developed and tested herein. Through a heated converging-diverging microchannel, thermal convection and thermophoresis induced by regional heating are shown to significantly elongate single DNA molecules; they are visualized via a confocal laser scanning microscopy. In addition, electrophoretic stretching is also implemented to examine the hybrid effect on the conformation and dynamics of single DNA molecules. The physical properties of the DNA molecules are secured via experimental measurements.

Theoretical Investigation of Single-Molecule Sensing Using Nanotube-Enhanced Circular Dichroism.

PubMed

Silva, Jaime; Milne, Bruce F; Nogueira, Fernando

2018-06-19

First-principles calculations have been used to investigate the potential use of circular dichroism (CD) spectroscopy in single-molecule sensing. Using a real-space implementation of time-dependent density functional theory (TDDFT), several systems involving single-walled carbon nanotubes (SWCNT) and small molecules have been studied to evaluate their CD response. Large induced CD (ICD) effects, differing for each test molecule, were observed in all SWCNT-molecule complexes. As the SWCNT used in this study shows no intrinsic CD response, the ICD spectra are the result of interaction with the small molecules. This finding is general and independent of the (a)chiral nature of the adsorbed molecule. Our results indicate that it is possible to design a system that uses SWCNT for detection of molecules using the change in CD spectrum of the system induced by adsorption of the molecule onto the SWCNT surface.

Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances

NASA Astrophysics Data System (ADS)

Moerner, William E.

The initial steps toward optical detection and spectroscopy of single molecules arose out of the study of spectral hole-burning in inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989. In the early 1990s, many fascinating physical effects were observed for individual molecules such as spectral diffusion, optical switching, vibrational spectra, and magnetic resonance of a single molecular spin. Since the mid-1990s when experiments moved to room temperature, a wide variety of biophysical effects may be explored, and a number of physical phenomena from the low temperature studies have analogs at high temperature. Recent advances worldwide cover a huge range, from in vitro studies of enzymes, proteins, and oligonucleotides, to observations in real time of a single protein performing a specific function inside a living cell. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation of individual fluorophores leads naturally to localization beyond the optical diffraction limit. Combining this with active optical control of the number of emitting molecules leads to superresolution imaging, a new frontier for optical microscopy beyond the optical diffraction limit and for chemical design of photoswitchable fluorescent labels. Finally, to study one molecule in aqueous solution without surface perturbations, a new electrokinetic trap is described (the ABEL trap) which can trap single small biomolecules without the need for large dielectric beads.

Improved Dye Stability in Single-Molecule Fluorescence Experiments

NASA Astrophysics Data System (ADS)

EcheverrÍa Aitken, Colin; Marshall, R. Andrew; Pugi, Joseph D.

Complex biological systems challenge existing single-molecule methods. In particular, dye stability limits observation time in singlemolecule fluorescence applications. Current approaches to improving dye performance involve the addition of enzymatic oxygen scavenging systems and small molecule additives. We present an enzymatic oxygen scavenging system that improves dye stability in single-molecule experiments. Compared to the currently-employed glucose-oxidase/catalase system, the protocatechuate-3,4-dioxygenase system achieves lower dissolved oxygen concentration and stabilizes single Cy3, Cy5, and Alexa488 fluorophores. Moreover, this system possesses none of the limitations associated with the glucose oxidase/catalase system. We also tested the effects of small molecule additives in this system. Biological reducing agents significantly destabilize the Cy5 fluorophore as a function of reducing potential. In contrast, anti-oxidants stabilize the Cy3 and Alexa488 fluorophores. We recommend use of the protocatechuate-3,4,-dioxygenase system with antioxidant additives, and in the absence of biological reducing agents. This system should have wide application to single-molecule fluorescence experiments.

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

NASA Astrophysics Data System (ADS)

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.

Video-rate nanoscopy enabled by sCMOS camera-specific single-molecule localization algorithms

PubMed Central

Huang, Fang; Hartwich, Tobias M. P.; Rivera-Molina, Felix E.; Lin, Yu; Duim, Whitney C.; Long, Jane J.; Uchil, Pradeep D.; Myers, Jordan R.; Baird, Michelle A.; Mothes, Walther; Davidson, Michael W.; Toomre, Derek; Bewersdorf, Joerg

2013-01-01

Newly developed scientific complementary metal–oxide–semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition in single-molecule switching nanoscopy (SMSN) while simultaneously increasing the effective quantum efficiency. However, sCMOS-intrinsic pixel-dependent readout noise substantially reduces the localization precision and introduces localization artifacts. Here we present algorithms that overcome these limitations and provide unbiased, precise localization of single molecules at the theoretical limit. In combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at up to 32 reconstructed images/second (recorded at 1,600–3,200 camera frames/second) in both fixed and living cells. PMID:23708387

Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

Simple method of DNA stretching on glass substrate for fluorescence image and spectroscopy

NASA Astrophysics Data System (ADS)

Neupane, Guru P.; Dhakal, Krishna P.; Lee, Hyunsoo; Guthold, Martin; Joseph, Vincent S.; Hong, Jong-Dal; Kim, Jeongyong

2013-05-01

Study of biological molecule DNA has contributed to developing many breaking thoughts and wide applications in multidisciplinary fields, such as genomic, medical, sensing and forensic fields. Stretching of DNA molecules is an important supportive tool for AFM or spectroscopic studies of DNA in a single molecular level. In this article, we established a simple method of DNA stretching (to its full length) that occurred on a rotating negatively-charged surface of glass substrate. The isolation of a single DNA molecule was attained by the two competitive forces on DNA molecules, that is, the electrostatic attraction developed between the positively charged YOYO-1 stained DNA and the negatively charged substrate, and the centrifugal force of the rotating substrate, which separates the DNA aggregates into the single molecule. Density of stretched DNA molecules was controlled by selecting the specific parameters such as spinning time and rates, loading volume of DNA-dye complex solution etc. The atomic force microscopy image exhibited a single DNA molecule on the negatively-charged substrate in an isolated state. Further, the photoluminescence spectra of a single DNA molecule stained with YOYO-1 were achieved using the method developed in the present study, which is strongly believed to effectively support the spectroscopic analysis of DNA in a single molecular level.

Stiffness, working stroke, and force of single-myosin molecules in skeletal muscle: elucidation of these mechanical properties via nonlinear elasticity evaluation.

PubMed

Kaya, Motoshi; Higuchi, Hideo

2013-11-01

In muscles, the arrays of skeletal myosin molecules interact with actin filaments and continuously generate force at various contraction speeds. Therefore, it is crucial for myosin molecules to generate force collectively and minimize the interference between individual myosin molecules. Knowledge of the elasticity of myosin molecules is crucial for understanding the molecular mechanisms of muscle contractions because elasticity directly affects the working and drag (resistance) force generation when myosin molecules are positively or negatively strained. The working stroke distance is also an important mechanical property necessary for elucidation of the thermodynamic efficiency of muscle contractions at the molecular level. In this review, we focus on these mechanical properties obtained from single-fiber and single-molecule studies and discuss recent findings associated with these mechanical properties. We also discuss the potential molecular mechanisms associated with reduction of the drag effect caused by negatively strained myosin molecules.

Detection and Spectroscopy of Single Pentacene Molecules in a p- Terphenyl Crystal by Means of Fluorescence Excitation

DTIC Science & Technology

1991-07-22

suggesting that the effect may be ite td’ phonon-assisted transitions of local degrees of freedom around the pentacene defect whose source remains to...the effect may be due to phonon-assisted transitions of local degrees of freedom around the pentacene defect whose source remains to be identified...single molecule of pentacene in p-terphenyl is an exquisitely sensitive probe of the detailed local environment around the impurity molecule. It appears

Far-field photostable optical nanoscopy (PHOTON) for real-time super-resolution single-molecular imaging of signaling pathways of single live cells

NASA Astrophysics Data System (ADS)

Huang, Tao; Browning, Lauren M.; Xu, Xiao-Hong Nancy

2012-04-01

Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11739h

Experimental Evidence for Quantum Interference and Vibrationally Induced Decoherence in Single-Molecule Junctions

NASA Astrophysics Data System (ADS)

Ballmann, Stefan; Härtle, Rainer; Coto, Pedro B.; Elbing, Mark; Mayor, Marcel; Bryce, Martin R.; Thoss, Michael; Weber, Heiko B.

2012-08-01

We analyze quantum interference and decoherence effects in single-molecule junctions both experimentally and theoretically by means of the mechanically controlled break junction technique and density-functional theory. We consider the case where interference is provided by overlapping quasidegenerate states. Decoherence mechanisms arising from electronic-vibrational coupling strongly affect the electrical current flowing through a single-molecule contact and can be controlled by temperature variation. Our findings underline the universal relevance of vibrations for understanding charge transport through molecular junctions.

Experimental evidence for quantum interference and vibrationally induced decoherence in single-molecule junctions.

PubMed

Ballmann, Stefan; Härtle, Rainer; Coto, Pedro B; Elbing, Mark; Mayor, Marcel; Bryce, Martin R; Thoss, Michael; Weber, Heiko B

2012-08-03

We analyze quantum interference and decoherence effects in single-molecule junctions both experimentally and theoretically by means of the mechanically controlled break junction technique and density-functional theory. We consider the case where interference is provided by overlapping quasidegenerate states. Decoherence mechanisms arising from electronic-vibrational coupling strongly affect the electrical current flowing through a single-molecule contact and can be controlled by temperature variation. Our findings underline the universal relevance of vibrations for understanding charge transport through molecular junctions.

Superconducting molybdenum-rhenium electrodes for single-molecule transport studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaudenzi, R.; Island, J. O.; Bruijckere, J. de

2015-06-01

We demonstrate that electronic transport through single molecules or molecular ensembles, commonly based on gold (Au) electrodes, can be extended to superconducting electrodes by combining gold with molybdenum-rhenium (MoRe). This combination induces proximity-effect superconductivity in the gold to temperatures of at least 4.6 K and magnetic fields of 6 T, improving on previously reported aluminum based superconducting nanojunctions. As a proof of concept, we show three-terminal superconductive transport measurements through an individual Fe{sub 4} single-molecule magnet.

Connection between the conformation and emission properties of poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] single molecules during thermal annealing

NASA Astrophysics Data System (ADS)

Ou, Jiemei; Yang, Yuzhao; Lin, Wensheng; Yuan, Zhongke; Gan, Lin; Lin, Xiaofeng; Chen, Xudong; Chen, Yujie

2015-03-01

We investigated the transitions of conformations and their effects on emission properties of poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) single molecules in PMMA matrix during thermal annealing process. Total internal reflection fluorescence microscopy measurements reveal the transformation from collapsed conformations to extended, highly ordered rod-like structures of MEH-PPV single molecules during thermal annealing. The blue shifts in the ensemble single molecule PL spectra support our hypnosis. The transition occurs as the annealing temperature exceeds 100 °C, implying that an annealing temperature near the glass transition temperature Tg of matrix is ideal for the control and optimization of blend polymer films.

Communication: Coordinate-dependent diffusivity from single molecule trajectories

NASA Astrophysics Data System (ADS)

Berezhkovskii, Alexander M.; Makarov, Dmitrii E.

2017-11-01

Single-molecule observations of biomolecular folding are commonly interpreted using the model of one-dimensional diffusion along a reaction coordinate, with a coordinate-independent diffusion coefficient. Recent analysis, however, suggests that more general models are required to account for single-molecule measurements performed with high temporal resolution. Here, we consider one such generalization: a model where the diffusion coefficient can be an arbitrary function of the reaction coordinate. Assuming Brownian dynamics along this coordinate, we derive an exact expression for the coordinate-dependent diffusivity in terms of the splitting probability within an arbitrarily chosen interval and the mean transition path time between the interval boundaries. This formula can be used to estimate the effective diffusion coefficient along a reaction coordinate directly from single-molecule trajectories.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lykkebo, Jacob; Solomon, Gemma C., E-mail: gsolomon@nano.ku.dk; Romano, Giuseppe

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, whichmore » 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.« less

Single Molecule Effects of Osteogenesis Imperfecta Mutations in Tropocollagen Protein Domains

DTIC Science & Technology

2008-12-02

Single molecule effects of osteogenesis imperfecta mutations in tropocollagen protein domains Alfonso Gautieri,1,2 Simone Vesentini,2 Alberto...2008 proteinscience.org Abstract: Osteogenesis imperfecta (OI) is a genetic disease characterized by fragile bones, skeletal deformities and, in severe...diagnosis and treatment, an effort referred to as materiomics. Keywords: steered molecular dynamics; osteogenesis imperfecta ; Young’s modulus; collagen

Nanopore extended field-effect transistor for selective single-molecule biosensing.

PubMed

Ren, Ren; Zhang, Yanjun; Nadappuram, Binoy Paulose; Akpinar, Bernice; Klenerman, David; Ivanov, Aleksandar P; Edel, Joshua B; Korchev, Yuri

2017-09-19

There has been a significant drive to deliver nanotechnological solutions to biosensing, yet there remains an unmet need in the development of biosensors that are affordable, integrated, fast, capable of multiplexed detection, and offer high selectivity for trace analyte detection in biological fluids. Herein, some of these challenges are addressed by designing a new class of nanoscale sensors dubbed nanopore extended field-effect transistor (nexFET) that combine the advantages of nanopore single-molecule sensing, field-effect transistors, and recognition chemistry. We report on a polypyrrole functionalized nexFET, with controllable gate voltage that can be used to switch on/off, and slow down single-molecule DNA transport through a nanopore. This strategy enables higher molecular throughput, enhanced signal-to-noise, and even heightened selectivity via functionalization with an embedded receptor. This is shown for selective sensing of an anti-insulin antibody in the presence of its IgG isotype.Efficient detection of single molecules is vital to many biosensing technologies, which require analytical platforms with high selectivity and sensitivity. Ren et al. combine a nanopore sensor and a field-effect transistor, whereby gate voltage mediates DNA and protein transport through the nanopore.

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

Contact and Length Dependent Effects in Single-Molecule Electronics

NASA Astrophysics Data System (ADS)

Hines, Thomas

Understanding charge transport in single molecules covalently bonded to electrodes is a fundamental goal in the field of molecular electronics. In the past decade, it has become possible to measure charge transport on the single-molecule level using the STM break junction method. Measurements on the single-molecule level shed light on charge transport phenomena which would otherwise be obfuscated by ensemble measurements of groups of molecules. This thesis will discuss three projects carried out using STM break junction. In the first project, the transition between two different charge transport mechanisms is reported in a set of molecular wires. The shortest wires show highly length dependent and temperature invariant conductance behavior, whereas the longer wires show weakly length dependent and temperature dependent behavior. This trend is consistent with a model whereby conduction occurs by coherent tunneling in the shortest wires and by incoherent hopping in the longer wires. Measurements are supported with calculations and the evolution of the molecular junction during the pulling process is investigated. The second project reports controlling the formation of single-molecule junctions by means of electrochemically reducing two axial-diazonium terminal groups on a molecule, thereby producing direct Au-C covalent bonds in-situ between the molecule and gold electrodes. Step length analysis shows that the molecular junction is significantly more stable, and can be pulled over a longer distance than a comparable junction created with amine anchoring bonds. The stability of the junction is explained by the calculated lower binding energy associated with the direct Au-C bond compared with the Au-N bond. Finally, the third project investigates the role that molecular conformation plays in the conductance of oligothiophene single-molecule junctions. Ethyl substituted oligothiophenes were measured and found to exhibit temperature dependent conductance and transition voltage for molecules with between two and six repeat units. While the molecule with only one repeat unit shows temperature invariant behavior. Density functional theory calculations show that at higher temperatures the oligomers with multiple repeat units assume a more planar conformation, which increases the conjugation length and decreases the effective energy barrier of the junction.

Multi-Scale Modeling to Improve Single-Molecule, Single-Cell Experiments

NASA Astrophysics Data System (ADS)

Munsky, Brian; Shepherd, Douglas

2014-03-01

Single-cell, single-molecule experiments are producing an unprecedented amount of data to capture the dynamics of biological systems. When integrated with computational models, observations of spatial, temporal and stochastic fluctuations can yield powerful quantitative insight. We concentrate on experiments that localize and count individual molecules of mRNA. These high precision experiments have large imaging and computational processing costs, and we explore how improved computational analyses can dramatically reduce overall data requirements. In particular, we show how analyses of spatial, temporal and stochastic fluctuations can significantly enhance parameter estimation results for small, noisy data sets. We also show how full probability distribution analyses can constrain parameters with far less data than bulk analyses or statistical moment closures. Finally, we discuss how a systematic modeling progression from simple to more complex analyses can reduce total computational costs by orders of magnitude. We illustrate our approach using single-molecule, spatial mRNA measurements of Interleukin 1-alpha mRNA induction in human THP1 cells following stimulation. Our approach could improve the effectiveness of single-molecule gene regulation analyses for many other process.

Thermoelectric effect and its dependence on molecular length and sequence in single DNA molecules.

PubMed

Li, Yueqi; Xiang, Limin; Palma, Julio L; Asai, Yoshihiro; Tao, Nongjian

2016-04-15

Studying the thermoelectric effect in DNA is important for unravelling charge transport mechanisms and for developing relevant applications of DNA molecules. Here we report a study of the thermoelectric effect in single DNA molecules. By varying the molecular length and sequence, we tune the charge transport in DNA to either a hopping- or tunnelling-dominated regimes. The thermoelectric effect is small and insensitive to the molecular length in the hopping regime. In contrast, the thermoelectric effect is large and sensitive to the length in the tunnelling regime. These findings indicate that one may control the thermoelectric effect in DNA by varying its sequence and length. We describe the experimental results in terms of hopping and tunnelling charge transport models.

Thermoelectric effect and its dependence on molecular length and sequence in single DNA molecules

PubMed Central

Li, Yueqi; Xiang, Limin; Palma, Julio L.; Asai, Yoshihiro; Tao, Nongjian

2016-01-01

Studying the thermoelectric effect in DNA is important for unravelling charge transport mechanisms and for developing relevant applications of DNA molecules. Here we report a study of the thermoelectric effect in single DNA molecules. By varying the molecular length and sequence, we tune the charge transport in DNA to either a hopping- or tunnelling-dominated regimes. The thermoelectric effect is small and insensitive to the molecular length in the hopping regime. In contrast, the thermoelectric effect is large and sensitive to the length in the tunnelling regime. These findings indicate that one may control the thermoelectric effect in DNA by varying its sequence and length. We describe the experimental results in terms of hopping and tunnelling charge transport models. PMID:27079152

Tuning charge and correlation effects for a single molecule on a graphene device

DOE PAGES

Wickenburg, Sebastian; Lu, Jiong; Lischner, Johannes; ...

2016-11-25

The ability to understand and control the electronic properties of individual molecules in a device environment is crucial for developing future technologies at the nanometre scale and below. Achieving this, however, requires the creation of three-terminal devices that allow single molecules to be both gated and imaged at the atomic scale. We have accomplished this by integrating a graphene field effect transistor with a scanning tunnelling microscope, thus allowing gate-controlled charging and spectroscopic interrogation of individual tetrafluoro-tetracyanoquinodimethane molecules. We observe a non-rigid shift in the molecule’s lowest unoccupied molecular orbital energy (relative to the Dirac point) as a function ofmore » gate voltage due to graphene polarization effects. Our results show that electron–electron interactions play an important role in how molecular energy levels align to the graphene Dirac point, and may significantly influence charge transport through individual molecules incorporated in graphene-based nanodevices.« less

Amplified emission and lasing in a plasmonic nanolaser with many three-level molecules

NASA Astrophysics Data System (ADS)

Zhang, Yuan; Mølmer, Klaus

2018-01-01

Steady-state plasmonic lasing is studied theoretically for a system consisting of many dye molecules arranged regularly around a gold nanosphere. A three-level model with realistic molecular dissipation is employed to analyze the performance as a function of the pump field amplitude and number of molecules. Few molecules and moderate pumping produce a single narrow emission peak because the excited molecules transfer energy to a single dipole plasmon mode by amplified spontaneous emission. Under strong pumping, the single peak splits into broader and weaker emission peaks because two molecular excited levels interfere with each other through coherent coupling with the pump field and with the dipole plasmon field. A large number of molecules gives rise to a Poisson-like distribution of plasmon number states with a large mean number characteristic of lasing action. These characteristics of lasing, however, deteriorate under strong pumping because of the molecular interference effect.

Photonic-plasmonic hybrid single-molecule nanosensor measures the effect of fluorescent labels on DNA-protein dynamics

PubMed Central

Liang, Feng; Guo, Yuzheng; Hou, Shaocong; Quan, Qimin

2017-01-01

Current methods to study molecular interactions require labeling the subject molecules with fluorescent reporters. However, the effect of the fluorescent reporters on molecular dynamics has not been quantified because of a lack of alternative methods. We develop a hybrid photonic-plasmonic antenna-in-a-nanocavity single-molecule biosensor to study DNA-protein dynamics without using fluorescent labels. Our results indicate that the fluorescein and fluorescent protein labels decrease the interaction between a single DNA and a protein due to weakened electrostatic interaction. Although the study is performed on the DNA-XPA system, the conclusion has a general implication that the traditional fluorescent labeling methods might be misestimating the molecular interactions. PMID:28560341

Thermal bleaching in single fluorescent molecules under two-photon excitation regime

NASA Astrophysics Data System (ADS)

Chirico, Giuseppe; Cannone, Fabio; Baldini, Giancarlo; Diaspro, Alberto

2003-07-01

Single molecule spectroscopy often requires the immobilization of the molecules onto solid or quasi-solid substrates and the use of relatively high excitation intensity We have studied the fluorescence emission of four common dyes used for bio-imaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level under two-photon excitation regime. We focus on two-photon excitation thermal effects on the stability of the single molecules, influencing the internal photo-dynamics and the total duration of the fluorescent emission. Single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output till a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, Indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution suggesting that bleaching is not due to photo-bleaching. Moreover it shows a correlation to the amount of absorbed power not re-irradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon excitation process. This study should be extended to different trapping media of interest in single molecule basic research and applications, such as silica and polyacrylamide gels or nanosctructured polyelectrolyte matrices. We think that the observed behavior and the correlations found to the molecular chemical and physical parameters, may be of some help for the design of molecules with switching on-off behavior of longer duration.

ADMET biosensors: up-to-date issues and strategies.

PubMed

Fang, Yan; Offenhaeusser, Andrease

2004-12-01

This insight review introduces the new concepts, theories, technology, instruments, frontier issues, and key strategies of ADMET (absorption, distribution, metabolism, elimination, and toxicity) biosensors, from the fermi to the quantum levels. Information about ADMET, originating from one author's invention, a patented pharmacotherapy for rescuing cardio-cerebral vascular stunning and regulating vascular endothelial growth-factor signaling at the post-genomic level, can be detected by a new generation of ADMET biosensor. This is a single-cell/single-molecule field-effect transistor (FET) hybrid system, where single molecules or single cells are assembled at the FET surface in a high density array manner via complementary metal-oxide-semiconductor (CMOS)-compatible technologies. Within a given nanometer distance, ADMET-mediated oxidation-reduction (redox) potentials, electrochemistry responses, and electron transfer processes can be simultaneously and directly probed by the gates of field-effect transistor arrays. The nanometer details of the functional coupling principles and characterization technologies of DNA single-molecule/single-cell FETs, as well as the design of lab-on-a-chip instruments, are indicated. Four frontier issues and key strategies are elucidated in detail. This can lead to innovative technology for high-throughout screening of labs-on-chips to resolve the pharmaceutical industry's current bottleneck via novel, FET-based drug discovery and single-molecule/single-cell screening methods, which can bring about a pharmaceutical industry revolution in the 21st century.

In situ superexchange electron transfer through a single molecule: a rectifying effect.

PubMed

Kornyshev, Alexei A; Kuznetsov, Alexander M; Ulstrup, Jens

2006-05-02

An increasingly comprehensive body of literature is being devoted to single-molecule bridge-mediated electronic nanojunctions, prompted by their prospective applications in molecular electronics and single-molecule analysis. These junctions may operate in gas phase or electrolyte solution (in situ). For biomolecules, the latter is much closer to their native environment. Convenient target molecules are aromatic molecules, peptides, oligonucleotides, transition metal complexes, and, broadly, molecules with repetitive units, for which the conducting orbitals are energetically well below electronic levels of the solvent. A key feature for these junctions is rectification in the current-voltage relation. A common view is that asymmetric molecules or asymmetric links to the electrodes are needed to acquire rectification. However, as we show here, this requirement could be different in situ, where a structurally symmetric system can provide rectification because of the Debye screening of the electric field in the nanogap if the screening length is smaller than the bridge length. The Galvani potentials of each electrode can be varied independently and lead to a transistor effect. We explore this behavior for the superexchange mechanism of electron transport, appropriate for a wide class of molecules. We also include the effect of conformational fluctuations on the lowest unoccupied molecular orbital (LUMO) energy levels; that gives rise to non-Arrhenius temperature dependence of the conductance, affected by the molecule length. Our study offers an analytical formula for the current-voltage characteristics that demonstrates all these features. A detailed physical interpretation of the results is given with a discussion of reported experimental data.

Toward the Space-Time Limit

NASA Astrophysics Data System (ADS)

Rios, Laura

A chemical reaction is fundamentally initiated by the restructuring of a chemical bond. Chemical reactions occur so quickly that their exact trajectory is unknown. To unlock the secret, first one would seek to know the inner working of a single molecule, and therein, a single chemical bond. However, the task is no small feat. Single molecule studies require exquisite spatial resolution afforded by relatively new technologies, and ultrafast laser techniques. The overarching theme of my dissertation will be the path towards achieving the space-time limit in chemistry: namely, the ability to record the structural changes of individual molecules during a reaction, one event at a time. A scanning tunneling microscope (STM) is used to image the molecules and manipulate their electronic environments. STM has the capacity to create topographical images of molecules with Angstrom ($10-10 m - the size of an atom) resolution, and can also probe the molecule electronically by use of a tunneling current (It). STM images reflect the changes in the potential energy surface (PES), and help us understand how molecules interact with surfaces and each other, thereby accessing the fundamental problem of catalysis and chemical reactions. In addition to seeing the molecule, we use Raman spectroscopy to track its molecular changes with chemical specificity. I combine these experimental tools to investigate tip-enhanced Raman spectra (TERS) of single molecules within the confines of a STM. These methods were used to report the conformational change of a single azobenzene-thiol derivative molecule. Although we were able to definitely isolate a single molecule signature, imaging the single molecule in real space and time proved elusive. Additionally, I report on a conductance switch based on the observable change of the topographic STM images of a radical anion mediated by the spin flip of a single electron on a single molecule. This is effectively the smallest achievable architecture of molecular electronics, negating the need for heat dissipation in small systems. A related work found how physisorption potentials of molecules to metals could be experimentally visually verified and modeled by STM, thus allowing us to use the STM tip as a driver for molecular motion on surfaces. Throughtout this work, we noted that a dominant feature of single molecule chemistry are intensity and spectral fluctuations that are difficult to characterize, as the molecule contorts wildly when it experiences distinct and powerful electromagnetic fields and field gradients. This much is evident in the last experiment, and chapter, of this thesis. Raman spectra associated with cobalt (II) tetraphenyl porphyrin (CoTPP) axially coordinated with bipyridyl ethylene (BPE) were captured with Raman mapping with nanometer resolution. However, the stochastic apperance of Raman lines and low resolution images made it difficult to ascertain which molecule we captured. The preliminary results as well as follow-up control experiments are discussed. While each experiment constitutes in and of itself an important, individual contribution, their sum establishes the principles of seeing single-molecule chemistry.

Preface: Special Topic on Single-Molecule Biophysics

NASA Astrophysics Data System (ADS)

Makarov, Dmitrii E.; Schuler, Benjamin

2018-03-01

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.

Effect of adsorbate electrophilicity and spiky uneven surfaces on single gold nanourchin-based localized surface plasmon resonance sensors

NASA Astrophysics Data System (ADS)

Kim, Geun Wan; Ha, Ji Won

2018-04-01

We present single particle studies on gold nanourchins (AuNUs) for their use as localized surface plasmon resonance (LSPR) biosensors under dark-field (DF) microscopy. First, the LSPR wavelength of single AuNUs was red-shifted as thiol molecules were attached onto the surface. AuNUs with sharp tips showed higher sensitivity for detecting thiol molecules than gold nanospheres (AuNSs) of similar size. Second, the degree of red shift was affected by the electrophilicity of adsorbate molecules on the nanoparticle surface. Last, real-time monitoring of molecular binding events on single AuNUs was achieved with introducing 1 μM of 4-aminothiophenol.

Different catalytic effects of a single water molecule: the gas-phase reaction of formic acid with hydroxyl radical in water vapor.

PubMed

Anglada, Josep M; Gonzalez, Javier

2009-12-07

The effect of a single water molecule on the reaction mechanism of the gas-phase reaction between formic acid and the hydroxyl radical was investigated with high-level quantum mechanical calculations using DFT-B3LYP, MP2 and CCSD(T) theoretical approaches in concert with the 6-311+G(2df,2p) and aug-cc-pVTZ basis sets. The reaction between HCOOH and HO has a very complex mechanism involving a proton-coupled electron transfer process (pcet), two hydrogen-atom transfer reactions (hat) and a double proton transfer process (dpt). The hydroxyl radical predominantly abstracts the acidic hydrogen of formic acid through a pcet mechanism. A single water molecule affects each one of these reaction mechanisms in different ways, depending on the way the water interacts. Very interesting is also the fact that our calculations predict that the participation of a single water molecule results in the abstraction of the formyl hydrogen of formic acid through a hydrogen atom transfer process (hat).

Tuning Charge and Correlation Effects for a Single Molecule on a Graphene Device

NASA Astrophysics Data System (ADS)

Tsai, Hsin-Zon; Wickenburg, Sebastian; Lu, Jiong; Lischner, Johannes; Omrani, Arash A.; Riss, Alexander; Karrasch, Christoph; Jung, Han Sae; Khajeh, Ramin; Wong, Dillon; Watanabe, Kenji; Taniguchi, Takashi; Zettl, Alex; Louie, Steven G.; Crommie, Michael F.

Controlling electronic devices down to the single molecule level is a grand challenge of nanotechnology. Single-molecules have been integrated into devices capable of tuning electronic response, but a drawback for these systems is that their microscopic structure remains unknown due to inability to image molecules in the junction region. Here we present a combined STM and nc-AFM study demonstrating gate-tunable control of the charge state of individual F4TCNQ molecules at the surface of a graphene field effect transistor. This is different from previous studies in that the Fermi level of the substrate was continuously tuned across the molecular orbital energy level. Using STS we have determined the resulting energy level evolution of the LUMO, its associated vibronic modes, and the graphene Dirac point (ED). We show that the energy difference between ED and the LUMO increases as EF is moved away from ED due to electron-electron interactions that renormalize the molecular quasiparticle energy. This is attributed to gate-tunable image-charge screening in graphene and corroborated by ab initio calculations.

Effect of impurity molecules on the low-temperature vibrational dynamics of polyisobutylene: Investigation by single-molecule spectroscopy

NASA Astrophysics Data System (ADS)

Eremchev, I. Yu.; Naumov, A. V.; Vainer, Yu. G.; Kador, L.

2009-05-01

The influence of impurity chromophore molecules—tetra-tert-butylterrylene (TBT) and dibenzo-anthanthrene (DBATT)—on the vibrational dynamics of the amorphous polymer polyisobutylene (PIB) has been studied via single-molecule spectroscopy. The measurements were performed in the temperature region of 7-30 K, where the interaction of the chromophores with quasilocalized low-frequency vibrational modes (LFMs) determines the observed spectral line broadening. The analysis of the individual temperature dependences of the linewidths for a large number of single probe molecules yielded effective frequency values of those LFMs which are located near the respective chromophores. In this way the distributions of the LFM frequencies were measured for the two systems, and they were found to be similar. Moreover, they are in good agreement with the vibrational density of states as measured in pure PIB by inelastic neutron scattering. This allows us to conclude that, at least in the case of PIB, doping with low concentrations of the nonpolar and neutral molecules TBT and DBATT does not affect the vibrational dynamics of the matrix markedly.

Single-molecule spectroscopy for plastic electronics: materials analysis from the bottom-up.

PubMed

Lupton, John M

2010-04-18

pi-conjugated polymers find a range of applications in electronic devices. These materials are generally highly disordered in terms of chain length and chain conformation, besides being influenced by a variety of chemical and physical defects. Although this characteristic can be of benefit in certain device applications, disorder severely complicates materials analysis. Accurate analytical techniques are, however, crucial to optimising synthetic procedures and assessing overall material purity. Fortunately, single-molecule spectroscopic techniques have emerged as an unlikely but uniquely powerful approach to unraveling intrinsic material properties from the bottom up. Building on the success of such techniques in the life sciences, single-molecule spectroscopy is finding increasing applicability in materials science, effectively enabling the dissection of the bulk down to the level of the individual molecular constituent. This article reviews recent progress in single molecule spectroscopy of conjugated polymers as used in organic electronics.

High thermopower of mechanically stretched single-molecule junctions

PubMed Central

Tsutsui, Makusu; Morikawa, Takanori; He, Yuhui; Arima, Akihide

2015-01-01

Metal-molecule-metal junction is a promising candidate for thermoelectric applications that utilizes quantum confinement effects in the chemically defined zero-dimensional atomic structure to achieve enhanced dimensionless figure of merit ZT. A key issue in this new class of thermoelectric nanomaterials is to clarify the sensitivity of thermoelectricity on the molecular junction configurations. Here we report simultaneous measurements of the thermoelectric voltage and conductance on Au-1,4-benzenedithiol (BDT)-Au junctions mechanically-stretched in-situ at sub-nanoscale. We obtained the average single-molecule conductance and thermopower of 0.01 G0 and 15 μV/K, respectively, suggesting charge transport through the highest occupied molecular orbital. Meanwhile, we found the single-molecule thermoelectric transport properties extremely-sensitive to the BDT bridge configurations, whereby manifesting the importance to design the electrode-molecule contact motifs for optimizing the thermoelectric performance of molecular junctions. PMID:26112999

A density functional theory for association of fluid molecules with a functionalized surface: fluid-wall single and double bonding.

PubMed

Haghmoradi, Amin; Wang, Le; Chapman, Walter G

2017-02-01

In this manuscript we extend Wertheim's two-density formalism beyond its first order to model a system of fluid molecules with a single association site close to a planar hard wall with association sites on its surface in a density functional theory framework. The association sites of the fluid molecules are small enough that they can form only one bond, while the wall association sites are large enough to bond with more than one fluid molecule. The effects of temperature and of bulk fluid and wall site densities on the fluid density profile, extent of association, and competition between single and double bonding of fluid segments at the wall sites versus distance from the wall are presented. The theory predictions are compared with new Monte Carlo simulation results and they are in good agreement. The theory captures the surface coverage over wide ranges of temperature and bulk density by introducing the effect of steric hindrance in fluid association at a wall site.

DNA confinement in nanochannels: physics and biological applications

NASA Astrophysics Data System (ADS)

Reisner, Walter; Pedersen, Jonas N.; Austin, Robert H.

2012-10-01

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement—including the effect of varying ionic strength—and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

Too Hot for Photon-Assisted Transport: Hot-Electrons Dominate Conductance Enhancement in Illuminated Single-Molecule Junctions.

PubMed

Fung, E-Dean; Adak, Olgun; Lovat, Giacomo; Scarabelli, Diego; Venkataraman, Latha

2017-02-08

We investigate light-induced conductance enhancement in single-molecule junctions via photon-assisted transport and hot-electron transport. Using 4,4'-bipyridine bound to Au electrodes as a prototypical single-molecule junction, we report a 20-40% enhancement in conductance under illumination with 980 nm wavelength radiation. We probe the effects of subtle changes in the transmission function on light-enhanced current and show that discrete variations in the binding geometry result in a 10% change in enhancement. Importantly, we prove theoretically that the steady-state behavior of photon-assisted transport and hot-electron transport is identical but that hot-electron transport is the dominant mechanism for optically induced conductance enhancement in single-molecule junctions when the wavelength used is absorbed by the electrodes and the hot-electron relaxation time is long. We confirm this experimentally by performing polarization-dependent conductance measurements of illuminated 4,4'-bipyridine junctions. Finally, we perform lock-in type measurements of optical current and conclude that currents due to laser-induced thermal expansion mask optical currents. This work provides a robust experimental framework for studying mechanisms of light-enhanced transport in single-molecule junctions and offers tools for tuning the performance of organic optoelectronic devices by analyzing detailed transport properties of the molecules involved.

DNA confinement in nanochannels: physics and biological applications.

PubMed

Reisner, Walter; Pedersen, Jonas N; Austin, Robert H

2012-10-01

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement--including the effect of varying ionic strength--and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

Quantum transport of the single metallocene molecule

NASA Astrophysics Data System (ADS)

Yu, Jing-Xin; Chang, Jing; Wei, Rong-Kai; Liu, Xiu-Ying; Li, Xiao-Dong

2016-10-01

The Quantum transport of three single metallocene molecule is investigated by performing theoretical calculations using the non-equilibrium Green's function method combined with density functional theory. We find that the three metallocen molecules structure become stretched along the transport direction, the distance between two Cp rings longer than the other theory and experiment results. The lager conductance is found in nickelocene molecule, the main transmission channel is the electron coupling between molecule and the electrodes is through the Ni dxz and dyz orbitals and the s, dxz, dyz of gold. This is also confirmed by the highest occupied molecular orbital resonance at Fermi level. In addition, negative differential resistance effect is found in the ferrocene, cobaltocene molecules, this is also closely related with the evolution of the transmission spectrum under applied bias.

Cucurbituril mediated single molecule detection and identification via recognition tunneling.

PubMed

Xiao, Bohuai; Liang, Feng; Liu, Simin; Im, JongOne; Li, Yunchuan; Liu, Jing; Zhang, Bintian; Zhou, Jianghao; He, Jin; Chang, Shuai

2018-06-08

Recognition tunneling (RT) is an emerging technique for investigating single molecules in a tunnel junction. We have previously demonstrated its capability of single molecule detection and identification, as well as probing the dynamics of intermolecular bonding at the single molecule level. Here by introducing cucurbituril as a new class of recognition molecule, we demonstrate a powerful platform for electronically investigating the host-guest chemistry at single molecule level. In this report, we first investigated the single molecule electrical properties of cucurbituril in a tunnel junction. Then we studied two model guest molecules, aminoferrocene and amantadine, which were encapsulated by cucurbituril. Small differences in conductance and lifetime can be recognized between the host-guest complexes with the inclusion of different guest molecules. By using a machine learning algorithm to classify the RT signals in a hyper dimensional space, the accuracy of guest molecule recognition can be significantly improved, suggesting the possibility of using cucurbituril molecule for single molecule identification. This work enables a new class of recognition molecule for RT technique and opens the door for detecting a vast variety of small molecules by electrical measurements.

Revealing time bunching effect in single-molecule enzyme conformational dynamics.

PubMed

Lu, H Peter

2011-04-21

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.

Superradiance Effects in the Linear and Nonlinear Optical Response of Quantum Dot Molecules

NASA Astrophysics Data System (ADS)

Sitek, A.; Machnikowski, P.

2008-11-01

We calculate the linear optical response from a single quantum dot molecule and the nonlinear, four-wave-mixing response from an inhomogeneously broadened ensemble of such molecules. We show that both optical signals are affected by the coupling-dependent superradiance effect and by optical interference between the two polarizations. As a result, the linear and nonlinear responses are not identical.

Evaluation of the Kinetic Property of Single-Molecule Junctions by Tunneling Current Measurements.

PubMed

Harashima, Takanori; Hasegawa, Yusuke; Kiguchi, Manabu; Nishino, Tomoaki

2018-01-01

We investigated the formation and breaking of single-molecule junctions of two kinds of dithiol molecules by time-resolved tunneling current measurements in a metal nanogap. The resulting current trajectory was statistically analyzed to determine the single-molecule conductance and, more importantly, to reveal the kinetic property of the single-molecular junction. These results suggested that combining a measurement of the single-molecule conductance and statistical analysis is a promising method to uncover the kinetic properties of the single-molecule junction.

Simulation of single-molecule trapping in a nanochannel

PubMed Central

Robinson, William Neil; Davis, Lloyd M.

2010-01-01

The detection and trapping of single fluorescent molecules in solution within a nanochannel is studied using numerical simulations. As optical forces are insufficient for trapping molecules much smaller than the optical wavelength, a means for sensing a molecule’s position along the nanochannel and adjusting electrokinetic motion to compensate diffusion is assessed. Fluorescence excitation is provided by two adjacently focused laser beams containing temporally interleaved laser pulses. Photon detection is time-gated, and the displacement of the molecule from the middle of the two foci alters the count rates collected in the two detection channels. An algorithm for feedback control of the electrokinetic motion in response to the timing of photons, to reposition the molecule back toward the middle for trapping and to rapidly reload the trap after a molecule photobleaches or escapes, is evaluated. While accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware, the algorithm is shown to be effective for trapping fast-diffusing single-chromophore molecules within a micron-sized confocal region. Studies show that there is an optimum laser power for which loss of molecules from the trap due to either photobleaching or shot-noise fluctuations is minimized. PMID:20799801

REVIEWS OF TOPICAL PROBLEMS Electron-vibration interaction in tunneling processes through single molecules

NASA Astrophysics Data System (ADS)

Arseev, Petr I.; Maslova, N. S.

2011-02-01

It is shown how effective Hamiltonians are constructed in the framework of the adiabatic approach to the electron-vibration interaction in electron tunneling through single molecules. Methods for calculating tunneling characteristics are discussed and possible features resulting from the electron-vibration coupling are described. The intensity of vibrations excited by a tunneling current in various systems is examined.

Discriminating Intercalative Effects of Threading Intercalator Nogalamycin, from Classical Intercalator Daunomycin, Using Single Molecule Atomic Force Spectroscopy.

PubMed

Banerjee, T; Banerjee, S; Sett, S; Ghosh, S; Rakshit, T; Mukhopadhyay, R

2016-01-01

DNA threading intercalators are a unique class of intercalating agents, albeit little biophysical information is available on their intercalative actions. Herein, the intercalative effects of nogalamycin, which is a naturally-occurring DNA threading intercalator, have been investigated by high-resolution atomic force microscopy (AFM) and spectroscopy (AFS). The results have been compared with those of the well-known chemotherapeutic drug daunomycin, which is a non-threading classical intercalator bearing structural similarity to nogalamycin. A comparative AFM assessment revealed a greater increase in DNA contour length over the entire incubation period of 48 h for nogalamycin treatment, whereas the contour length increase manifested faster in case of daunomycin. The elastic response of single DNA molecules to an externally applied force was investigated by the single molecule AFS approach. Characteristic mechanical fingerprints in the overstretching behaviour clearly distinguished the nogalamycin/daunomycin-treated dsDNA from untreated dsDNA-the former appearing less elastic than the latter, and the nogalamycin-treated DNA distinguished from the daunomycin-treated DNA-the classically intercalated dsDNA appearing the least elastic. A single molecule AFS-based discrimination of threading intercalation from the classical type is being reported for the first time.

Discriminating Intercalative Effects of Threading Intercalator Nogalamycin, from Classical Intercalator Daunomycin, Using Single Molecule Atomic Force Spectroscopy

PubMed Central

Sett, S.; Ghosh, S.; Rakshit, T.; Mukhopadhyay, R.

2016-01-01

DNA threading intercalators are a unique class of intercalating agents, albeit little biophysical information is available on their intercalative actions. Herein, the intercalative effects of nogalamycin, which is a naturally-occurring DNA threading intercalator, have been investigated by high-resolution atomic force microscopy (AFM) and spectroscopy (AFS). The results have been compared with those of the well-known chemotherapeutic drug daunomycin, which is a non-threading classical intercalator bearing structural similarity to nogalamycin. A comparative AFM assessment revealed a greater increase in DNA contour length over the entire incubation period of 48 h for nogalamycin treatment, whereas the contour length increase manifested faster in case of daunomycin. The elastic response of single DNA molecules to an externally applied force was investigated by the single molecule AFS approach. Characteristic mechanical fingerprints in the overstretching behaviour clearly distinguished the nogalamycin/daunomycin-treated dsDNA from untreated dsDNA—the former appearing less elastic than the latter, and the nogalamycin-treated DNA distinguished from the daunomycin-treated DNA—the classically intercalated dsDNA appearing the least elastic. A single molecule AFS-based discrimination of threading intercalation from the classical type is being reported for the first time. PMID:27183010

Orientation of surfactant self-assembled aggregates on graphite

NASA Astrophysics Data System (ADS)

Sammalkorpi, Maria; Hynninen, Antti-Pekka; Panagiotopoulos, Athanassios Z.; Haataja, Mikko

2007-03-01

Micellar aggregates on surfaces can provide a self-healing corrosion protection or lubrication layer. It has been observed experimentally that on a single crystal surface this layer often consists of oriented hemi-cylindrical micelles which are aligned with the underlying crystal lattice (``orientation effect''). A key feature of this self-assembly process is the interplay between detergent--detergent and detergent--surface interactions. Since the dimensions of the detergent molecules and the unit cell of the surface are typically quite different, the origins of this orientation effect remain unclear. Here we address the question and present the results of Molecular Dynamics simulations of sodium dodecyl sulfate (SDS) self-aggregation on graphite. We employ both single-molecule and multi-molecule simulations of SDS to unravel the origins of the orientation effect. We report that the underlying graphite surface is sufficient to impose orientational bias on individual SDS molecules diffusing on the surface. This produces collective effects that give rise to the oriented hemi-micelles.

Modeling single molecule junction mechanics as a probe of interface bonding

NASA Astrophysics Data System (ADS)

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-acceptor bond formation at the junction interfaces. The force extension characteristic of longer molecules such as diaminooctane, where the dipole interaction effects drop to a negligible level, accurately fit to the renormalized single-bond potential form. The results suggest that measured force extension characteristics for single molecule junctions could be analyzed with a modified potential form that accounts for the energy stored in deformable mechanical components in series.

Modeling single molecule junction mechanics as a probe of interface bonding

DOE PAGES

Hybertsen, Mark S.

2017-03-07

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. We analyzed a set of exemplary model junction structures 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 tomore » 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, then, 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-acceptor bond formation at the junction interfaces. The force extension characteristic of longer molecules such as diaminooctane, where the dipole interaction effects drop to a negligible level, accurately fit to the renormalized single-bond potential form. Our results suggest that measured force extension characteristics for single molecule junctions could be analyzed with a modified potential form that accounts for the energy stored in deformable mechanical components in series.« less

Modeling single molecule junction mechanics as a probe of interface bonding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hybertsen, Mark S.

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. We analyzed a set of exemplary model junction structures 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 tomore » 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, then, 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-acceptor bond formation at the junction interfaces. The force extension characteristic of longer molecules such as diaminooctane, where the dipole interaction effects drop to a negligible level, accurately fit to the renormalized single-bond potential form. Our results suggest that measured force extension characteristics for single molecule junctions could be analyzed with a modified potential form that accounts for the energy stored in deformable mechanical components in series.« less

Controlled chain polymerisation and chemical soldering for single-molecule electronics.

PubMed

Okawa, Yuji; Akai-Kasaya, Megumi; Kuwahara, Yuji; Mandal, Swapan K; Aono, Masakazu

2012-05-21

Single functional molecules offer great potential for the development of novel nanoelectronic devices with capabilities beyond today's silicon-based devices. To realise single-molecule electronics, the development of a viable method for connecting functional molecules to each other using single conductive polymer chains is required. The method of initiating chain polymerisation using the tip of a scanning tunnelling microscope (STM) is very useful for fabricating single conductive polymer chains at designated positions and thereby wiring single molecules. In this feature article, developments in the controlled chain polymerisation of diacetylene compounds and the properties of polydiacetylene chains are summarised. Recent studies of "chemical soldering", a technique enabling the covalent connection of single polydiacetylene chains to single functional molecules, are also introduced. This represents a key step in advancing the development of single-molecule electronics.

A comprehensive study of extended tetrathiafulvalene cruciform molecules for molecular electronics: synthesis and electrical transport measurements.

PubMed

Parker, Christian R; Leary, Edmund; Frisenda, Riccardo; Wei, Zhongming; Jennum, Karsten S; Glibstrup, Emil; Abrahamsen, Peter Bæch; Santella, Marco; Christensen, Mikkel A; Della Pia, Eduardo Antonio; Li, Tao; Gonzalez, Maria Teresa; Jiang, Xingbin; Morsing, Thorbjørn J; Rubio-Bollinger, Gabino; Laursen, Bo W; Nørgaard, Kasper; van der Zant, Herre; Agrait, Nicolas; Nielsen, Mogens Brøndsted

2014-11-26

Cruciform-like molecules with two orthogonally placed π-conjugated systems have in recent years attracted significant interest for their potential use as molecular wires in molecular electronics. Here we present synthetic protocols for a large selection of cruciform molecules based on oligo(phenyleneethynylene) (OPE) and tetrathiafulvalene (TTF) scaffolds, end-capped with acetyl-protected thiolates as electrode anchoring groups. The molecules were subjected to a comprehensive study of their conducting properties as well as their photophysical and electrochemical properties in solution. The complex nature of the molecules and their possible binding in different configurations in junctions called for different techniques of conductance measurements: (1) conducting-probe atomic force microscopy (CP-AFM) measurements on self-assembled monolayers (SAMs), (2) mechanically controlled break-junction (MCBJ) measurements, and (3) scanning tunneling microscopy break-junction (STM-BJ) measurements. The CP-AFM measurements showed structure-property relationships from SAMs of series of OPE3 and OPE5 cruciform molecules; the conductance of the SAM increased with the number of dithiafulvene (DTF) units (0, 1, 2) along the wire, and it increased when substituting two arylethynyl end groups of the OPE3 backbone with two DTF units. The MCBJ and STM-BJ studies on single molecules both showed that DTFs decreased the junction formation probability, but, in contrast, no significant influence on the single-molecule conductance was observed. We suggest that the origins of the difference between SAM and single-molecule measurements lie in the nature of the molecule-electrode interface as well as in effects arising from molecular packing in the SAMs. This comprehensive study shows that for complex molecules care should be taken when directly comparing single-molecule measurements and measurements of SAMs and solid-state devices thereof.

Strong plasmonic enhancement of single molecule photostability in silver dimer optical antennas

NASA Astrophysics Data System (ADS)

Kaminska, Izabela; Vietz, Carolin; Cuartero-González, Álvaro; Tinnefeld, Philip; Fernández-Domínguez, Antonio I.; Acuna, Guillermo P.

2018-02-01

Photobleaching is an effect terminating the photon output of fluorophores, limiting the duration of fluorescence-based experiments. Plasmonic nanoparticles (NPs) can increase the overall fluorophore photostability through an enhancement of the radiative rate. In this work, we use the DNA origami technique to arrange a single fluorophore in the 12-nm gap of a silver NP dimer and study the number of emitted photons at the single molecule level. Our findings yielded a 30× enhancement in the average number of photons emitted before photobleaching. Numerical simulations are employed to rationalize our results. They reveal the effect of silver oxidation on decreasing the radiative rate enhancement.

Single-Molecule Counting of Point Mutations by Transient DNA Binding

NASA Astrophysics Data System (ADS)

Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

2017-03-01

High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

Kondo blockade due to quantum interference in single-molecule junctions

PubMed Central

Mitchell, Andrew K.; Pedersen, Kim G. L.; Hedegård, Per; Paaske, Jens

2017-01-01

Molecular electronics offers unique scientific and technological possibilities, resulting from both the nanometre scale of the devices and their reproducible chemical complexity. Two fundamental yet different effects, with no classical analogue, have been demonstrated experimentally in single-molecule junctions: quantum interference due to competing electron transport pathways, and the Kondo effect due to entanglement from strong electronic interactions. Here we unify these phenomena, showing that transport through a spin-degenerate molecule can be either enhanced or blocked by Kondo correlations, depending on molecular structure, contacting geometry and applied gate voltages. An exact framework is developed, in terms of which the quantum interference properties of interacting molecular junctions can be systematically studied and understood. We prove that an exact Kondo-mediated conductance node results from destructive interference in exchange-cotunneling. Nonstandard temperature dependences and gate-tunable conductance peaks/nodes are demonstrated for prototypical molecular junctions, illustrating the intricate interplay of quantum effects beyond the single-orbital paradigm. PMID:28492236

Compressive Force Spectroscopy: From Living Cells to Single Proteins.

PubMed

Wang, Jiabin; Liu, Meijun; Shen, Yi; Sun, Jielin; Shao, Zhifeng; Czajkowsky, Daniel Mark

2018-03-23

One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.

Reconstructing Folding Energy Landscapes by Single-Molecule Force Spectroscopy

PubMed Central

Woodside, Michael T.; Block, Steven M.

2015-01-01

Folding may be described conceptually in terms of trajectories over a landscape of free energies corresponding to different molecular configurations. In practice, energy landscapes can be difficult to measure. Single-molecule force spectroscopy (SMFS), whereby structural changes are monitored in molecules subjected to controlled forces, has emerged as a powerful tool for probing energy landscapes. We summarize methods for reconstructing landscapes from force spectroscopy measurements under both equilibrium and nonequilibrium conditions. Other complementary, but technically less demanding, methods provide a model-dependent characterization of key features of the landscape. Once reconstructed, energy landscapes can be used to study critical folding parameters, such as the characteristic transition times required for structural changes and the effective diffusion coefficient setting the timescale for motions over the landscape. We also discuss issues that complicate measurement and interpretation, including the possibility of multiple states or pathways and the effects of projecting multiple dimensions onto a single coordinate. PMID:24895850

Structure–property relationships in atomic-scale junctions: Histograms and beyond

DOE PAGES

Mark S. Hybertsen; Venkataraman, Latha

2016-03-03

Over the past 10 years, there has been tremendous progress in the measurement, modeling and understanding of structure–function relationships in single molecule junctions. Numerous research groups have addressed significant scientific questions, directed both to conductance phenomena at the single molecule level and to the fundamental chemistry that controls junction functionality. Many different functionalities have been demonstrated, including single-molecule diodes, optically and mechanically activated switches, and, significantly, physical phenomena with no classical analogues, such as those based on quantum interference effects. Experimental techniques for reliable and reproducible single molecule junction formation and characterization have led to this progress. In particular, themore » scanning tunneling microscope based break-junction (STM-BJ) technique has enabled rapid, sequential measurement of large numbers of nanoscale junctions allowing a statistical analysis to readily distinguish reproducible characteristics. Furthermore, harnessing fundamental link chemistry has provided the necessary chemical control over junction formation, enabling measurements that revealed clear relationships between molecular structure and conductance characteristics.« less

Structure–property relationships in atomic-scale junctions: Histograms and beyond

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mark S. Hybertsen; Venkataraman, Latha

Over the past 10 years, there has been tremendous progress in the measurement, modeling and understanding of structure–function relationships in single molecule junctions. Numerous research groups have addressed significant scientific questions, directed both to conductance phenomena at the single molecule level and to the fundamental chemistry that controls junction functionality. Many different functionalities have been demonstrated, including single-molecule diodes, optically and mechanically activated switches, and, significantly, physical phenomena with no classical analogues, such as those based on quantum interference effects. Experimental techniques for reliable and reproducible single molecule junction formation and characterization have led to this progress. In particular, themore » scanning tunneling microscope based break-junction (STM-BJ) technique has enabled rapid, sequential measurement of large numbers of nanoscale junctions allowing a statistical analysis to readily distinguish reproducible characteristics. Furthermore, harnessing fundamental link chemistry has provided the necessary chemical control over junction formation, enabling measurements that revealed clear relationships between molecular structure and conductance characteristics.« less

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

PubMed Central

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

DNA combing on low-pressure oxygen plasma modified polysilsesquioxane substrates for single-molecule studies

PubMed Central

Sriram, K. K.; Chang, Chun-Ling; Rajesh Kumar, U.; Chou, Chia-Fu

2014-01-01

Molecular combing and flow-induced stretching are the most commonly used methods to immobilize and stretch DNA molecules. While both approaches require functionalization steps for the substrate surface and the molecules, conventionally the former does not take advantage of, as the latter, the versatility of microfluidics regarding robustness, buffer exchange capability, and molecule manipulation using external forces for single molecule studies. Here, we demonstrate a simple one-step combing process involving only low-pressure oxygen (O2) plasma modified polysilsesquioxane (PSQ) polymer layer to facilitate both room temperature microfluidic device bonding and immobilization of stretched single DNA molecules without molecular functionalization step. Atomic force microscopy and Kelvin probe force microscopy experiments revealed a significant increase in surface roughness and surface potential on low-pressure O2 plasma treated PSQ, in contrast to that with high-pressure O2 plasma treatment, which are proposed to be responsible for enabling effective DNA immobilization. We further demonstrate the use of our platform to observe DNA-RNA polymerase complexes and cancer drug cisplatin induced DNA condensation using wide-field fluorescence imaging. PMID:25332730

Optimization of cell morphology measurement via single-molecule tracking PALM.

PubMed

Frost, Nicholas A; Lu, Hsiangmin E; Blanpied, Thomas A

2012-01-01

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments.

Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape.

PubMed

Ferreon, Allan Chris M; Moosa, Mahdi Muhammad; Gambin, Yann; Deniz, Ashok A

2012-10-30

Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson's disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 21 [urea][TMAO] ratio has a net neutral effect on the protein's dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments.

Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape

PubMed Central

Ferreon, Allan Chris M.; Moosa, Mahdi Muhammad; Deniz, Ashok A.

2012-01-01

Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson’s disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 2∶1 [urea]∶[TMAO] ratio has a net neutral effect on the protein’s dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments. PMID:22826265

Single-molecule dynamics in nanofabricated traps

NASA Astrophysics Data System (ADS)

Cohen, Adam

2009-03-01

The Anti-Brownian Electrokinetic trap (ABEL trap) provides a means to immobilize a single fluorescent molecule in solution, without surface attachment chemistry. The ABEL trap works by tracking the Brownian motion of a single molecule, and applying feedback electric fields to induce an electrokinetic motion that approximately cancels the Brownian motion. We present a new design for the ABEL trap that allows smaller molecules to be trapped and more information to be extracted from the dynamics of a single molecule than was previously possible. In particular, we present strategies for extracting dynamically fluctuating mobilities and diffusion coefficients, as a means to probe dynamic changes in molecular charge and shape. If one trapped molecule is good, many trapped molecules are better. An array of single molecules in solution, each immobilized without surface attachment chemistry, provides an ideal test-bed for single-molecule analyses of intramolecular dynamics and intermolecular interactions. We present a technology for creating such an array, using a fused silica plate with nanofabricated dimples and a removable cover for sealing single molecules within the dimples. With this device one can watch the shape fluctuations of single molecules of DNA or study cooperative interactions in weakly associating protein complexes.

Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules

DOE PAGES

Lee, Somin Eunice; Chen, Qian; Bhat, Ramray; ...

2015-06-03

Plasmon rulers, consisting of pairs of gold nanoparticles, allow single-molecule analysis without photobleaching or blinking; however, current plasmon rulers are irreversible, restricting detection to only single events. Here, we present a reversible plasmon ruler, comprised of coupled gold nanoparticles linked by a single aptamer, capable of binding individual secreted molecules with high specificity. We show that the binding of target secreted molecules to the reversible plasmon ruler is characterized by single-molecule sensitivity, high specificity, and reversibility. Lastly, such reversible plasmon rulers should enable dynamic and adaptive live-cell measurement of secreted single molecules in their local microenvironment.

Single-Molecule Plasmon Sensing: Current Status and Future Prospects

PubMed Central

2017-01-01

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. PMID:28762723

Effects of electrostatic screening on the conformation of single DNA molecules confined in a nanochannel

NASA Astrophysics Data System (ADS)

Zhang, Ce; Zhang, Fang; van Kan, Jeroen A.; van der Maarel, Johan R. C.

2008-06-01

Single T4-DNA molecules were confined in rectangular-shaped channels with a depth of 300 nm and a width in the range of 150-300 nm casted in a poly(dimethylsiloxane) nanofluidic chip. The extensions of the DNA molecules were measured with fluorescence microscopy as a function of the ionic strength and composition of the buffer as well as the DNA intercalation level by the YOYO-1 dye. The data were interpreted with the scaling theory for a wormlike polymer in good solvent, including the effects of confinement, charge, and self-avoidance. It was found that the elongation of the DNA molecules with decreasing ionic strength can be interpreted in terms of an increase of the persistence length. Self-avoidance effects on the extension are moderate, due to the small correlation length imposed by the channel cross-sectional diameter. Intercalation of the dye results in an increase of the DNA contour length and a partial neutralization of the DNA charge, but besides effects of electrostatic origin it has no significant effect on the bare bending rigidity. In the presence of divalent cations, the DNA molecules were observed to contract, but they do not collapse into a condensed structure. It is proposed that this contraction results from a divalent counterion mediated attractive force between the segments of the DNA molecule.

Bond-equilibrium theory of liquid Se-Te alloys. II. Effect of singly attached ring molecules

NASA Astrophysics Data System (ADS)

Cutler, Melvin; Bez, Wolfgang G.

1981-06-01

A statistical-mechanical theory for bond equilibrium of chain polymers containing threefold (3F) and onefold (1F) bond defects is extended to include the effects of free ring molecules and ring molecules attached to chains by a single 3F atom. Positively charged singly attached rings are shown to play a key role in bond equilibrium in liquid Sex Te1-x by permitting the formation of ion pairs in which both constituents are effectively chain terminators, thus decreasing the average polymer size. The theory is applied to explain the behavior of the paramagnetic susceptibility, χp, and electronic transport as affected by the Fermi energy EF. It is found that the increase in χp with the concentration of Te is primarily the result of the smaller energy for breaking Te bonds. In addition, attached rings play an important role in determining the effect of temperature on χp. At x<~0.5, the concentrations of both free and attached rings becomes small at high T because of the high concentration of bond defects.

Choosing the right fluorophore for single-molecule fluorescence studies in a lipid environment.

PubMed

Zhang, Zhenfu; Yomo, Dan; Gradinaru, Claudiu

2017-07-01

Nonspecific interactions between lipids and fluorophores can alter the outcomes of single-molecule spectroscopy of membrane proteins in live cells, liposomes or lipid nanodiscs and of cytosolic proteins encapsulated in liposomes or tethered to supported lipid bilayers. To gain insight into these effects, we examined interactions between 9 dyes that are commonly used as labels for single-molecule fluorescence (SMF) and 6 standard lipids including cationic, zwitterionic and anionic types. The diffusion coefficients of dyes in the absence and presence of set amounts of lipid vesicles were measured by fluorescence correlation spectroscopy (FCS). The partition coefficients and the free energies of partitioning for different fluorophore-lipid pairs were obtained by global fitting of the titration FCS curves. Lipids with different charges, head groups and degrees of chain saturation were investigated, and interactions with dyes are discussed in terms of hydrophobic, electrostatic and steric contributions. Fluorescence imaging of individual fluorophores adsorbed on supported lipid bilayers provides visualization and additional quantification of the strength of dye-lipid interaction in the context of single-molecule measurements. By dissecting fluorophore-lipid interactions, our study provides new insights into setting up single-molecule fluorescence spectroscopy experiments with minimal interference from interactions between fluorescent labels and lipids in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Negative differential conductance and super-Poissonian shot noise in single-molecule magnet junctions

PubMed Central

Xue, Hai-Bin; Liang, Jiu-Qing; Liu, Wu-Ming

2015-01-01

Molecular spintroinic device based on a single-molecule magnet is one of the ultimate goals of semiconductor nanofabrication technologies. It is thus necessary to understand the electron transport properties of a single-molecule magnet junction. Here we study the negative differential conductance and super-Poissonian shot noise properties of electron transport through a single-molecule magnet weakly coupled to two electrodes with either one or both of them being ferromagnetic. We predict that the negative differential conductance and super-Poissonian shot noise, which can be tuned by a gate voltage, depend sensitively on the spin polarization of the source and drain electrodes. In particular, the shot noise in the negative differential conductance region can be enhanced or decreased originating from the different formation mechanisms of negative differential conductance. The effective competition between fast and slow transport channels is responsible for the observed negative differential conductance and super-Poissonian shot noise. In addition, we further discuss the skewness and kurtosis properties of transport current in the super-Poissonian shot noise regions. Our findings suggest a tunable negative differential conductance molecular device, and the predicted properties of high-order current cumulants are very interesting for a better understanding of electron transport through single-molecule magnet junctions. PMID:25736094

Negative differential conductance and super-Poissonian shot noise in single-molecule magnet junctions.

PubMed

Xue, Hai-Bin; Liang, Jiu-Qing; Liu, Wu-Ming

2015-03-04

Molecular spintroinic device based on a single-molecule magnet is one of the ultimate goals of semiconductor nanofabrication technologies. It is thus necessary to understand the electron transport properties of a single-molecule magnet junction. Here we study the negative differential conductance and super-Poissonian shot noise properties of electron transport through a single-molecule magnet weakly coupled to two electrodes with either one or both of them being ferromagnetic. We predict that the negative differential conductance and super-Poissonian shot noise, which can be tuned by a gate voltage, depend sensitively on the spin polarization of the source and drain electrodes. In particular, the shot noise in the negative differential conductance region can be enhanced or decreased originating from the different formation mechanisms of negative differential conductance. The effective competition between fast and slow transport channels is responsible for the observed negative differential conductance and super-Poissonian shot noise. In addition, we further discuss the skewness and kurtosis properties of transport current in the super-Poissonian shot noise regions. Our findings suggest a tunable negative differential conductance molecular device, and the predicted properties of high-order current cumulants are very interesting for a better understanding of electron transport through single-molecule magnet junctions.

Single-molecule study of thymidine glycol and i-motif through the alpha-hemolysin ion channel

NASA Astrophysics Data System (ADS)

He, Lidong

Nanopore-based devices have emerged as a single-molecule detection and analysis tool for a wide range of applications. Through electrophoretically driving DNA molecules across a nanosized pore, a lot of information can be received, including unfolding kinetics and DNA-protein interactions. This single-molecule method has the potential to sequence kilobase length DNA polymers without amplification or labeling, approaching "the third generation" genome sequencing for around $1000 within 24 hours. alpha-Hemolysin biological nanopores have the advantages of excellent stability, low-noise level, and precise site-directed mutagenesis for engineering this protein nanopore. The first work presented in this thesis established the current signal of the thymidine glycol lesion in DNA oligomers through an immobilization experiment. The thymidine glycol enantiomers were differentiated from each other by different current blockage levels. Also, the effect of bulky hydrophobic adducts to the current blockage was investigated. Secondly, the alpha-hemolysin nanopore was used to study the human telomere i-motif and RET oncogene i-motif at a single-molecule level. In Chapter 3, it was demonstrated that the alpha-hemolysin nanopore can differentiate an i-motif form and single-strand DNA form at different pH values based on the same sequence. In addition, it shows potential to differentiate the folding topologies generated from the same DNA sequence.

Unconventional Current Scaling and Edge Effects for Charge Transport through Molecular Clusters

PubMed Central

2017-01-01

Metal–molecule–metal junctions are the key components of molecular electronics circuits. Gaining a microscopic understanding of their conducting properties is central to advancing the field. In the present contribution, we highlight the fundamental differences between single-molecule and ensemble junctions focusing on the fundamentals of transport through molecular clusters. In this way, we elucidate the collective behavior of parallel molecular wires, bridging the gap between single molecule and large-area monolayer electronics, where even in the latter case transport is usually dominated by finite-size islands. On the basis of first-principles charge-transport simulations, we explain why the scaling of the conductivity of a junction has to be distinctly nonlinear in the number of molecules it contains. Moreover, transport through molecular clusters is found to be highly inhomogeneous with pronounced edge effects determined by molecules in locally different electrostatic environments. These effects are most pronounced for comparably small clusters, but electrostatic considerations show that they prevail also for more extended systems. PMID:29043825

Ordered array of CoPc-vacancies filled with single-molecule rotors

NASA Astrophysics Data System (ADS)

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.

Quantum-Sequencing: Fast electronic single DNA molecule sequencing

NASA Astrophysics Data System (ADS)

Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

2014-03-01

A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

Molecular vibrations in metal-single-molecule-metal junctions

NASA Astrophysics Data System (ADS)

Yokota, Kazumichi; Taniguchi, Masateru; Kawai, Tomoji

2010-03-01

Molecular vibrations in a metal-single-molecule-metal junction were studied based on density functional theory using a single benzenedithiolate molecule connected between gold clusters. We found that the difference in vibrational energy between an isolated benzenedithiol and the single-molecule junction is less than 3% in the energy range above 540 cm -1, where sulfur atoms contribute little to molecular vibrations. The finding implies that we can predict the peak energy in the inelastic electron tunneling spectrum of the single-molecule junction in the high energy range by vibrational analyses of isolated molecules.

Spin effects in transport through single-molecule magnets in the sequential and cotunneling regimes

NASA Astrophysics Data System (ADS)

Misiorny, Maciej; Weymann, Ireneusz; Barnaś, Józef

2009-06-01

We analyze the stationary spin-dependent transport through a single-molecule magnet weakly coupled to external ferromagnetic leads. Using the real-time diagrammatic technique, we calculate the sequential and cotunneling contributions to current, tunnel magnetoresistance, and Fano factor in both linear and nonlinear response regimes. We show that the effects of cotunneling are predominantly visible in the blockade regime and lead to enhancement of tunnel magnetoresistance (TMR) above the Julliere value, which is accompanied with super-Poissonian shot noise due to bunching of inelastic cotunneling processes through different virtual spin states of the molecule. The effects of external magnetic field and the role of type and strength of exchange interaction between the LUMO level and the molecule’s spin are also considered. When the exchange coupling is ferromagnetic, we find an enhanced TMR, while in the case of antiferromagnetic coupling we predict a large negative TMR effect.

Nonlinear thermoelectric transport in single-molecule junctions: the effect of electron-phonon interactions.

PubMed

Zimbovskaya, Natalya A

2016-07-27

In this paper, we theoretically analyze steady-state thermoelectric transport through a single-molecule junction with a vibrating bridge. The thermally induced charge current in the system is explored using a nonequilibrium Green function formalism. We study the combined effects of Coulomb interactions between charge carriers on the bridge and electron-phonon interactions on the thermocurrent beyond the linear response regime. It is shown that electron-vibron interactions may significantly affect both the magnitude and the direction of the thermocurrent, and vibrational signatures may appear.

Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis.

PubMed

Zhu, Zhi; Yang, Chaoyong James

2017-01-17

Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/cell detection, single cell sequencing, and molecular evolution.

An optical conveyor for molecules.

PubMed

Weinert, Franz M; Braun, Dieter

2009-12-01

Trapping single ions under vacuum allows for precise spectroscopy in atomic physics. The confinement of biological molecules in bulk water is hindered by the lack of comparably strong forces. Molecules have been immobilized to surfaces, however often with detrimental effects on their function. Here, we optically trap molecules by creating the microscale analogue of a conveyor belt: a bidirectional flow is combined with a perpendicular thermophoretic molecule drift. Arranged in a toroidal geometry, the conveyor accumulates a hundredfold excess of 5-base DNA within seconds. The concentrations of the trapped DNA scale exponentially with length, reaching trapping potential depths of 14 kT for 50 bases. The mechanism does not require microfluidics, electrodes, or surface modifications. As a result, the trap can be dynamically relocated. The optical conveyor can be used to enhance diffusion-limited surface reactions, redirect cellular signaling, observe individual biomolecules over a prolonged time, or approach single-molecule chemistry in bulk water.

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

PubMed Central

von Diezmann, Alex; Shechtman, Yoav; Moerner, W. E.

2017-01-01

Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers, or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information of single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field-dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems. PMID:28151646

Characterization of photoactivated singlet oxygen damage in single-molecule optical trap experiments.

PubMed

Landry, Markita P; McCall, Patrick M; Qi, Zhi; Chemla, Yann R

2009-10-21

Optical traps or "tweezers" use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments-the most common biological application of optical tweezers-and may guide the development of more robust experimental protocols.

Characterization of Photoactivated Singlet Oxygen Damage in Single-Molecule Optical Trap Experiments

PubMed Central

Landry, Markita P.; McCall, Patrick M.; Qi, Zhi; Chemla, Yann R.

2009-01-01

Abstract Optical traps or “tweezers” use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments—the most common biological application of optical tweezers—and may guide the development of more robust experimental protocols. PMID:19843445

Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

NASA Astrophysics Data System (ADS)

Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

Single-molecule detection: applications to ultrasensitive biochemical analysis

NASA Astrophysics Data System (ADS)

Castro, Alonso; Shera, E. Brooks

1995-06-01

Recent developments in laser-based detection of fluorescent molecules have made possible the implementation of very sensitive techniques for biochemical analysis. We present and discuss our experiments on the applications of our recently developed technique of single-molecule detection to the analysis of molecules of biological interest. These newly developed methods are capable of detecting and identifying biomolecules at the single-molecule level of sensitivity. In one case, identification is based on measuring fluorescence brightness from single molecules. In another, molecules are classified by determining their electrophoretic velocities.

Single-molecule fluorescence study of the inhibition of the oncogenic functionality of STAT3

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Badali, Daniel; Fletcher, Steven; Avadisian, Miriam; Gunning, Patrick; Gradinaru, Claudiu

2009-06-01

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.

Spectroscopic characterization of Venus at the single molecule level.

PubMed

David, Charlotte C; Dedecker, Peter; De Cremer, Gert; Verstraeten, Natalie; Kint, Cyrielle; Michiels, Jan; Hofkens, Johan

2012-02-01

Venus is a recently developed, fast maturating, yellow fluorescent protein that has been used as a probe for in vivo applications. In the present work the photophysical characteristics of Venus were analyzed spectroscopically at the bulk and single molecule level. Through time-resolved single molecule measurements we found that single molecules of Venus display pronounced fluctuations in fluorescence emission, with clear fluorescence on- and off-times. These fluorescence intermittencies were found to occupy a broad range of time scales, ranging from milliseconds to several seconds. Such long off-times can complicate the analysis of single molecule counting experiments or single-molecule FRET experiments. This journal is © The Royal Society of Chemistry and Owner Societies 2012

Single molecule data under scrutiny. Comment on "Extracting physics of life at the molecular level: A review of single-molecule data analyses" by W. Colomb & S.K. Sarkar

NASA Astrophysics Data System (ADS)

Wohland, Thorsten

2015-06-01

Single Molecule Detection and Spectroscopy have grown from their first beginnings into mainstream, mature research areas that are widely applied in the biological sciences. However, despite the advances in technology and the application of many single molecule techniques even in in vivo settings, the data analysis of single molecule experiments is complicated by noise, systematic errors, and complex underlying processes that are only incompletely understood. Colomb and Sarkar provide in this issue an overview of single molecule experiments and the accompanying problems in data analysis, which have to be overcome for a proper interpretation of the experiments [1].

Single Molecule Electronics and Devices

PubMed Central

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

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

PubMed

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, we develop an integrated current measurement system and an accurate data processing method for nanopore sensing. The unique geometric structure of a biological nanopore offers a distinct advantage as a nanosensor for single-molecule sensing. The construction of the pore entrance is responsible for capturing the target molecule, while the lumen region determines the translocation process of the single molecule. Since the capture of the target molecule is predominantly diffusion-limited, it is expected that the capture ability of the nanopore toward the target analyte could be effectively enhanced by site-directed mutations of key amino acids with desirable groups. Additionally, changing the side chains inside the wall of the biological nanopore could optimize the geometry of the pore and realize an optimal interaction between the single-molecule interface and the analyte. These improvements would allow for high spatial and current resolution of nanopore sensors, which would ensure the possibility of dynamic study of single biomolecules, including their metastable conformations, charge distributions, and interactions. In the future, data analysis with powerful algorithms will make it possible to automatically and statistically extract detailed information while an analyte translocates through the pore. We conclude that these improvements could have tremendous potential applications for nanopore sensing in the near future.

Reversible gating of smart plasmonic molecular traps using thermoresponsive polymers for single-molecule detection

PubMed Central

Zheng, Yuanhui; Soeriyadi, Alexander H.; Rosa, Lorenzo; Ng, Soon Hock; Bach, Udo; Justin Gooding, J.

2015-01-01

Single-molecule surface-enhanced Raman spectroscopy (SERS) has attracted increasing interest for chemical and biochemical sensing. Many conventional substrates have a broad distribution of SERS enhancements, which compromise reproducibility and result in slow response times for single-molecule detection. Here we report a smart plasmonic sensor that can reversibly trap a single molecule at hotspots for rapid single-molecule detection. The sensor was fabricated through electrostatic self-assembly of gold nanoparticles onto a gold/silica-coated silicon substrate, producing a high yield of uniformly distributed hotspots on the surface. The hotspots were isolated with a monolayer of a thermoresponsive polymer (poly(N-isopropylacrylamide)), which act as gates for molecular trapping at the hotspots. The sensor shows not only a good SERS reproducibility but also a capability to repetitively trap and release molecules for single-molecular sensing. The single-molecule sensitivity is experimentally verified using SERS spectral blinking and bianalyte methods. PMID:26549539

Effects of chemically induced contraction of a coordination polyhedron on the dynamical magnetism of bis(phthalocyaninato)disprosium, a single-4f-ionic single-molecule magnet with a Kramers ground state.

PubMed

Ishikawa, Naoto; Mizuno, Yoshifumi; Takamatsu, Satoshi; Ishikawa, Tadahiko; Koshihara, Shin-ya

2008-11-17

Chemically induced longitudinal contraction of the square-antiprism coordination polyhedron of a peripherically substituted bis(phthalocyaninato)dysprosiumate(III), a dysprosium-based single-4f-ionic single-molecule magnet having a J z = +/- (13)/ 2 Kramers doublet ground state, resulted in drastic changes in dynamical magnetism including a doubling of the energy barrier, a 2-order-of-magnitude decrease of the spin reversal rate, a significant rise of the blocking temperature, and the first observation of the emergence of a large remanent magnetization.

Field-induced inversion of resonant tunneling currents through single molecule junctions and the directional photo-electric effect

NASA Astrophysics Data System (ADS)

Kuperman, Maayan; Peskin, Uri

2017-03-01

It has been known for several decades that the electric current through tunneling junctions is affected by irradiation. In particular, photon-assisted currents by asymmetric irradiation of the two leads was demonstrated and studied extensively in tunneling junctions of different compositions and for different radiation wavelengths. In this work, this phenomenon is revisited in the context of single molecule junctions. Restricting the theoretical discussion to adiabatic periodic driving of one lead with respect to the other within a non-interacting electron formulation, the main features of specific molecules are encoded in the discrete electronic energy levels. The detailed level structure of the molecule is shown to yield new effects in the presence of asymmetric driving of the leads. In particular, when the field-free tunneling process is dominated by a single electronic level, the electric current can be suppressed to zero or flow against the direction of an applied static bias. In the presence of a second electronic level, a directional photo-electric effect is predicted, where not only the magnitude but also the direction of the steady state electric current through the tunneling junction can be changed by a monotonous increase of the field intensity. These effects are analyzed and explained by outlying the relevant theory, using analytic expressions in the wide-band limit, as well as numerical simulations beyond this limit.

Effects of Water on the Single-Chain Elasticity of Poly(U) RNA.

PubMed

Luo, Zhonglong; Cheng, Bo; Cui, Shuxun

2015-06-09

Water, the dominant component under the physiological condition, is a complicated solvent which greatly affects the properties of solute molecules. Here, we utilize atomic force microscope-based single-molecule force spectroscopy to study the influence of water on the single-molecule elasticity of an unstructured single-stranded RNA (poly(U)). In nonpolar solvents, RNA presents its inherent elasticity, which is consistent with the theoretical single-chain elasticity calculated by quantum mechanics calculations. In aqueous buffers, however, an additional energy of 1.88 kJ/mol·base is needed for the stretching of the ssRNA chain. This energy is consumed by the bound water rearrangement (Ew) during chain elongation. Further experimental results indicate that the Ew value is uncorrelated to the salt concentrations and stretching velocity. The results obtained in an 8 M guanidine·HCl solution provide more evidence that the bound water molecules around RNA give rise to the observed deviation between aqueous and nonaqueous environments. Compared to synthetic water-soluble polymers, the value of Ew of RNA is much lower. The weak interference of water is supposed to be the precondition for the RNA secondary structure to exist in aqueous solution.

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

PubMed

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

Direct single-molecule dynamic detection of chemical reactions.

PubMed

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.

Direct single-molecule dynamic detection of chemical reactions

PubMed Central

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

2018-01-01

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. PMID:29487914

The disclosure of mesoscale behaviour of a 3d-SMM monolayer on Au(111) through a multilevel approach.

PubMed

Fernandez Garcia, Guglielmo; Lunghi, Alessandro; Totti, Federico; Sessoli, Roberta

2018-02-22

Here we present a computational study of a full- and a half-monolayer of a Fe 4 single molecule magnet ([Fe 4 (L) 2 (dpm) 6 ], where H 3 L = 2-hydroxymethyl-2-phenylpropane-1,3-diol and Hdpm = dipivaloylmethane, Fe 4 Ph) on an unreconstructed surface of Au(111). This has been possible through the application of an integrated approach, which allows the explicit inclusion of the packing effects in the classical dynamics to be used in a second step in periodic and non-periodic high level DFT calculations. In this way we can obtain access to mesoscale geometrical data and verify how they can influence the magnetic properties of interest of the single Fe 4 molecule. The proposed approach allows to overcome the ab initio state-of-the-art approaches used to study Single Molecule Magnets (SMMs), which are based on the study of one single adsorbed molecule and cannot represent effects on the scale of a monolayer. Indeed, we show here that it is possible to go beyond the computational limitations inherent to the use, for such complex systems, of accurate calculation techniques (e.g. ab initio molecular dynamics) without losing the level of accuracy necessary to gain new detailed insights, hardly reachable at the experimental level. Indeed, long-range and edge effects on the Fe 4 structures and their easy axis of magnetization orientations have been evidenced as their different contributions to the overall macroscopic behavior.

[Biophysics of single molecules].

PubMed

Serdiuk, I N; Deriusheva, E I

2011-01-01

The modern methods of research of biological molecules whose application led to the development of a new field of science, biophysics of single molecules, are reviewed. The measurement of the characteristics of single molecules enables one to reveal their individual features, and it is just for this reason that much more information can be obtained from one molecule than from the entire ensample of molecules. The high sensitivity of the methods considered in detail makes it possible to come close to the solution of the basic problem of practical importance, namely, the determination of the nucleotide sequence of a single DNA molecule.

Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy.

PubMed

Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

2016-07-01

In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers.

Creation of stable molecular junctions with a custom-designed scanning tunneling microscope.

PubMed

Lee, Woochul; Reddy, Pramod

2011-12-02

The scanning tunneling microscope break junction (STMBJ) technique is a powerful approach for creating single-molecule junctions and studying electrical transport in them. However, junctions created using the STMBJ technique are usually mechanically stable for relatively short times (<1 s), impeding detailed studies of their charge transport characteristics. Here, we report a custom-designed scanning tunneling microscope that enables the creation of metal-single molecule-metal junctions that are mechanically stable for more than 1 minute at room temperature. This stability is achieved by a design that minimizes thermal drift as well as the effect of environmental perturbations. The utility of this instrument is demonstrated by performing transition voltage spectroscopy-at the single-molecule level-on Au-hexanedithiol-Au, Au-octanedithiol-Au and Au-decanedithiol-Au junctions.

Atomistic theory of excitonic fine structure in InAs/InP nanowire quantum dot molecules

NASA Astrophysics Data System (ADS)

Świderski, M.; Zieliński, M.

2017-03-01

Nanowire quantum dots have peculiar electronic and optical properties. In this work we use atomistic tight binding to study excitonic spectra of artificial molecules formed by a double nanowire quantum dot. We demonstrate a key role of atomistic symmetry and nanowire substrate orientation rather than cylindrical shape symmetry of a nanowire and a molecule. In particular for [001 ] nanowire orientation we observe a nonvanishing bright exciton splitting for a quasimolecule formed by two cylindrical quantum dots of different heights. This effect is due to interdot coupling that effectively reduces the overall symmetry, whereas single uncoupled [001 ] quantum dots have zero fine structure splitting. We found that the same double quantum dot system grown on [111 ] nanowire reveals no excitonic fine structure for all considered quantum dot distances and individual quantum dot heights. Further we demonstrate a pronounced, by several orders of magnitude, increase of the dark exciton optical activity in a quantum dot molecule as compared to a single quantum dot. For [111 ] systems we also show spontaneous localization of single particle states in one of nominally identical quantum dots forming a molecule, which is mediated by strain and origins from the lack of the vertical inversion symmetry in [111 ] nanostructures of overall C3 v symmetry. Finally, we study lowering of symmetry due to alloy randomness that triggers nonzero excitonic fine structure and the dark exciton optical activity in realistic nanowire quantum dot molecules of intermixed composition.

Effects of a Single Water Molecule on the Reaction Barrier of Interstellar CO2 Formation Reaction.

PubMed

Tachikawa, Hiroto; Kawabata, Hiroshi

2016-08-25

The mechanism by which CO2 is formed in the interstellar space remains a mystery. The most likely reaction is collision between CO and OH; however, previous theoretical works have shown that the activation barrier for CO2 formation is high enough to prevent the reaction at the low thermal conditions of space (∼10 K). The effects of single water molecule on the reaction barrier of CO2 formation from reaction between CO and OH have been investigated here by means of ab initio calculation. The barrier height along the lowest-energy pathway in the reaction between CO and OH in the absence of the H2O molecule was calculated to be 2.3 kcal/mol when CCSD(T) energy corrections are combined with the MP2 basis set limit. In the case of the hydrated (H2O-CO-OH) system, the inclusion of a single H2O molecule into the system significantly decreased the barrier height to 0.2 kcal/mol. This suggests that CO2 can be formed when CO and OH react in the presence of H2O, even under thermal conditions as low as 10 K.

Nanotechnology Approaches to Studying Epigenetic Changes in Cancer

NASA Astrophysics Data System (ADS)

Riehn, Robert

2011-03-01

Placing polyelectrolytes into confined geometries has a profound effect on their molecular configuration. For instance, placing long DNA molecules into channels with a cross-section of about 100 nm 2 stretches them out to about 70% of their contour length. We are using this effect to map epigenetic changes on single DNA and chromatin strands. This mapping on single molecules becomes central in the study of the heterogeneity of cell population in cancer, since rapid change of epigenetic makeup, propagated through rare cancer stem cells, is a hallmark of its progression. We demonstrate the basic building blocks for the single-molecule epigenetic analysis of genomic sized DNA. In particular, we have achieved the mapping of methylated regions in DNA with heterogeneous 5-methyl cytosine modification using a specific fluorescent marker. We further show that chromatin with an intact histone structure can be stretched similar to DNA, and that the epigenetic state of histone tails can be detected using fluorescent antibodies.

Hydrophobic Hydration of Stimulus-Responsive Polyproteins Measured by Single Molecule Force Spectroscopy

NASA Astrophysics Data System (ADS)

Zauscher, Stefan

2007-03-01

We present a new procedure to reduce and analyze force-extension data obtained by single molecule force spectroscopy (SMFS). This approach allows, for the first time, to infer effects of solvent quality and minor changes in molecular architecture on molecular-elasticity of individual (bio)macromolecules. Specifically, we show how changes in the effective Kuhn segment length can be used to interpret the hydrophobic hydration behavior of elastin-like polypeptides (ELPs).Our results are intriguing as they suggest that SMFS in combination with our analysis procedure can be used to study the subtleties of polypeptide-water interactions on the single molecule level. We also report on the force-induced cis-trans isomerization of prolines, which are repeated every fifth residue in the main chain of ELPs. We present evidence for this mechanism by Monte Carlo simulations of the force-extension curves using an elastically coupled two-state system. Our results suggest that SMFS could be used to assay proline cis-trans isomerization in proteins and may thus have significant potential diagnostic utility.

A four-coordinate cobalt(II) single-ion magnet with coercivity and a very high energy barrier

PubMed Central

Rechkemmer, Yvonne; Breitgoff, Frauke D.; van der Meer, Margarethe; Atanasov, Mihail; Hakl, Michael; Orlita, Milan; Neugebauer, Petr; Sarkar, Biprajit; van Slageren, Joris

2016-01-01

Single-molecule magnets display magnetic bistability of molecular origin, which may one day be exploited in magnetic data storage devices. Recently it was realised that increasing the magnetic moment of polynuclear molecules does not automatically lead to a substantial increase in magnetic bistability. Attention has thus increasingly focussed on ions with large magnetic anisotropies, especially lanthanides. In spite of large effective energy barriers towards relaxation of the magnetic moment, this has so far not led to a big increase in magnetic bistability. Here we present a comprehensive study of a mononuclear, tetrahedrally coordinated cobalt(II) single-molecule magnet, which has a very high effective energy barrier and displays pronounced magnetic bistability. The combined experimental-theoretical approach enables an in-depth understanding of the origin of these favourable properties, which are shown to arise from a strong ligand field in combination with axial distortion. Our findings allow formulation of clear design principles for improved materials. PMID:26883902

A four-coordinate cobalt(II) single-ion magnet with coercivity and a very high energy barrier

NASA Astrophysics Data System (ADS)

Rechkemmer, Yvonne; Breitgoff, Frauke D.; van der Meer, Margarethe; Atanasov, Mihail; Hakl, Michael; Orlita, Milan; Neugebauer, Petr; Neese, Frank; Sarkar, Biprajit; van Slageren, Joris

2016-02-01

Single-molecule magnets display magnetic bistability of molecular origin, which may one day be exploited in magnetic data storage devices. Recently it was realised that increasing the magnetic moment of polynuclear molecules does not automatically lead to a substantial increase in magnetic bistability. Attention has thus increasingly focussed on ions with large magnetic anisotropies, especially lanthanides. In spite of large effective energy barriers towards relaxation of the magnetic moment, this has so far not led to a big increase in magnetic bistability. Here we present a comprehensive study of a mononuclear, tetrahedrally coordinated cobalt(II) single-molecule magnet, which has a very high effective energy barrier and displays pronounced magnetic bistability. The combined experimental-theoretical approach enables an in-depth understanding of the origin of these favourable properties, which are shown to arise from a strong ligand field in combination with axial distortion. Our findings allow formulation of clear design principles for improved materials.

Cooperative communication within and between single nanocatalysts

NASA Astrophysics Data System (ADS)

Zou, Ningmu; Zhou, Xiaochun; Chen, Guanqun; Andoy, Nesha May; Jung, Won; Liu, Guokun; Chen, Peng

2018-06-01

Enzymes often show catalytic allostery in which reactions occurring at different sites communicate cooperatively over distances of up to a few nanometres. Whether such effects can occur with non-biological nanocatalysts remains unclear, even though these nanocatalysts can undergo restructuring and molecules can diffuse over catalyst surfaces. Here we report that phenomenologically similar, but mechanistically distinct, cooperative effects indeed exist for nanocatalysts. Using spatiotemporally resolved single-molecule catalysis imaging, we find that catalytic reactions on a single Pd or Au nanocatalyst can communicate with each other, probably via hopping of positively charged holes on the catalyst surface, over 102 nanometres and with a temporal memory of 101 to 102 seconds, giving rise to positive cooperativity among its surface active sites. Similar communication is also observed between individual nanocatalysts, however it operates via a molecular diffusion mechanism involving negatively charged product molecules, and its communication distance is many micrometres. Generalization of these long-range intra- and interparticle catalytic communication mechanisms may introduce a novel conceptual framework for understanding nanoscale catalysis.

Real-time single-molecule imaging of quantum interference.

PubMed

Juffmann, Thomas; Milic, Adriana; Müllneritsch, Michael; Asenbaum, Peter; Tsukernik, Alexander; Tüxen, Jens; Mayor, Marcel; Cheshnovsky, Ori; Arndt, Markus

2012-03-25

The observation of interference patterns in double-slit experiments with massive particles is generally regarded as the ultimate demonstration of the quantum nature of these objects. Such matter-wave interference has been observed for electrons, neutrons, atoms and molecules and, in contrast to classical physics, quantum interference can be observed when single particles arrive at the detector one by one. The build-up of such patterns in experiments with electrons has been described as the "most beautiful experiment in physics". Here, we show how a combination of nanofabrication and nano-imaging allows us to record the full two-dimensional build-up of quantum interference patterns in real time for phthalocyanine molecules and for derivatives of phthalocyanine molecules, which have masses of 514 AMU and 1,298 AMU respectively. A laser-controlled micro-evaporation source was used to produce a beam of molecules with the required intensity and coherence, and the gratings were machined in 10-nm-thick silicon nitride membranes to reduce the effect of van der Waals forces. Wide-field fluorescence microscopy detected the position of each molecule with an accuracy of 10 nm and revealed the build-up of a deterministic ensemble interference pattern from single molecules that arrived stochastically at the detector. In addition to providing this particularly clear demonstration of wave-particle duality, our approach could also be used to study larger molecules and explore the boundary between quantum and classical physics.

Real-time single-molecule imaging of quantum interference

NASA Astrophysics Data System (ADS)

Juffmann, Thomas; Milic, Adriana; Müllneritsch, Michael; Asenbaum, Peter; Tsukernik, Alexander; Tüxen, Jens; Mayor, Marcel; Cheshnovsky, Ori; Arndt, Markus

2012-05-01

The observation of interference patterns in double-slit experiments with massive particles is generally regarded as the ultimate demonstration of the quantum nature of these objects. Such matter-wave interference has been observed for electrons, neutrons, atoms and molecules and, in contrast to classical physics, quantum interference can be observed when single particles arrive at the detector one by one. The build-up of such patterns in experiments with electrons has been described as the ``most beautiful experiment in physics''. Here, we show how a combination of nanofabrication and nano-imaging allows us to record the full two-dimensional build-up of quantum interference patterns in real time for phthalocyanine molecules and for derivatives of phthalocyanine molecules, which have masses of 514 AMU and 1,298 AMU respectively. A laser-controlled micro-evaporation source was used to produce a beam of molecules with the required intensity and coherence, and the gratings were machined in 10-nm-thick silicon nitride membranes to reduce the effect of van der Waals forces. Wide-field fluorescence microscopy detected the position of each molecule with an accuracy of 10 nm and revealed the build-up of a deterministic ensemble interference pattern from single molecules that arrived stochastically at the detector. In addition to providing this particularly clear demonstration of wave-particle duality, our approach could also be used to study larger molecules and explore the boundary between quantum and classical physics.

Role of Solvation Effects in Protein Denaturation: From Thermodynamics to Single Molecules and Back

PubMed Central

England, Jeremy L.; Haran, Gilad

2011-01-01

Protein stability often is studied in vitro through the use of urea and guanidinium chloride, chemical cosolvents that disrupt protein native structure. Much controversy still surrounds the underlying mechanism by which these molecules denature proteins. Here we review current thinking on various aspects of chemical denaturation. We begin by discussing classic models of protein folding and how the effects of denaturants may fit into this picture through their modulation of the collapse, or coil-globule transition, which typically precedes folding. Subsequently, we examine recent molecular dynamics simulations that have shed new light on the possible microscopic origins of the solvation effects brought on by denaturants. It seems likely that both denaturants operate by facilitating solvation of hydrophobic regions of proteins. Finally, we present recent single-molecule fluorescence studies of denatured proteins, the analysis of which corroborates the role of denaturants in shifting the equilibrium of the coil-globule transition. PMID:21219136

Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation.

PubMed

Zhang, Hui; Guo, Peixuan

2014-05-15

Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The single molecule photobleaching (SMPB) technology for direct counting developed by our team (Shu et al., 2007 [18]; Zhang et al., 2007 [19]) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described. Copyright © 2014 Elsevier Inc. All rights reserved.

Single-molecule strong coupling at room temperature in plasmonic nanocavities

NASA Astrophysics Data System (ADS)

Chikkaraddy, Rohit; de Nijs, Bart; Benz, Felix; Barrow, Steven J.; Scherman, Oren A.; Rosta, Edina; Demetriadou, Angela; Fox, Peter; Hess, Ortwin; Baumberg, Jeremy J.

2016-07-01

Photon emitters placed in an optical cavity experience an environment that changes how they are coupled to the surrounding light field. In the weak-coupling regime, the extraction of light from the emitter is enhanced. But more profound effects emerge when single-emitter strong coupling occurs: mixed states are produced that are part light, part matter, forming building blocks for quantum information systems and for ultralow-power switches and lasers. Such cavity quantum electrodynamics has until now been the preserve of low temperatures and complicated fabrication methods, compromising its use. Here, by scaling the cavity volume to less than 40 cubic nanometres and using host-guest chemistry to align one to ten protectively isolated methylene-blue molecules, we reach the strong-coupling regime at room temperature and in ambient conditions. Dispersion curves from more than 50 such plasmonic nanocavities display characteristic light-matter mixing, with Rabi frequencies of 300 millielectronvolts for ten methylene-blue molecules, decreasing to 90 millielectronvolts for single molecules—matching quantitative models. Statistical analysis of vibrational spectroscopy time series and dark-field scattering spectra provides evidence of single-molecule strong coupling. This dressing of molecules with light can modify photochemistry, opening up the exploration of complex natural processes such as photosynthesis and the possibility of manipulating chemical bonds.

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sotthewes, Kai; Heimbuch, René, E-mail: r.heimbuch@utwente.nl; Kumar, Avijit

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 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 dropmore » 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.« less

Antibody-Mediated Small Molecule Detection Using Programmable DNA-Switches.

PubMed

Rossetti, Marianna; Ippodrino, Rudy; Marini, Bruna; Palleschi, Giuseppe; Porchetta, Alessandro

2018-06-13

The development of rapid, cost-effective, and single-step methods for the detection of small molecules is crucial for improving the quality and efficiency of many applications ranging from life science to environmental analysis. Unfortunately, current methodologies still require multiple complex, time-consuming washing and incubation steps, which limit their applicability. In this work we present a competitive DNA-based platform that makes use of both programmable DNA-switches and antibodies to detect small target molecules. The strategy exploits both the advantages of proximity-based methods and structure-switching DNA-probes. The platform is modular and versatile and it can potentially be applied for the detection of any small target molecule that can be conjugated to a nucleic acid sequence. Here the rational design of programmable DNA-switches is discussed, and the sensitive, rapid, and single-step detection of different environmentally relevant small target molecules is demonstrated.

Label-free in-flow detection of single DNA molecules using glass nanopipettes.

PubMed

Gong, Xiuqing; Patil, Amol V; Ivanov, Aleksandar P; Kong, Qingyuan; Gibb, Thomas; Dogan, Fatma; deMello, Andrew J; Edel, Joshua B

2014-01-07

With the view of enhancing the functionality of label-free single molecule nanopore-based detection, we have designed and developed a highly robust, mechanically stable, integrated nanopipette-microfluidic device which combines the recognized advantages of microfluidic systems and the unique properties/advantages of nanopipettes. Unlike more typical planar solid-state nanopores, which have inherent geometrical constraints, nanopipettes can be easily positioned at any point within a microfluidic channel. This is highly advantageous, especially when taking into account fluid flow properties. We show that we are able to detect and discriminate between DNA molecules of varying lengths when motivated through a microfluidic channel, upon the application of appropriate voltage bias across the nanopipette. The effects of applied voltage and volumetric flow rates have been studied to ascertain translocation event frequency and capture rate. Additionally, by exploiting the advantages associated with microfluidic systems (such as flow control and concomitant control over analyte concentration/presence), we show that the technology offers a new opportunity for single molecule detection and recognition in microfluidic devices.

Observation des cycles enzymatiques des ADN topoisomérases par micromanipulation de molécules individuelles

NASA Astrophysics Data System (ADS)

Strick, Terence R.; Charvin, Gilles; Dekker, Nynke H.; Allemand, Jean-François; Bensimon, David; Croquette, Vincent

In this article, we describe single-molecule assays using magnetic traps and we applied these assays to topoisomerase enzymes which unwind and disentangle DNA molecules. First, the elasticity of single DNA molecule is characterized using the magnetic trap. We show that a twisting constraint may be easily applied and that its effect upon DNA may be measured accurately. Then we describe how the topoisomerase activity may be observed at the single-molecule level giving direct access to the important biological parameters of the enzyme such as velocity and processivity. Furthermore, individual cycles of unwinding can be observed in real time. This permits an accurate characterization of the enzyme's biochemical cycle. The data treatment required to identify and analyze individual topoisomerization cycles will be presented in detail. This analysis is applicable to a wide variety of molecular motors. To cite this article: T.R. Strick et al., C. R. Physique 3 (2002) 595-618.

Imaging and sizing of single DNA molecules on a mobile phone.

PubMed

Wei, Qingshan; Luo, Wei; Chiang, Samuel; Kappel, Tara; Mejia, Crystal; Tseng, Derek; Chan, Raymond Yan Lok; Yan, Eddie; Qi, Hangfei; Shabbir, Faizan; Ozkan, Haydar; Feng, Steve; Ozcan, Aydogan

2014-12-23

DNA imaging techniques using optical microscopy have found numerous applications in biology, chemistry and physics and are based on relatively expensive, bulky and complicated set-ups that limit their use to advanced laboratory settings. Here we demonstrate imaging and length quantification of single molecule DNA strands using a compact, lightweight and cost-effective fluorescence microscope installed on a mobile phone. In addition to an optomechanical attachment that creates a high contrast dark-field imaging setup using an external lens, thin-film interference filters, a miniature dovetail stage and a laser-diode for oblique-angle excitation, we also created a computational framework and a mobile phone application connected to a server back-end for measurement of the lengths of individual DNA molecules that are labeled and stretched using disposable chips. Using this mobile phone platform, we imaged single DNA molecules of various lengths to demonstrate a sizing accuracy of <1 kilobase-pairs (kbp) for 10 kbp and longer DNA samples imaged over a field-of-view of ∼2 mm2.

Giant Suppression of Photobleaching for Single Molecule Detection via the Purcell Effect

DTIC Science & Technology

2013-11-18

the molecule dissipates energy to emit another photon (spontaneous emission, or fluorescence, with rate kf) or to heat (intrinsic nonradiative process...enhancement gives rise to both enhanced radiation and enhanced nonradiation (energy dissipation due to Ohmic losses). The enhancement of

Single Molecule Enzymology via Nanoelectronic Circuits

NASA Astrophysics Data System (ADS)

Collins, Philip

Traditional single-molecule techniques rely on fluorescence or force transduction to monitor conformational changes and biochemical activity. Recent demonstrations of single-molecule monitoring with electronic transistors are poised to add to the single-molecule research toolkit. The transistor-based technique is sensitive to the motion of single charged side chain residues and can transduce those motions with microsecond resolution, opening the doors to single-molecule enzymology with unprecedented resolution. Furthermore, the solid-state platform provides opportunities for parallelization in arrays and long-duration monitoring of one molecule's activity or processivity, all without the limitations caused by photo-oxidation or mutagenic fluorophore incorporation. This presentation will review some of these advantages and their particular application to DNA polymerase I processing single-stranded DNA templates. This research was supported financially by the NIH NCI (R01 CA133592-01), the NIH NIGMS (1R01GM106957-01) and the NSF (DMR-1104629 and ECCS-1231910).

Finding a Single Molecule in a Haystack: Optical Detection and Spectroscopy of Single Absorbers in Solids

DTIC Science & Technology

1989-08-18

CODES 18 SUBJECT TERMS (Continue on reverse if necessary and identify by block number) FIELD GROUP SUB-GROUP Single Molecule Detection Pentacene in p...and 10 additional pentacene molecules. This may be accomplished by- a combination of laser FM spectroscopy and either Stark or ultrasonic double...6099 408-927-2426 ABSTRACT: Single-absorber optical spectroscopy in solids is described for the case of finding a single pentacene molecule in a

Differences in single and aggregated nanoparticle plasmon spectroscopy.

PubMed

Singh, Pushkar; Deckert-Gaudig, Tanja; Schneidewind, Henrik; Kirsch, Konstantin; van Schrojenstein Lantman, Evelien M; Weckhuysen, Bert M; Deckert, Volker

2015-02-07

Vibrational spectroscopy usually provides structural information averaged over many molecules. We report a larger peak position variation and reproducibly smaller FWHM of TERS spectra compared to SERS spectra indicating that the number of molecules excited in a TERS experiment is extremely low. Thus, orientational averaging effects are suppressed and micro ensembles are investigated. This is shown for a thiophenol molecule adsorbed on Au nanoplates and nanoparticles.

Tuning the thermal conductance of molecular junctions with interference effects

NASA Astrophysics Data System (ADS)

Klöckner, J. C.; Cuevas, J. C.; Pauly, F.

2017-12-01

We present an ab initio study of the role of interference effects in the thermal conductance of single-molecule junctions. To be precise, using a first-principles transport method based on density functional theory, we analyze the coherent phonon transport in single-molecule junctions made of several benzene and oligo(phenylene ethynylene) derivatives. We show that the thermal conductance of these junctions can be tuned via the inclusion of substituents, which induces destructive interference effects and results in a decrease of the thermal conductance with respect to the unmodified molecules. In particular, we demonstrate that these interference effects manifest as antiresonances in the phonon transmission, whose energy positions can be tuned by varying the mass of the substituents. Our work provides clear strategies for the heat management in molecular junctions and, more generally, in nanostructured metal-organic hybrid systems, which are important to determine how these systems can function as efficient energy-conversion devices such as thermoelectric generators and refrigerators.

Investigating single molecule adhesion by atomic force spectroscopy.

PubMed

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.

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

PubMed Central

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

Electron transport in single molecules: from benzene to graphene.

PubMed

Chen, F; Tao, N J

2009-03-17

Electron movement within and between molecules--that is, electron transfer--is important in many chemical, electrochemical, and biological processes. Recent advances, particularly in scanning electrochemical microscopy (SECM), scanning-tunneling microscopy (STM), and atomic force microscopy (AFM), permit the study of electron movement within single molecules. In this Account, we describe electron transport at the single-molecule level. We begin by examining the distinction between electron transport (from semiconductor physics) and electron transfer (a more general term referring to electron movement between donor and acceptor). The relation between these phenomena allows us to apply our understanding of single-molecule electron transport between electrodes to a broad range of other electron transfer processes. Electron transport is most efficient when the electron transmission probability via a molecule reaches 100%; the corresponding conductance is then 2e(2)/h (e is the charge of the electron and h is the Planck constant). This ideal conduction has been observed in a single metal atom and a string of metal atoms connected between two electrodes. However, the conductance of a molecule connected to two electrodes is often orders of magnitude less than the ideal and strongly depends on both the intrinsic properties of the molecule and its local environment. Molecular length, means of coupling to the electrodes, the presence of conjugated double bonds, and the inclusion of possible redox centers (for example, ferrocene) within the molecular wire have a pronounced effect on the conductance. This complex behavior is responsible for diverse chemical and biological phenomena and is potentially useful for device applications. Polycyclic aromatic hydrocarbons (PAHs) afford unique insight into electron transport in single molecules. The simplest one, benzene, has a conductance much less than 2e(2)/h due to its large LUMO-HOMO gap. At the other end of the spectrum, graphene sheets and carbon nanotubes--consisting of infinite numbers of aromatic rings--have small or even zero energy gaps between the conduction and valence bands. Between these two limits are intermediate molecules with rich properties, such as perylene derivatives made of seven aromatic rings; the properties of these types of molecules have yet to be fully explored. Studying PAHs is important not only in answering fundamental questions about electron transport but also in the ongoing quest for molecular-scale electronic devices. This line of research also helps bridge the gap between electron transfer phenomena in small redox molecules and electron transport properties in nanostructures.

Effect of Chain Conformation on the Single-Molecule Melting Force in Polymer Single Crystals: Steered Molecular Dynamics Simulations Study.

PubMed

Feng, Wei; Wang, Zhigang; Zhang, Wenke

2017-02-28

Understanding the relationship between polymer chain conformation as well as the chain composition within the single crystal and the mechanical properties of the corresponding single polymer chain will facilitate the rational design of high performance polymer materials. Here three model systems of polymer single crystals, namely poly(ethylene oxide) (PEO), polyethylene (PE), and nylon-66 (PA66) have been chosen to study the effects of chain conformation, helical (PEO) versus planar zigzag conformation (PE, PA66), and chain composition (PE versus PA66) on the mechanical properties of a single polymer chain. To do that, steered molecular dynamics simulations were performed on those polymer single crystals by pulling individual polymer chains out of the crystals. Our results show that the patterns of force-extension curve as well as the chain moving mode are closely related to the conformation of the polymer chain in the single crystal. In addition, hydrogen bonds can enhance greatly the force required to stretch the polymer chain out of the single crystal. The dynamic breaking and reformation of multivalent hydrogen bonds have been observed for the first time in PA66 at the single molecule level.

Statistical inference in single molecule measurements of protein adsorption

NASA Astrophysics Data System (ADS)

Armstrong, Megan J.; Tsitkov, Stanislav; Hess, Henry

2018-02-01

Significant effort has been invested into understanding the dynamics of protein adsorption on surfaces, in particular to predict protein behavior at the specialized surfaces of biomedical technologies like hydrogels, nanoparticles, and biosensors. Recently, the application of fluorescent single molecule imaging to this field has permitted the tracking of individual proteins and their stochastic contribution to the aggregate dynamics of adsorption. However, the interpretation of these results is complicated by (1) the finite time available to observe effectively infinite adsorption timescales and (2) the contribution of photobleaching kinetics to adsorption kinetics. Here, we perform a protein adsorption simulation to introduce specific survival analysis methods that overcome the first complication. Additionally, we collect single molecule residence time data from the adsorption of fibrinogen to glass and use survival analysis to distinguish photobleaching kinetics from protein adsorption kinetics.

Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

PubMed

Kong, Muwen; Beckwitt, Emily C; Springall, Luke; Kad, Neil M; Van Houten, Bennett

2017-01-01

Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair. © 2017 Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2015-08-01

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.

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

PubMed

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.

Analyzing Single-Molecule Time Series via Nonparametric Bayesian Inference

PubMed Central

Hines, Keegan E.; Bankston, John R.; Aldrich, Richard W.

2015-01-01

The ability to measure the properties of proteins at the single-molecule level offers an unparalleled glimpse into biological systems at the molecular scale. The interpretation of single-molecule time series has often been rooted in statistical mechanics and the theory of Markov processes. While existing analysis methods have been useful, they are not without significant limitations including problems of model selection and parameter nonidentifiability. To address these challenges, we introduce the use of nonparametric Bayesian inference for the analysis of single-molecule time series. These methods provide a flexible way to extract structure from data instead of assuming models beforehand. We demonstrate these methods with applications to several diverse settings in single-molecule biophysics. This approach provides a well-constrained and rigorously grounded method for determining the number of biophysical states underlying single-molecule data. PMID:25650922

Tracking the ultrafast motion of a single molecule by femtosecond orbital imaging

PubMed Central

Yu, Ping; Repp, Jascha; Huber, Rupert

2017-01-01

Watching a single molecule move on its intrinsic time scale—one of the central goals of modern nanoscience—calls for measurements that combine ultrafast temporal resolution1–8 with atomic spatial resolution9–30. Steady-state experiments achieve the requisite spatial resolution, as illustrated by direct imaging of individual molecular orbitals using scanning tunnelling microscopy9–11 or the acquisition of tip-enhanced Raman and luminescence spectra with sub-molecular resolution27–29. But tracking the dynamics of a single molecule directly in the time domain faces the challenge that single-molecule excitations need to be confined to an ultrashort time window. A first step towards overcoming this challenge has combined scanning tunnelling microscopy with so-called ‘lightwave electronics”1–8, which uses the oscillating carrier wave of tailored light pulses to directly manipulate electronic motion on time scales faster even than that of a single cycle of light. Here we use such ultrafast terahertz scanning tunnelling microscopy to access a state-selective tunnelling regime, where the peak of a terahertz electric-field waveform transiently opens an otherwise forbidden tunnelling channel through a single molecular state and thereby removes a single electron from an individual pentacene molecule’s highest occupied molecular orbital within a time window shorter than one oscillation cycle of the terahertz wave. We exploit this effect to record ~100 fs snapshot images of the structure of the orbital involved, and to reveal through pump-probe measurements coherent molecular vibrations at terahertz frequencies directly in the time domain and with sub-angstrom spatial resolution. We anticipate that the combination of lightwave electronics1–8 and atomic resolution of our approach will open the door to controlling electronic motion inside individual molecules at optical clock rates. PMID:27830788

On the uncertainty in single molecule fluorescent lifetime and energy emission measurements

NASA Technical Reports Server (NTRS)

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

1995-01-01

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 square 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% 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 probably 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 nose 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.

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

NASA Technical Reports Server (NTRS)

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.

Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase.

PubMed

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.

A mechanical model of bacteriophage DNA ejection

NASA Astrophysics Data System (ADS)

Arun, Rahul; Ghosal, Sandip

2017-08-01

Single molecule experiments on bacteriophages show an exponential scaling for the dependence of mobility on the length of DNA within the capsid. It has been suggested that this could be due to the ;capstan mechanism; - the exponential amplification of friction forces that result when a rope is wound around a cylinder as in a ship's capstan. Here we describe a desktop experiment that illustrates the effect. Though our model phage is a million times larger, it exhibits the same scaling observed in single molecule experiments.

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?

NASA Astrophysics Data System (ADS)

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.

Optical Modification of a Single Impurity Molecule in a Solid

DTIC Science & Technology

1991-10-17

have led to direct observations of the lifetime-limited homogeneous Iinewidth of a single pentacene molecule as well as the surprising observation of...advances in the optical detection and spectroscopy of single impurity centers in solids. For the system composed of pentacene impurity molecules in the...limited homogcncous linewidth of a single pentacene molecule as well as the surprising observation of spontaneous spectral diffusion in a crystal

Solvent dependence of the activation energy of attachment determined by single molecule observations of surfactant adsorption.

PubMed

Honciuc, Andrei; Baptiste, Denver Jn; Campbell, Ian P; Schwartz, Daniel K

2009-07-07

Single-molecule total internal reflection fluorescence microscopy was used to obtain real-time images of fluorescently labeled hexadecanoic (palmitic) acid molecules as they adsorbed at the interface between fused silica and three different solvents: hexadecane (HD), tetrahydrofuran (THF), and water. These solvents were chosen to explore the effect of solvent polarity on the activation energy associated with the attachment rate, i.e., the rate at which molecules were transferred to the surface from the near-surface layer. Direct counting of single-molecule events, made under steady-state conditions at extremely low coverage, provided direct, model-independent measurements of this attachment rate, in contrast with conventional ensemble-averaged methods, which are influenced by bulk transport and competing detachment processes. We found that the attachment rate increased with increasing temperature for all solvents. Arrhenius analyses gave activation energies of 5+/-2 kJ/mol for adsorption from HD, 10+/-2 kJ/mol for adsorption from THF, and 19+/-2 kJ/mol for adsorption from water. These energies increased systematically with the solvent polarity and, therefore, with the expected strength of the solvent-substrate interaction. We hypothesize that the adsorption of amphiphilic solute molecules from solution can be regarded as a competitive exchange between solute molecules and surface-bound solvent. In this scenario, adsorption is an activated process, and the activation energy for attachment is associated with the solvent-substrate interaction energy.

An integrated optics microfluidic device for detecting single DNA molecules.

PubMed

Krogmeier, Jeffrey R; Schaefer, Ian; Seward, George; Yantz, Gregory R; Larson, Jonathan W

2007-12-01

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.

Counterion accumulation effects on a suspension of DNA molecules: Equation of state and pressure-driven denaturation

NASA Astrophysics Data System (ADS)

Nicasio-Collazo, Luz Adriana; Delgado-González, Alexandra; Hernández-Lemus, Enrique; Castañeda-Priego, Ramón

2017-04-01

The study of the effects associated with the electrostatic properties of DNA is of fundamental importance to understand both its molecular properties at the single molecule level, like the rigidity of the chain, and its interaction with other charged bio-molecules, including other DNA molecules; such interactions are crucial to maintain the thermodynamic stability of the intra-cellular medium. In the present work, we combine the Poisson-Boltzmann mean-field theory with an irreversible thermodynamic approximation to analyze the effects of counterion accumulation inside DNA on both the denaturation profile of the chain and the equation of state of the suspension. To this end, we model the DNA molecule as a porous charged cylinder immersed in an aqueous solution. These thermo-electrostatic effects are explicitly studied in the particular case of some genes for which damage in their sequence is associated with diffuse large B-cell lymphoma.

Syntheses, Characterizations, and Applications of Molecular Metal Wires and Functional Nanomaterials

DTIC Science & Technology

2009-12-22

challenges Conductance of single molecules Concluding Remarks...values of single -molecule conductance for such pentaruthenium EMACs, other triruthenium and trirhodium EMACs (Dalton Trans. 2009, 2623) were obtained...a voltage-activated switch, or a single -molecule transistor. Among the molecules reported in this research field,1 EMACs2 are particularly

Optical-nanofiber-based interface for single molecules

NASA Astrophysics Data System (ADS)

Skoff, Sarah M.; Papencordt, David; Schauffert, Hardy; Bayer, Bernhard C.; Rauschenbeutel, Arno

2018-04-01

Optical interfaces for quantum emitters are a prerequisite for implementing quantum networks. Here, we couple single molecules to the guided modes of an optical nanofiber. The molecules are embedded within a crystal that provides photostability and, due to the inhomogeneous broadening, a means to spectrally address single molecules. Single molecules are excited and detected solely via the nanofiber interface without the requirement of additional optical access. In this way, we realize a fully fiber-integrated system that is scalable and may become a versatile constituent for quantum hybrid systems.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Xiaoliang Sunney

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,more » 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 specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.« less

Single Molecule Spectral Diffusion in a Solid Detected Via Fluorescence Spectroscopy

DTIC Science & Technology

1991-10-15

other local fields) at the position of the molecule, the spectral jumps may occur because the class II pentacene molecules are coupled to an...and identify by block number) FIELD jGROUP SUB-GROUP_ Single molecule spectroscopy Precision detection Spectral diffusion, Pentacene in p-terphenyl 19...significant increases in detection sensitivity for single pentacene molecules in crystals of p-terphenyl at low temperatures. With the increased signal to

IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure

PubMed Central

Zhang, Lei; Ren, Gang

2012-01-01

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules. PMID:22291925

Dynamic fluctuations in single-molecule biophysics experiments. Comment on "Extracting physics of life at the molecular level: A review of single-molecule data analyses" by W. Colomb and S.K. Sarkar

NASA Astrophysics Data System (ADS)

Krapf, Diego

2015-06-01

Single-molecule biophysics includes the study of isolated molecules and that of individual molecules within living cells. In both cases, dynamic fluctuations at the nanoscale play a critical role. Colomb and Sarkar emphasize how different noise sources affect the analysis of single molecule data [1]. Fluctuations in biomolecular systems arise from two very different mechanisms. On one hand thermal fluctuations are a predominant feature in the behavior of individual molecules. On the other hand, non-Gaussian fluctuations can arise from inter- and intramolecular interactions [2], spatial heterogeneities [3], non-Poisson external perturbations [4] and complex non-linear dynamics in general [5,6].

Non-equilibrium transport and spin dynamics in single-molecule magnets

NASA Astrophysics Data System (ADS)

Moldoveanu, V.; Dinu, I. V.; Tanatar, B.

2015-11-01

The time-dependent transport through single-molecule magnets (SMM) coupled to magnetic or non-magnetic electrodes is studied in the framework of the generalized Master equation (GME) method. We calculate the transient currents which develop when the molecule is smoothly coupled to the source and drain electrodes. The signature of the electrically induced magnetic switching on these transient currents is investigated. Our simulations show that the magnetic switching of the molecular spin can be read indirectly from the transient currents if one lead is magnetic and it is much faster if the leads have opposite spin polarizations. We identify effects of the transverse anisotropy on the dynamics of molecular states.

Bias voltage induced resistance switching effect in single-molecule magnets' tunneling junction.

PubMed

Zhang, Zhengzhong; Jiang, Liang

2014-09-12

An electric-pulse-induced reversible resistance change effect in a molecular magnetic tunneling junction, consisting of a single-molecule magnet (SMM) sandwiched in one nonmagnetic and one ferromagnetic electrode, is theoretically investigated. By applying a time-varying bias voltage, the SMM's spin orientation can be manipulated with large bias voltage pulses. Moreover, the different magnetic configuration at high-resistance/low-resistance states can be 'read out' by utilizing relative low bias voltage. This device scheme can be implemented with current technologies (Khajetoorians et al 2013 Science 339 55) and has potential application in molecular spintronics and high-density nonvolatile memory devices.

Single-molecule toroics in Ising-type lanthanide molecular clusters.

PubMed

Ungur, Liviu; Lin, Shuang-Yan; Tang, Jinkui; Chibotaru, Liviu F

2014-01-01

Single-molecule toroics (SMTs) are defined, by analogy with single-molecule magnets, as bistable molecules with a toroidal magnetic state, and seem to be most promising for future applications in quantum computing and information storage and use as multiferroic materials with magnetoelectric effect. As an interdisciplinary research area that spans chemistry, physics and material sciences, synthetic chemists have produced systems suitable for detailed study by physicists and materials scientists, while ab initio calculations have been playing a major role in the detection of toroidal magnetization and the advancement of this field. In this tutorial review, we demonstrate the research developed in the fascinating and challenging field of molecular-based SMTs with particular focus on how recent studies tend to address the issue of toroidal arrangement of the magnetic moment in these systems. Herein, nine typical SMTs are summarized, showing that the assembly of wheel-shaped complexes with the high symmetry of the molecule unit and strong intra-molecular dipolar interactions using strong anisotropy metal ions represents the most promising route toward the design of a toroidal moment. Furthermore, the linkage of such robust toroidal moment units with ferromagnetic type through appropriate bridging ligands enhances the toroidal magnetic moment per unit cell.

How to probe transverse magnetic anisotropy of a single-molecule magnet by electronic transport?

NASA Astrophysics Data System (ADS)

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.

Computer systems for annotation of single molecule fragments

DOEpatents

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.

Extracting Models in Single Molecule Experiments

NASA Astrophysics Data System (ADS)

Presse, Steve

2013-03-01

Single molecule experiments can now monitor the journey of a protein from its assembly near a ribosome to its proteolytic demise. Ideally all single molecule data should be self-explanatory. However data originating from single molecule experiments is particularly challenging to interpret on account of fluctuations and noise at such small scales. Realistically, basic understanding comes from models carefully extracted from the noisy data. Statistical mechanics, and maximum entropy in particular, provide a powerful framework for accomplishing this task in a principled fashion. Here I will discuss our work in extracting conformational memory from single molecule force spectroscopy experiments on large biomolecules. One clear advantage of this method is that we let the data tend towards the correct model, we do not fit the data. I will show that the dynamical model of the single molecule dynamics which emerges from this analysis is often more textured and complex than could otherwise come from fitting the data to a pre-conceived model.

Smart SERS Hot Spots: Single Molecules Can Be Positioned in a Plasmonic Nanojunction Using Host-Guest Chemistry.

PubMed

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

2018-04-04

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.

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

PubMed

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.

Zero-phonon-line emission of single molecules for applications in quantum information processing

NASA Astrophysics Data System (ADS)

Kiraz, Alper; Ehrl, M.; Mustecaplioglu, O. E.; Hellerer, T.; Brauchle, C.; Zumbusch, A.

2005-07-01

A single photon source which generates transform limited single photons is highly desirable for applications in quantum optics. Transform limited emission guarantees the indistinguishability of the emitted single photons. This, in turn brings groundbreaking applications in linear optics quantum information processing within an experimental reach. Recently, self-assembled InAs quantum dots and trapped atoms have successfully been demonstrated as such sources for highly indistinguishable single photons. Here, we demonstrate that nearly transform limited zero-phonon-line (ZPL) emission from single molecules can be obtained by using vibronic excitation. Furthermore we report the results of coincidence detection experiments at the output of a Michelson-type interferometer. These experiments reveal Hong-Ou-Mandel correlations as a proof of the indistinguishability of the single photons emitted consecutively from a single molecule. Therefore, single molecules constitute an attractive alternative to single InAs quantum dots and trapped atoms for applications in linear optics quantum information processing. Experiments were performed with a home-built confocal microscope keeping the sample in a superfluid liquid Helium bath at 1.4K. We investigated terrylenediimide (TDI) molecules highly diluted in hexadecane (Shpol'skii matrix). A continuous wave single mode dye laser was used for excitation of vibronic transitions of individual molecules. From the integral fluorescence, the ZPL of single molecules was selected with a spectrally narrow interference filter. The ZPL emission was then sent to a scanning Fabry-Perot interferometer for linewidth measurements or a Michelson-type interferometer for coincidence detection.

Zero-mode waveguide nanophotonic structures for single molecule characterization

NASA Astrophysics Data System (ADS)

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.

Photophysical Behaviors of Single Fluorophores Localized on Zinc Oxide Nanostructures

PubMed Central

Fu, Yi; Zhang, Jian; Lakowicz, Joseph R.

2012-01-01

Single-molecule fluorescence spectroscopy has now been widely used to investigate complex dynamic processes which would normally be obscured in an ensemble-averaged measurement. In this report we studied photophysical behaviors of single fluorophores in proximity to zinc oxide nanostructures by single-molecule fluorescence spectroscopy and time-correlated single-photon counting (TCSPC). Single fluorophores on ZnO surfaces showed enhanced fluorescence brightness to various extents compared with those on glass; the single-molecule time trajectories also illustrated pronounced fluctuations of emission intensities, with time periods distributed from milliseconds to seconds. We attribute fluorescence fluctuations to the interfacial electron transfer (ET) events. The fluorescence fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the interfacial electron transfer reaction processes. PMID:23109903

Single molecule quantum-confined Stark effect measurements of semiconductor nanoparticles at room temperature

NASA Astrophysics Data System (ADS)

Park, Kyoung Won; Deutsch, Zvicka; Li, J. Jack; Oron, Dan; Weiss, Shimon

2013-02-01

We investigate the quantum confined Stark effect (QCSE) of various nanoparticles (NPs) on the single molecule level at room temperature. We tested 8 different NPs with different geometry, material composition and electronic structure, and measured their QCSE by single molecule spectroscopy. This study reveals that suppressing the Coulomb interaction force between electron and hole by asymmetric type-II interface is critical for an enhanced QCSE. For example, ZnSe-CdS and CdSe(Te)-CdS-CdZnSe asymmetric nanorods (type-II) display respectively twice and more than three times larger QCSE than that of simple type-I nanorods (CdSe). In addition, wavelength blue-shift of QCSE and roughly linear Δλ-F (emission wavelength shift vs. the applied electric field) relation are observed for the type-II nanorods. Experimental results (Δλ-F or ΔE-F) are successfully reproduced by self-consistent quantum mechanical calculation. Intensity reduction in blue-shifted spectrum is also accounted for. Both calculations and experiments suggest that the magnitude of the QCSE is predominantly determined by the degree of initial charge separation in these structures.

Combining single-molecule manipulation and single-molecule detection.

PubMed

Cordova, Juan Carlos; Das, Dibyendu Kumar; Manning, Harris W; Lang, Matthew J

2014-10-01

Single molecule force manipulation combined with fluorescence techniques offers much promise in revealing mechanistic details of biomolecular machinery. Here, we review force-fluorescence microscopy, which combines the best features of manipulation and detection techniques. Three of the mainstay manipulation methods (optical traps, magnetic traps and atomic force microscopy) are discussed with respect to milestones in combination developments, in addition to highlight recent contributions to the field. An overview of additional strategies is discussed, including fluorescence based force sensors for force measurement in vivo. Armed with recent exciting demonstrations of this technology, the field of combined single-molecule manipulation and single-molecule detection is poised to provide unprecedented views of molecular machinery. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Manipulating, Reacting, and Constructing Single Molecules with a Scanning Tunneling Microscope Tip

NASA Astrophysics Data System (ADS)

Hla, S.-W.

The fascinating advances in atom and molecule manipulation with the scanning tunneling microscope (STM) tip allow scientists to fabricate artificial atomic scale structures, to study local quantum phenomena, or to probe physical and chemical properties of single atoms and molecules on surfaces. Recent achievements in individual synthesis of single molecules with the STM tip further open up an entirely new opportunities in nanoscience and technology. The STM manipulation techniques usef ul in the molecular construction are reviewed and prospects for future opportunities of single molecule chemical engineering and their possible implications to nano-scale science and technology are discussed.

A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads

PubMed Central

Iwaki, M.; Wickham, S. F.; Ikezaki, K.; Yanagida, T.; Shih, W. M.

2016-01-01

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes. PMID:27941751

Biochemical and single-molecule analyses of the RNA silencing suppressing activity of CrPV-1A

PubMed Central

Watanabe, Mariko; Iwakawa, Hiro-oki; Tadakuma, Hisashi

2017-01-01

Abstract Viruses often encode viral silencing suppressors (VSSs) to counteract the hosts’ RNA silencing activity. The cricket paralysis virus 1A protein (CrPV-1A) is a unique VSS that binds to a specific Argonaute protein (Ago)—the core of the RNA-induced silencing complex (RISC)—in insects to suppress its target cleavage reaction. However, the precise molecular mechanism of CrPV-1A action remains unclear. Here we utilized biochemical and single-molecule imaging approaches to analyze the effect of CrPV-1A during target recognition and cleavage by Drosophila Ago2-RISC. Our results suggest that CrPV-1A obstructs the initial target searching by Ago2-RISC via base pairing in the seed region. The combination of biochemistry and single-molecule imaging may help to pave the way for mechanistic understanding of VSSs with diverse functions. PMID:28977639

A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads.

PubMed

Iwaki, M; Wickham, S F; Ikezaki, K; Yanagida, T; Shih, W M

2016-12-12

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

NASA Astrophysics Data System (ADS)

Hu, Xiaohu; Hong, Liang; Dean Smith, Micholas; Neusius, Thomas; Cheng, Xiaolin; Smith, Jeremy C.

2016-02-01

Internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behaviour with effective relaxation times existing over many decades in time, from ps up to ~102 s (refs ,,,). Here, using molecular dynamics simulations, we show that, on timescales from 10-12 to 10-5 s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of the energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behaviour persists up to timescales approaching the in vivo lifespan of individual protein molecules.

Largely Enhanced Single-molecule Fluorescence in Plasmonic Nanogaps formed by Hybrid Silver Nanostructures

PubMed Central

Zhang, Jian; Lakowicz, Joseph R.

2013-01-01

It has been suggested that narrow gaps between metallic nanostructures can be practical for producing large field enhancement. We design a hybrid silver nanostructure geometry in which fluorescent emitters are sandwiched between silver nanoparticles and silver island film (SIF). A desired number of polyelectrolyte layers are deposited on the SIF surface before the self-assembly of a second silver nanoparticle layer. Layer-by-layer configuration provides a well-defined dye position. It allows us to study the photophyical behaviors of fluorophores in the resulting gap at the single molecule level. The enhancement factor of a fluorophore located in the gap is much higher than those on silver surfaces alone and on glass. These effects may be used for increased detectability of single molecules bound to surfaces which contain metallic structures for either biophysical studies or high sensitivity assays. PMID:23373787

Debye screening in single-molecule carbon nanotube field-effect sensors.

PubMed

Sorgenfrei, Sebastian; Chiu, Chien-Yang; Johnston, Matthew; Nuckolls, Colin; Shepard, Kenneth L

2011-09-14

Point-functionalized carbon nanotube field-effect transistors can serve as highly sensitive detectors for biomolecules. With a probe molecule covalently bound to a defect in the nanotube sidewall, two-level random telegraph noise (RTN) in the conductance of the device is observed as a result of a charged target biomolecule binding and unbinding at the defect site. Charge in proximity to the defect modulates the potential (and transmission) of the conductance-limiting barrier created by the defect. In this Letter, we study how these single-molecule electronic sensors are affected by ionic screening. Both charge in proximity to the defect site and buffer concentration are found to affect RTN amplitude in a manner that follows from simple Debye length considerations. RTN amplitude is also dependent on the potential of the electrolyte gate as applied to the reference electrode; at high enough gate potentials, the target DNA is completely repelled and RTN is suppressed.

Single-molecule spectromicroscopy: a route towards sub-wavelength refractometry.

PubMed

Anikushina, T A; Gladush, M G; Gorshelev, A A; Naumov, A V

2015-01-01

We suggest a novel approach for spatially resolved probing of local fluctuations of the refractive index n in solids by means of single-molecule (SM) spectroscopy. It is based on the dependence T1(n) of the effective radiative lifetime T1 of dye centres in solids on n due to the local-field effects. Detection of SM zero-phonon lines at low temperatures gives the values of the SM natural spectral linewidth (which is inversely proportional to T1) and makes it possible to reveal the distribution of the local n values in solids. Here we demonstrate this possibility on the example of amorphous polyethylene and polycrystalline naphthalene doped with terrylene. In particular, we show that the obtained distributions of lifetime limited spectral linewidths of terrylene molecules embedded into these matrices are due to the spatial fluctuations of the refractive index local values.

Debye screening in single-molecule carbon nanotube field-effect transistors

PubMed Central

Sorgenfrei, Sebastian; Chiu, Chien-yang; Johnston, Matthew; Nuckolls, Colin; Shepard, Kenneth L.

2013-01-01

Point-functionalized carbon nanotube field-effect transistors can serve as highly sensitive detectors for biomolecules. With a probe molecule covalently bound to a defect in the nanotube sidewall, two-level random telegraph noise (RTN) in the conductance of the device is observed as a result of a charged target biomolecule binding and unbinding at the defect site. Charge in proximity to the defect modulates the potential (and transmission) of the conductance-limiting barrier created by the defect. In this Letter, we study how these single-molecule electronic sensors are affected by ionic screening. Both charge in proximity to the defect site and buffer concentration are found to affect RTN amplitude in a manner that follows from simple Debye length considerations. RTN amplitude is also dependent on the potential of the electrolyte gate as applied to the reference electrode; at high enough repulsive potentials, the target DNA is completely repelled and RTN is suppressed. PMID:21806018

Ultracold Nonreactive Molecules in an Optical Lattice: Connecting Chemistry to Many-Body Physics.

PubMed

Doçaj, Andris; Wall, Michael L; Mukherjee, Rick; Hazzard, Kaden R A

2016-04-01

We derive effective lattice models for ultracold bosonic or fermionic nonreactive molecules (NRMs) in an optical lattice, analogous to the Hubbard model that describes ultracold atoms in a lattice. In stark contrast to the Hubbard model, which is commonly assumed to accurately describe NRMs, we find that the single on-site interaction parameter U is replaced by a multichannel interaction, whose properties we elucidate. Because this arises from complex short-range collisional physics, it requires no dipolar interactions and thus occurs even in the absence of an electric field or for homonuclear molecules. We find a crossover between coherent few-channel models and fully incoherent single-channel models as the lattice depth is increased. We show that the effective model parameters can be determined in lattice modulation experiments, which, consequently, measure molecular collision dynamics with a vastly sharper energy resolution than experiments in a free-space ultracold gas.

Effect of the substitution of F on the photoswitching behavior in single molecular device

NASA Astrophysics Data System (ADS)

Bian, Baoan; Zheng, Yapeng; Yuan, Peipei; Liao, Bin; Chen, Wei; An, Xiuhua; Mo, Xiaotong; Ding, Yuqiang

2017-09-01

We carry out first-principles calculations based on density functional theory and non-equilibrium Green's function to investigate the electronic transport properties of a 5-arylidenehydantoin molecule sandwiched between two Au electrodes. A reversible switching behavior between E and Z isomerization can be observed in the device through light irradiation, and their currents display different characteristic. Furthermore, it is found that the substitution of F in the molecule enlarges the switching ratio of device. The different characteristics of currents for E/Z forms and E/Z with the substitution of F are discussed by the transmission spectra and the molecular projected self-consistent Hamiltonian states. We discuss the change of Fermi level alignment due to the substitution of F, and the polarization effect under bias. We find the negative differential resistance effect in the E form with the substitution of F, which is explained by change of molecule-electrode coupling with the varied bias. The results suggest that the 5-arylidenehydantoin molecule with the substitution of F that improves the performance of device, becoming one of the methods for improving single molecular photoswitching performance in the future.

Single-Molecule Tracking Study of the Permeability and Transverse Width of Individual Cylindrical Microdomains in Solvent-Swollen Polystyrene-block-poly(ethylene oxide) Films.

PubMed

Sapkota, Dol Raj; Tran-Ba, Khanh-Hoa; Elwell-Cuddy, Trevor; Higgins, Daniel A; Ito, Takashi

2016-12-01

Understanding the properties of solvent-swollen block copolymer (BCP) microdomains is important for better solvent-based control of microdomain morphology, orientation, and permeability. In this study, single-molecule tracking (SMT) was explored to assess the permeability and transverse width of individual cylindrical microdomains in solvent-swollen polystyrene-block-poly(ethylene oxide) (PS-b-PEO) films. PS-b-PEO films comprising shear-elongated cylindrical PEO microdomains were prepared by sandwiching its benzene or tetrahydrofuran (THF) solution between two glass substrates. SMT measurements were performed at different drying times to investigate the effects of solvent evaporation on the microdomain properties. SMT data showed one-dimensional (1D) motions of single fluorescent molecules (sulforhodamine B) based on their diffusion within the cylindrical microdomains. Microdomain permeability and transverse width were assessed from the single-molecule diffusion coefficients (D SMT ) and transverse variance of the 1D trajectories (σ δ 2 ), respectively. The D SMT and σ δ 2 values from individual 1D trajectories were widely distributed with no evidence of correlation on a single molecule basis, possibly because the individual microdomains in a film were swollen to different extents. On average, microdomain permeability (D) and effective radius (r) gradually decreased within the first 3 days of drying due to solvent evaporation, and changed negligibly thereafter. PS-b-PEO films prepared from THF solutions exhibited larger changes in D and r as compared with those from benzene solutions due to the better swelling of the PEO microdomains by THF. Importantly, changes in D were more prominent than those in r, suggesting that the permeability of the PEO microdomains is very susceptible to the presence of solvent. These results reveal the unique capability of SMT to assess the properties of individual cylindrical microdomains in a solvent-swollen BCP film.

One-by-one single-molecule detection of mutated nucleobases by monitoring tunneling current using a DNA tip.

PubMed

Bui, Phuc Tan; Nishino, Tomoaki; Shiigi, Hiroshi; Nagaoka, Tsutomu

2015-01-31

A DNA molecule was utilized as a probe tip to achieve single-molecule genetic diagnoses. Hybridization of the probe and target DNAs resulted in electron tunneling along the emergent double-stranded DNA. Simple stationary monitoring of the tunneling current leads to single-molecule DNA detection and discovery of base mismatches and methylation.

Extracting physics of life at the molecular level: A review of single-molecule data analyses.

PubMed

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.

Inferring diffusion in single live cells at the single-molecule level

PubMed Central

Robson, Alex; Burrage, Kevin; Leake, Mark C.

2013-01-01

The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single-molecule and single-cell level can add significant insight into understanding molecular architectures of diffusing molecules and the nanoscale environment in which the molecules diffuse. The tool of choice for monitoring dynamic molecular localization in live cells is fluorescence microscopy, especially so combining total internal reflection fluorescence with the use of fluorescent protein (FP) reporters in offering exceptional imaging contrast for dynamic processes in the cell membrane under relatively physiological conditions compared with competing single-molecule techniques. There exist several different complex modes of diffusion, and discriminating these from each other is challenging at the molecular level owing to underlying stochastic behaviour. Analysis is traditionally performed using mean square displacements of tracked particles; however, this generally requires more data points than is typical for single FP tracks owing to photophysical instability. Presented here is a novel approach allowing robust Bayesian ranking of diffusion processes to discriminate multiple complex modes probabilistically. It is a computational approach that biologists can use to understand single-molecule features in live cells. PMID:23267182

Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA.

PubMed

Ren, Hang; Cheyne, Cameron G; Fleming, Aaron M; Burrows, Cynthia J; White, Henry S

2018-04-18

Measurement of single-molecule reactions can elucidate microscopic mechanisms that are often hidden from ensemble analysis. Herein, we report the acid-base titration of a single DNA duplex confined within the wild-type α-hemolysin (α-HL) nanopore for up to 3 h, while monitoring the ionic current through the nanopore. Modulation between two states in the current-time trace for duplexes containing the C:C mismatch in proximity to the latch constriction of α-HL is attributed to the base flipping of the C:C mismatch. As the pH is lowered, the rate for the C:C mismatch to flip from the intra-helical state to the extra-helical state ( k intra-extra ) decreases, while the rate for base flipping from the extra-helical state to the intra-helical state ( k extra-intra ) remains unchanged. Both k intra-extra and k extra-intra are on the order of 1 × 10 -2 s -1 to 1 × 10 -1 s -1 and remain stable over the time scale of the measurement (several hours). Analysis of the pH-dependent kinetics of base flipping using a hidden Markov kinetic model demonstrates that protonation/deprotonation occurs while the base pair is in the intra-helical state. We also demonstrate that the rate of protonation is limited by transport of H + into the α-HL nanopore. Single-molecule kinetic isotope experiments exhibit a large kinetic isotope effect (KIE) for k intra-extra ( k H / k D ≈ 5) but a limited KIE for k extra-intra ( k H / k D ≈ 1.3), supporting our model. Our experiments correspond to the longest single-molecule measurements performed using a nanopore, and demonstrate its application in interrogating mechanisms of single-molecule reactions in confined geometries.

Quantitative Aspects of Single Molecule Microscopy

PubMed Central

Ober, Raimund J.; Tahmasbi, Amir; Ram, Sripad; Lin, Zhiping; Ward, E. Sally

2015-01-01

Single molecule microscopy is a relatively new optical microscopy technique that allows the detection of individual molecules such as proteins in a cellular context. This technique has generated significant interest among biologists, biophysicists and biochemists, as it holds the promise to provide novel insights into subcellular processes and structures that otherwise cannot be gained through traditional experimental approaches. Single molecule experiments place stringent demands on experimental and algorithmic tools due to the low signal levels and the presence of significant extraneous noise sources. Consequently, this has necessitated the use of advanced statistical signal and image processing techniques for the design and analysis of single molecule experiments. In this tutorial paper, we provide an overview of single molecule microscopy from early works to current applications and challenges. Specific emphasis will be on the quantitative aspects of this imaging modality, in particular single molecule localization and resolvability, which will be discussed from an information theoretic perspective. We review the stochastic framework for image formation, different types of estimation techniques and expressions for the Fisher information matrix. We also discuss several open problems in the field that demand highly non-trivial signal processing algorithms. PMID:26167102

Discriminating cascading processes in nonlinear optics: A QED analysis based on their molecular and geometric origin

NASA Astrophysics Data System (ADS)

Bennett, Kochise; Chernyak, Vladimir Y.; Mukamel, Shaul

2017-03-01

The nonlinear optical response of a system of molecules often contains contributions whereby the products of lower-order processes in two separate molecules give signals that appear on top of a genuine direct higher-order process with a single molecule. These many-body contributions are known as cascading and complicate the interpretation of multidimensional stimulated Raman and other nonlinear signals. In a quantum electrodynamic treatment, these cascading processes arise from second-order expansion in the molecular coupling to vacuum modes of the radiation field, i.e., single-photon exchange between molecules, which also gives rise to other collective effects. We predict the relative phase of the direct and cascading nonlinear signals and its dependence on the microscopic dynamics as well as the sample geometry. This phase may be used to identify experimental conditions for distinguishing the direct and cascading signals by their phase. Higher-order cascading processes involving the exchange of several photons between more than two molecules are discussed.

Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy.

PubMed

Yang, Darren; Wong, Wesley P

2018-01-01

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as high cost, low throughput, technical difficulty, and strict infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope (CFM) adapted to a commercial centrifuge enables use by nonspecialists, and integration with DNA nanoswitches facilitates both reliable measurements and repeated molecular interrogation. Here, we provide detailed protocols for constructing the CFM, creating DNA nanoswitch samples, and carrying out single-molecule force measurements.

Single molecule detection, thermal fluctuation and life

PubMed Central

YANAGIDA, Toshio; ISHII, Yoshiharu

2017-01-01

Single molecule detection has contributed to our understanding of the unique mechanisms of life. Unlike artificial man-made machines, biological molecular machines integrate thermal noises rather than avoid them. For example, single molecule detection has demonstrated that myosin motors undergo biased Brownian motion for stepwise movement and that single protein molecules spontaneously change their conformation, for switching to interactions with other proteins, in response to thermal fluctuation. Thus, molecular machines have flexibility and efficiency not seen in artificial machines. PMID:28190869

Nonlinearity, resonance, charging, and motion at the atomic scale studied with scanning tunneling microscopes

NASA Astrophysics Data System (ADS)

Tu, Xiuwen

2008-10-01

Several novel phenomena at the single-atom and single-molecule level occurring on the surfaces of single crystals were studied with home-built low temperature scanning tunneling microscopes. The results revealed intriguing properties of single atoms and single molecules, including nonlinearity, resonance, charging, and motion. First, negative differential resistance (NDR) was observed in the dI/dV spectra for single copper-phthalocyanine (CuPc) molecules adsorbed on one- and two-layer sodium bromide (NaBr), but not for single CuPc molecules adsorbed on three-layer NaBr, all grown on a NiAl(110) surface. This transition from NDR to the absence of NDR was explained as the result of competing effects in the double-barrier tunnel junction (DBTJ) and was reproduced in a calculation based on a resonant-tunneling model. Second, the nonlinearity of the STM junction due to a single manganese (Mn) atom or MnCO molecule adsorbed on a NiAl(110) surface was used to rectify microwave irradiation. The resulting rectification current was shown to be sensitive to the spin-splitting of the electronic states of the Mn atom and to the vibrations of the MnCO molecule. Next, the ordering of cesium (Cs) atoms adsorbed on a Au(111) surface and a NiAl(110) surface was imaged in real space. Because of charge transfer to the substrates, Cs adatoms were positively charged on both surfaces. Even at 12 K, Cs adatoms were able to move and adjust according to coverage. On Au(111), the Cs first layer had a quasi-hexagonal lattice and islands of the second Cs layer did not appear until the first was completed. On NiAl(110), a locally disordered Cs first layer was observed before a locally ordered layer appeared at higher coverages. The cation-pi interactions were then studied at the single molecular level. We were able to form cation-pi complexes such as Cs···DSB, Cs···DSB···Cs, Rb···DSB, and Rb···ZnEtiol controllably by manipulation with the STM tip. We could also separate these complexes controllably by voltage pulses. STM imaging and spectroscopy revealed precise information about the atomic and electronic structure of these cation-pi complexes. Finally, electron transport through single atoms and molecules in a double-barrier tunnel junction (DBTJ) was examined. Charge bistability was observed for single ZnEtioI molecules adsorbed in several different conformations on ultrathin aluminum oxide. A sudden decrease in local apparent barrier height (LABH) was observed at the onset of an adsorbate electronic orbital for single ZnEtioI molecules and Cs atoms supported by the ultrathin aluminum oxide. The resonant-tunneling model, which was proposed to explain the transition from NDR to the absence of NDR, was found useful in explaining the sudden decrease in LABH, too. NDR, bipolar tunneling, and vibronic states were also observed and discussed in the context of DBTJ.

Electron-molecule scattering in a strong laser field: Two-center interference effects

NASA Astrophysics Data System (ADS)

Dakić, J.; Habibović, D.; Čerkić, A.; Busuladžić, M.; Milošević, D. B.

2017-10-01

Laser-assisted scattering of electrons on diatomic molecules is considered using the S -matrix theory within the second Born approximation. The first term of the expansion in powers of the scattering potential corresponds to the direct or single laser-assisted scattering of electrons on molecular targets, while the second term of this expansion corresponds to the laser-assisted rescattering or double scattering. The rescattered electrons may have considerably higher energies in the final state than those that scattered only once. For multicenter polyatomic molecules scattering and rescattering may happen at any center and in any order. All these cases contribute to the scattering amplitude and the interference of different contributions leads to an increase or a decrease of the differential cross section in particular electron energy regions. For diatomic molecules there are two such contributions for single scattering and four contributions for double scattering. Analyzing the spectra of the scattered electrons, we find two interesting effects. For certain molecular orientations, the plateaus in the electron energy spectrum, characteristic of laser-assisted electron-atom scattering, are replaced by a sequence of gradually declining maxima, caused by the two-center interference effects. The second effect is the appearance of symmetric U -shaped structures in the angle-resolved energy spectra, which are described very well by the analytical formulas we provide.

Single-molecule dataset (SMD): a generalized storage format for raw and processed single-molecule data.

PubMed

Greenfeld, Max; van de Meent, Jan-Willem; Pavlichin, Dmitri S; Mabuchi, Hideo; Wiggins, Chris H; Gonzalez, Ruben L; Herschlag, Daniel

2015-01-16

Single-molecule techniques have emerged as incisive approaches for addressing a wide range of questions arising in contemporary biological research [Trends Biochem Sci 38:30-37, 2013; Nat Rev Genet 14:9-22, 2013; Curr Opin Struct Biol 2014, 28C:112-121; Annu Rev Biophys 43:19-39, 2014]. The analysis and interpretation of raw single-molecule data benefits greatly from the ongoing development of sophisticated statistical analysis tools that enable accurate inference at the low signal-to-noise ratios frequently associated with these measurements. While a number of groups have released analysis toolkits as open source software [J Phys Chem B 114:5386-5403, 2010; Biophys J 79:1915-1927, 2000; Biophys J 91:1941-1951, 2006; Biophys J 79:1928-1944, 2000; Biophys J 86:4015-4029, 2004; Biophys J 97:3196-3205, 2009; PLoS One 7:e30024, 2012; BMC Bioinformatics 288 11(8):S2, 2010; Biophys J 106:1327-1337, 2014; Proc Int Conf Mach Learn 28:361-369, 2013], it remains difficult to compare analysis for experiments performed in different labs due to a lack of standardization. Here we propose a standardized single-molecule dataset (SMD) file format. SMD is designed to accommodate a wide variety of computer programming languages, single-molecule techniques, and analysis strategies. To facilitate adoption of this format we have made two existing data analysis packages that are used for single-molecule analysis compatible with this format. Adoption of a common, standard data file format for sharing raw single-molecule data and analysis outcomes is a critical step for the emerging and powerful single-molecule field, which will benefit both sophisticated users and non-specialists by allowing standardized, transparent, and reproducible analysis practices.

Trap density of states in small-molecule organic semiconductors: A quantitative comparison of thin-film transistors with single crystals

NASA Astrophysics Data System (ADS)

Kalb, Wolfgang L.; Haas, Simon; Krellner, Cornelius; Mathis, Thomas; Batlogg, Bertram

2010-04-01

We show that it is possible to reach one of the ultimate goals of organic electronics: producing organic field-effect transistors with trap densities as low as in the bulk of single crystals. We studied the spectral density of localized states in the band gap [trap density of states (trap DOS)] of small-molecule organic semiconductors as derived from electrical characteristics of organic field-effect transistors or from space-charge-limited current measurements. This was done by comparing data from a large number of samples including thin-film transistors (TFT’s), single crystal field-effect transistors (SC-FET’s) and bulk samples. The compilation of all data strongly suggests that structural defects associated with grain boundaries are the main cause of “fast” hole traps in TFT’s made with vacuum-evaporated pentacene. For high-performance transistors made with small-molecule semiconductors such as rubrene it is essential to reduce the dipolar disorder caused by water adsorbed on the gate dielectric surface. In samples with very low trap densities, we sometimes observe a steep increase in the trap DOS very close (<0.15eV) to the mobility edge with a characteristic slope of 10-20 meV. It is discussed to what degree band broadening due to the thermal fluctuation of the intermolecular transfer integral is reflected in this steep increase in the trap DOS. Moreover, we show that the trap DOS in TFT’s with small-molecule semiconductors is very similar to the trap DOS in hydrogenated amorphous silicon even though polycrystalline films of small-molecules with van der Waals-type interaction on the one hand are compared with covalently bound amorphous silicon on the other hand.

A revised set of values of single-bond radii derived from the observed interatomic distances in metals by correction for bond number and resonance energy

PubMed Central

Pauling, Linus; Kamb, Barclay

1986-01-01

An earlier discussion [Pauling, L. (1947) J. Am. Chem. Soc. 69, 542] of observed bond lengths in elemental metals with correction for bond number and resonance energy led to a set of single-bond metallic radii with values usually somewhat less than the corresponding values obtained from molecules and complex ions. A theory of resonating covalent bonds has now been developed that permits calculation of the number of resonance structures per atom and of the effective resonance energy per bond. With this refined method of correcting the observed bond lengths for the effect of resonance energy, a new set of single-bond covalent radii, in better agreement with values from molecules and complex ions, has been constructed. PMID:16593698

Desolvation-Driven 100-Fold Slow-down of Tunneling Relaxation Rate in Co(II)-Dy(III) Single-Molecule Magnets through a Single-Crystal-to-Single-Crystal Process

NASA Astrophysics Data System (ADS)

Liu, Jun-Liang; Wu, Jie-Yi; Huang, Guo-Zhang; Chen, Yan-Cong; Jia, Jian-Hua; Ungur, Liviu; Chibotaru, Liviu F.; Chen, Xiao-Ming; Tong, Ming-Liang

2015-11-01

Single-molecule magnets (SMMs) are regarded as a class of promising materials for spintronic and ultrahigh-density storage devices. Tuning the magnetic dynamics of single-molecule magnets is a crucial challenge for chemists. Lanthanide ions are not only highly magnetically anisotropic but also highly sensitive to the changes in the coordination environments. We developed a feasible approach to understand parts of the magneto-structure correlations and propose to regulate the relaxation behaviors via rational design. A series of Co(II)-Dy(III)-Co(II) complexes were obtained using in situ synthesis; in this system of complexes, the relaxation dynamics can be greatly improved, accompanied with desolvation, via single-crystal to single-crystal transformation. The effective energy barrier can be increased from 293 cm-1 (422 K) to 416 cm-1 (600 K), and the tunneling relaxation time can be grown from 8.5 × 10-4 s to 7.4 × 10-2 s. These remarkable improvements are due to the change in the coordination environments of Dy(III) and Co(II). Ab initio calculations were performed to better understand the magnetic dynamics.

Extending Single-Molecule Microscopy Using Optical Fourier Processing

PubMed Central

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

Extending single-molecule microscopy using optical Fourier processing.

PubMed

Backer, Adam S; Moerner, W E

2014-07-17

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.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

PubMed

Bartke, Rebecca M; Cameron, Elizabeth L; Cristie-David, Ajitha S; Custer, Thomas C; Denies, Maxwell S; Daher, May; Dhakal, Soma; Ghosh, Soumi; Heinicke, Laurie A; Hoff, J Damon; Hou, Qian; Kahlscheuer, Matthew L; Karslake, Joshua; Krieger, Adam G; Li, Jieming; Li, Xiang; Lund, Paul E; Vo, Nguyen N; Park, Jun; Pitchiaya, Sethuramasundaram; Rai, Victoria; Smith, David J; Suddala, Krishna C; Wang, Jiarui; Widom, Julia R; Walter, Nils G

2015-05-01

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines. © 2014 Wiley Periodicals, Inc.

Single Molecules as Optical Probes for Structure and Dynamics

NASA Astrophysics Data System (ADS)

Orrit, Michel

Single molecules and single nanoparticles are convenient links between the nanoscale world and the laboratory. We discuss the limits for their optical detection by three different methods: fluorescence, direct absorption, and photothermal detection. We briefly review some recent illustrations of qualitatively new information gathered from single-molecule signals: intermittency of the fluorescence intensity, acoustic vibrations of nanoparticles (1-100 GHz) or of extended defects in molecular crystals (0.1-1 MHz), and dynamical heterogeneity in glass-forming molecular liquids. We conclude with an outlook of future uses of single-molecule methods in physical chemistry, soft matter, and material science.

Single-Molecule Electronics: Chemical and Analytical Perspectives.

PubMed

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.

Quantitative analysis of single-molecule superresolution images

PubMed Central

Coltharp, Carla; Yang, Xinxing; Xiao, Jie

2014-01-01

This review highlights the quantitative capabilities of single-molecule localization-based superresolution imaging methods. In addition to revealing fine structural details, the molecule coordinate lists generated by these methods provide the critical ability to quantify the number, clustering, and colocalization of molecules with 10 – 50 nm resolution. Here we describe typical workflows and precautions for quantitative analysis of single-molecule superresolution images. These guidelines include potential pitfalls and essential control experiments, allowing critical assessment and interpretation of superresolution images. PMID:25179006

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

NASA Astrophysics Data System (ADS)

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.

Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

PubMed Central

2009-01-01

Many studies of RNA folding and catalysis have revealed conformational heterogeneity, metastable folding intermediates, and long-lived states with distinct catalytic activities. We have developed a single-molecule imaging approach for investigating the functional heterogeneity of in vitro-evolved RNA aptamers. Monitoring the association of fluorescently labeled ligands with individual RNA aptamer molecules has allowed us to record binding events over the course of multiple days, thus providing sufficient statistics to quantitatively define the kinetic properties at the single-molecule level. The ligand binding kinetics of the highly optimized RNA aptamer studied here displays a remarkable degree of uniformity and lack of memory. Such homogeneous behavior is quite different from the heterogeneity seen in previous single-molecule studies of naturally derived RNA and protein enzymes. The single-molecule methods we describe may be of use in analyzing the distribution of functional molecules in heterogeneous evolving populations or even in unselected samples of random sequences. PMID:19572753

Probe-based measurement of lateral single-electron transfer between individual molecules

PubMed Central

Steurer, Wolfram; Fatayer, Shadi; Gross, Leo; Meyer, Gerhard

2015-01-01

The field of molecular electronics aims at using single molecules as functional building blocks for electronics components, such as switches, rectifiers or transistors. A key challenge is to perform measurements with atomistic control over the alignment of the molecule and its contacting electrodes. Here we use atomic force microscopy to examine charge transfer between weakly coupled pentacene molecules on insulating films with single-electron sensitivity and control over the atomistic details. We show that, in addition to the imaging capability, the probe tip can be used to control the charge state of individual molecules and to detect charge transfers to/from the tip, as well as between individual molecules. Our approach represents a novel route for molecular charge transfer studies with a host of opportunities, especially in combination with single atom/molecule manipulation and nanopatterning techniques. PMID:26387533

Probing molecular choreography through single-molecule biochemistry.

PubMed

van Oijen, Antoine M; Dixon, Nicholas E

2015-12-01

Single-molecule approaches are having a dramatic impact on views of how proteins work. The ability to observe molecular properties at the single-molecule level allows characterization of subpopulations and acquisition of detailed kinetic information that would otherwise be hidden in the averaging over an ensemble of molecules. In this Perspective, we discuss how such approaches have successfully been applied to in vitro-reconstituted systems of increasing complexity.

Single molecule image formation, reconstruction and processing: introduction.

PubMed

Ashok, Amit; Piestun, Rafael; Stallinga, Sjoerd

2016-07-01

The ability to image at the single molecule scale has revolutionized research in molecular biology. This feature issue presents a collection of articles that provides new insights into the fundamental limits of single molecule imaging and reports novel techniques for image formation and analysis.

Single Molecule and Collective Dynamics of Motor Protein Coupled with Mechano-Sensitive Chemical Reaction

NASA Astrophysics Data System (ADS)

Iwaki, Mitsuhiro; Marcucci, Lorenzo; Togashi, Yuichi; Yanagida, Toshio

2013-12-01

Motor proteins such as myosin and kinesin hydrolyze ATP into ADP and Pi to convert chemical energy into mechanical work. This resultsin various motile processes like muscle contraction, vesicle transport and cell division. Recent single molecule experiments have revealed that external load applied to these motor proteins perturb not only the mechanical motion, but the ATP hydrolysis cycle as well, making these molecules mechano-enzymes. Here, we describe our single molecule detection techniques to reveal the mechano-enzymatic properties of myosin and introduce recent progress from both experimental and theoretical approaches at the single- and multiple-molecule level.

Beyond experimental noise: Analyzing single-molecule data of heterogeneous systems. Comment on "Extracting physics of life at the molecular level: A review of single-molecule data analyses" by W. Colomb and S.K. Sarkar

NASA Astrophysics Data System (ADS)

Meroz, Yasmine

2015-06-01

In the 1980s the world witnessed the advent of single-molecule experiments. The first atomic resolution characterization of a surface was reported by scanning tunneling microscope (STM) in 1982 [1], followed by atomic force microscope (AFM) in 1986 [2]. The first optical detection and spectroscopy of a single molecule in a solid took place in 1989 [3,4], in a time where essentially all chemical experiments were made on bulk, i.e. averaging over millions of copies of the same molecule.

Observation of zero-point quantum fluctuations of a single-molecule magnet through the relaxation of its nuclear spin bath.

PubMed

Morello, A; Millán, A; de Jongh, L J

2014-03-21

A single-molecule magnet placed in a magnetic field perpendicular to its anisotropy axis can be truncated to an effective two-level system, with easily tunable energy splitting. The quantum coherence of the molecular spin is largely determined by the dynamics of the surrounding nuclear spin bath. Here we report the measurement of the nuclear spin-lattice relaxation rate 1/T1n in a single crystal of the single-molecule magnet Mn12-ac, at T ≈ 30 mK in perpendicular fields B⊥ up to 9 T. The relaxation channel at B ≈ 0 is dominated by incoherent quantum tunneling of the Mn12-ac spin S, aided by the nuclear bath itself. However for B⊥>5 T we observe an increase of 1/T1n by several orders of magnitude up to the highest field, despite the fact that the molecular spin is in its quantum mechanical ground state. This striking observation is a consequence of the zero-point quantum fluctuations of S, which allow it to mediate the transfer of energy from the excited nuclear spin bath to the crystal lattice at much higher rates. Our experiment highlights the importance of quantum fluctuations in the interaction between an "effective two-level system" and its surrounding spin bath.

Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

2015-07-28

Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging themore » ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.« less

Dynamic effects in friction and adhesion through cooperative rupture and formation of supramolecular bonds.

PubMed

Blass, Johanna; Albrecht, Marcel; Bozna, Bianca L; Wenz, Gerhard; Bennewitz, Roland

2015-05-07

We introduce a molecular toolkit for studying the dynamics in friction and adhesion from the single molecule level to effects of multivalency. As experimental model system we use supramolecular bonds established by the inclusion of ditopic adamantane connector molecules into two surface-bound cyclodextrin molecules, attached to a tip of an atomic force microscope (AFM) and to a flat silicon surface. The rupture force of a single bond does not depend on the pulling rate, indicating that the fast complexation kinetics of adamantane and cyclodextrin are probed in thermal equilibrium. In contrast, the pull-off force for a group of supramolecular bonds depends on the unloading rate revealing a non-equilibrium situation, an effect discussed as the combined action of multivalency and cantilever inertia effects. Friction forces exhibit a stick-slip characteristic which is explained by the cooperative rupture of groups of host-guest bonds and their rebinding. No dependence of friction on the sliding velocity has been observed in the accessible range of velocities due to fast rebinding and the negligible delay of cantilever response in AFM lateral force measurements.

Room temperature stable single molecule rectifiers with graphite electrodes

NASA Astrophysics Data System (ADS)

Rungger, Ivan; Kaliginedi, V.; Droghetti, A.; Ozawa, H.; Kuzume, A.; Haga, M.; Broekmann, P.; Rudnev, A. V.

In this combined theoretical and experimental study we present new molecular electronics device characteristics of unprecedented stability at room temperature by using electrodes based on highly oriented pyrolytic graphite with covalently attached molecules. To this aim, we explore the effect of the anchoring group chemistry on the charge transport properties of graphite/molecule contacts by means of the scanning tunneling microscopy break-junction technique and ab initio simulations. The theoretical approach to evaluate the conductance is based on density functional theory calculations combined with the non-equilibrium Greens function technique, as implemented in the Smeagol electron transport code. We also demonstrate a strong bias dependence and rectification of the single molecule conductance induced by the anchoring chemistry in combination with the very low density of states of graphite around the Fermi energy. We show that the direction of tunneling current rectification can be tuned by anchoring group chemistry.

The separation between the 5'-3' ends in long RNA molecules is short and nearly constant.

PubMed

Leija-Martínez, Nehemías; Casas-Flores, Sergio; Cadena-Nava, Rubén D; Roca, Joan A; Mendez-Cabañas, José A; Gomez, Eduardo; Ruiz-Garcia, Jaime

2014-12-16

RNA molecules play different roles in coding, decoding and gene expression regulation. Such roles are often associated to the RNA secondary or tertiary structures. The folding dynamics lead to multiple secondary structures of long RNA molecules, since an RNA molecule might fold into multiple distinct native states. Despite an ensemble of different structures, it has been theoretically proposed that the separation between the 5' and 3' ends of long single-stranded RNA molecules (ssRNA) remains constant, independent of their base content and length. Here, we present the first experimental measurements of the end-to-end separation in long ssRNA molecules. To determine this separation, we use single molecule Fluorescence Resonance Energy Transfer of fluorescently end-labeled ssRNA molecules ranging from 500 to 5500 nucleotides in length, obtained from two viruses and a fungus. We found that the end-to-end separation is indeed short, within 5-9 nm. It is remarkable that the separation of the ends of all RNA molecules studied remains small and similar, despite the origin, length and differences in their secondary structure. This implies that the ssRNA molecules are 'effectively circularized' something that might be a general feature of RNAs, and could result in fine-tuning for translation and gene expression regulation. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hybrid materials and methods for producing the same

DOEpatents

Luzzi, David E [Wallingford, PA; Smith, Brian W [Schelton, CT

2003-04-08

A hybrid material is provided which comprises a first single-walled nanotube having a lumen, and a fill molecule contained within the lumen of the single-walled nanotube. A method for producing the hybrid material is also provided wherein a single-walled nanotube is contacted with a fill molecule to cause the fill molecule to enter the lumen of the single-walled nanotube.

Hybrid materials and methods for producing the same

DOEpatents

Luzzi, David E [Wallingford, PA; Smith, Brian W [Philadelphia, PA

2008-02-19

A hybrid material is provided which comprises a first single-walled nanotube having a lumen, and a fill molecule contained within the lumen of the single-walled nanotube. A method for producing the hybrid material is also provided wherein a single-walled nanotube is contacted with a fill molecule to cause the fill molecule to enter the lumen of the single-walled nanotube.

Techniques for 3D tracking of single molecules with nanometer accuracy in living cells

NASA Astrophysics Data System (ADS)

Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

2013-06-01

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.

Single-molecule electrocatalysis by single-walled carbon nanotubes.

PubMed

Xu, Weilin; Shen, Hao; Kim, Yoon Ji; Zhou, Xiaochun; Liu, Guokun; Park, Jiwoong; Chen, Peng

2009-12-01

We report a single-molecule fluorescence study of electrocatalysis by single-walled carbon nanotubes (SWNTs) at single-reaction resolution. Applying super-resolution optical imaging, we find that the electrocatalysis occurs at discrete, nanometer-dimension sites on SWNTs. Single-molecule kinetic analysis leads to an electrocatalytic mechanism, allowing quantification of the reactivity and heterogeneity of individual reactive sites. Combined with conductivity measurements, this approach will be powerful to interrogate how the electronic structure of SWNTs affects the electrocatalytic interfacial charge transfer, a process fundamental to photoelectrochemical cells.

Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

NASA Astrophysics Data System (ADS)

Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

2011-03-01

A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

Measuring molecular motions inside single cells with improved analysis of single-particle trajectories

NASA Astrophysics Data System (ADS)

Rowland, David J.; Biteen, Julie S.

2017-04-01

Single-molecule super-resolution imaging and tracking can measure molecular motions inside living cells on the scale of the molecules themselves. Diffusion in biological systems commonly exhibits multiple modes of motion, which can be effectively quantified by fitting the cumulative probability distribution of the squared step sizes in a two-step fitting process. Here we combine this two-step fit into a single least-squares minimization; this new method vastly reduces the total number of fitting parameters and increases the precision with which diffusion may be measured. We demonstrate this Global Fit approach on a simulated two-component system as well as on a mixture of diffusing 80 nm and 200 nm gold spheres to show improvements in fitting robustness and localization precision compared to the traditional Local Fit algorithm.

Surface-enhanced Raman scattering on single-wall carbon nanotubes.

PubMed

Kneipp, Katrin; Kneipp, Harald; Dresselhaus, Mildred S; Lefrant, Serge

2004-11-15

Exploiting the effect of surface-enhanced Raman scattering (SERS), the Raman signal of single-wall carbon nanotubes (SWNTs) can be enhanced by up to 14 orders of magnitude when the tubes are in contact with silver or gold nanostructures and Raman scattering takes place predominantly in the enhanced local optical fields of the nanostructures. Such a level of enhancement offers exciting opportunities for ultrasensitive Raman studies on SWNTs and allows resonant and non-resonant Raman experiments to be done on single SWNTs at relatively high signal levels. Since the optical fields are highly localized within so-called "hot spots" on fractal silver colloidal clusters, lateral confinement of the Raman scattering can be as small as 5 nm, allowing spectroscopic selection of a single nanotube from a larger population. Moreover, since SWNTs are very stable "artificial molecules" with a high aspect ratio and a strong electron-phonon coupling, they are unique "test molecules" for investigating the SERS effect itself and for probing the "electromagnetic field contribution" and "charge transfer contribution" to the effect. SERS is also a powerful tool for monitoring the "chemical" interaction between the nanotube and the metal nanostructure.

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

PubMed Central

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

Biomimetic Nanoarchitectures for the Study of T Cell Activation with Single-Molecule Control

NASA Astrophysics Data System (ADS)

Cai, Haogang

Physical factors in the environment of a cell affect its function and behavior in a variety of ways. There is increasing evidence that, among these factors, the geometric arrangement of receptor ligands plays an important role in setting the conditions for critical cellular processes. The goal of this thesis is to develop new techniques for probing the role of extracellular ligand geometry, with a focus on T cell activation. In this work, top-down molecular-scale nanofabrication and bottom-up selective self-assembly were combined in order to present functional nanomaterials (primarily biomolecules) on a surface with precise spatial control and single-molecule resolution. Such biomolecule nanoarrays are becoming an increasingly important tool in surface-based in vitro assays for biosensing, molecular and cellular studies. The nanoarrays consist of metallic nanodots patterned on glass coverslips using electron beam and nanoimprint lithography, combined with self-aligned pattern transfer. The nanodots were then used as anchors for the immobilization of biological ligands, and backfilled with a protein-repellent passivation layer of polyethylene glycol. The passivation efficiency was improved to minimize nonspecific adsorption. In order to ensure true single-molecule control, we developed an on-chip protocol to measure the molecular occupancy of nanodot arrays based on fluorescence photobleaching, while accounting for quenching effects by plasmonic absorption. We found that the molecular occupancy can be interpreted as a packing problem, with the solution depending on the nanodot size and the concentration of self-assembly reagents, where the latter can be easily adjusted to control the molecular occupancy according to the dot size. The optimized nanoarrays were used as biomimetic architectures for the study of T cell activation with single-molecule control. T cell activation involves an elaborate arrangement of signaling, adhesion, and costimulatory molecules organized into a stereotypic geometric structure, known as the immunological synapse, between T cell and antigen-presenting cell. Novel bifunctionalization schemes were developed to better mimic the antigen-presenting surfaces. Nanoarrays were functionalized by single molecules of UCHT1 Fab', and served as individual T cell receptor binding sites. The adhesion molecule ICAM-1 was bound to either static PEG background, or a mobile supported lipid bilayer. The minimum geometric requirements (receptor clustering, spacing and stoichiometry) for T cell activation was probed by systematic variation of the nanoarray spacing and cluster size. Out-of-plane spatial control of the two key molecules by way of nanopillar arrays was used to adjust the membrane bending and steric effects, which were essential for the investigation of molecular segregation in T cell activation. The results provide insights into the complicated T cell activation mechanism, with translational implications toward adoptive immunotherapies for cancer and other diseases. This single-molecule platform serves as a novel and powerful tool for molecular and cellular biology, e.g., receptor-mediated signaling/adhesion, especially when multiple ligands or membrane deformation are involved.

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

PubMed

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.

Electrostatic placement of single ferritin molecules

NASA Astrophysics Data System (ADS)

Kumagai, Shinya; Yoshii, Shigeo; Yamada, Kiyohito; Matsukawa, Nozomu; Fujiwara, Isamu; Iwahori, Kenji; Yamashita, Ichiro

2006-04-01

We electrostatically placed a single ferritin molecule on a nanometric 3-aminopropyltriethoxysilane (APTES) pattern that was on an oxidized Si substrate. The numerical analysis of the total interaction free energy for ferritin predicted that a quadrilateral array of 15nm diameter APTES nanodisks placed at intervals of 100nm would accommodate a single molecule of ferritin in each disk under a Debye length of 14nm. The experiments we conducted conformed to theoretical predictions and we successfully placed a single ferritin molecule on each ATPES disk without ferritin adsorbing on the SiO2 substrate surface.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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,more » 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 Tuscany, provides an avenue for scientists from different disciplines to interact and brainstorm and promotes cross-disciplinary collaborations directed toward compelling biological problems.« less

Raman spectroscopy: Watching a molecule breathe

NASA Astrophysics Data System (ADS)

Piatkowski, Lukasz; Hugall, James T.; van Hulst, Niek F.

2014-08-01

Marrying the single-molecule detection ability of surface-enhanced Raman scattering with the extreme time resolution of ultrafast coherent spectroscopy enables the vibrations of a single molecule to be observed.

Single-Molecule Probing the Energy Landscape of Enzymatic Reaction and Non-Covalent Interactions

NASA Astrophysics Data System (ADS)

Lu, H. Peter; Hu, Dehong; Chen, Yu; Vorpagel, Erich R.

2002-03-01

We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions, characterizing mode-specific protein conformational dynamics. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. The overall reaction rates were found to vary widely from molecule-to-molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. The reaction steps subsequent to the initial binding were found to have homogeneous rates. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Combining the analysis of single-molecule experimental trajectories, MD simulation trajectories, and statistical modeling, we have revealed the nature of multiple intermediate states involved in the active enzyme-substrate complex formation and the associated conformational change mechanism and dynamics.

A quantum spin-probe molecular microscope

NASA Astrophysics Data System (ADS)

Perunicic, V. S.; Hill, C. D.; Hall, L. T.; Hollenberg, L. C. L.

2016-10-01

Imaging the atomic structure of a single biomolecule is an important challenge in the physical biosciences. Whilst existing techniques all rely on averaging over large ensembles of molecules, the single-molecule realm remains unsolved. Here we present a protocol for 3D magnetic resonance imaging of a single molecule using a quantum spin probe acting simultaneously as the magnetic resonance sensor and source of magnetic field gradient. Signals corresponding to specific regions of the molecule's nuclear spin density are encoded on the quantum state of the probe, which is used to produce a 3D image of the molecular structure. Quantum simulations of the protocol applied to the rapamycin molecule (C51H79NO13) show that the hydrogen and carbon substructure can be imaged at the angstrom level using current spin-probe technology. With prospects for scaling to large molecules and/or fast dynamic conformation mapping using spin labels, this method provides a realistic pathway for single-molecule microscopy.

Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

NASA Astrophysics Data System (ADS)

Biteen, Julie

2013-03-01

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

Fisher information theory for parameter estimation in single molecule microscopy: tutorial

PubMed Central

Chao, Jerry; Ward, E. Sally; Ober, Raimund J.

2016-01-01

Estimation of a parameter of interest from image data represents a task that is commonly carried out in single molecule microscopy data analysis. The determination of the positional coordinates of a molecule from its image, for example, forms the basis of standard applications such as single molecule tracking and localization-based superresolution image reconstruction. Assuming that the estimator used recovers, on average, the true value of the parameter, its accuracy, or standard deviation, is then at best equal to the square root of the Cramér-Rao lower bound. The Cramér-Rao lower bound can therefore be used as a benchmark in the evaluation of the accuracy of an estimator. Additionally, as its value can be computed and assessed for different experimental settings, it is useful as an experimental design tool. This tutorial demonstrates a mathematical framework that has been specifically developed to calculate the Cramér-Rao lower bound for estimation problems in single molecule microscopy and, more broadly, fluorescence microscopy. The material includes a presentation of the photon detection process that underlies all image data, various image data models that describe images acquired with different detector types, and Fisher information expressions that are necessary for the calculation of the lower bound. Throughout the tutorial, examples involving concrete estimation problems are used to illustrate the effects of various factors on the accuracy of parameter estimation, and more generally, to demonstrate the flexibility of the mathematical framework. PMID:27409706

Yeast cytochrome c integrated with electronic elements: a nanoscopic and spectroscopic study down to single-molecule level

NASA Astrophysics Data System (ADS)

Delfino, I.; Bonanni, B.; Andolfi, L.; Baldacchini, C.; Bizzarri, A. R.; Cannistraro, S.

2007-06-01

Various aspects of redox protein integration with nano-electronic elements are addressed by a multi-technique investigation of different yeast cytochrome c (YCC)-based hybrid systems. Three different immobilization strategies on gold via organic linkers are explored, involving either covalent bonding or electrostatic interaction. Specifically, Au surfaces are chemically modified by self-assembled monolayers (SAMs) exposing thiol-reactive groups, or by acid-oxidized single-wall carbon nanotubes (SWNTs). Atomic force microscopy and scanning tunnelling microscopy are employed to characterize the morphology and the electronic properties of single YCC molecules adsorbed on the modified gold surfaces. In each hybrid system, the protein molecules are stably assembled, in a native configuration. A standing-up arrangement of YCC on SAMs is suggested, together with an enhancement of the molecular conduction, as compared to YCC directly assembled on gold. The electrostatic interaction with functionalized SWNTs allows several YCC adsorption geometries, with a preferential high-spin haem configuration, as outlined by Raman spectroscopy. Moreover, the conduction properties of YCC, explored in different YCC nanojunctions by conductive atomic force microscopy, indicate the effectiveness of electrical conduction through the molecule and its dependence on the electrode material. The joint employment of several techniques confirms the key role of a well-designed immobilization strategy, for optimizing biorecognition capabilities and electrical coupling with conductive substrates at the single-molecule level, as a starting point for advanced applications in nano-biotechnology.

Single-molecule force-conductance spectroscopy of hydrogen-bonded complexes

NASA Astrophysics Data System (ADS)

Pirrotta, Alessandro; De Vico, Luca; Solomon, Gemma C.; Franco, Ignacio

2017-03-01

The emerging ability to study physical properties at the single-molecule limit highlights the disparity between what is observable in an ensemble of molecules and the heterogeneous contributions of its constituent parts. A particularly convenient platform for single-molecule studies are molecular junctions where forces and voltages can be applied to individual molecules, giving access to a series of electromechanical observables that can form the basis of highly discriminating multidimensional single-molecule spectroscopies. Here, we computationally examine the ability of force and conductance 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 receptor that can form up to six simultaneous hydrogen bonds. The simulations combine classical molecular dynamics of the mechanical deformation of the junction with non-equilibrium Green's function computations of the electronic transport. As shown, in these complexes hydrogen bonds mediate transport either by directly participating as a possible transport pathway or by stabilizing molecular conformations with enhanced conductance properties. Further, we observe that force-conductance correlations can be very sensitive to small changes in the chemical structure of the complexes and provide detailed information about the behavior of single molecules that cannot be gleaned from either measurement alone. In fact, there are regions during the elongation that are only mechanically active, others that are only conductance active, and regions where both force and conductance changes as the complex is mechanically manipulated. The implication is that force and conductance provide complementary information about the evolution of molecules in junctions that can be used to interrogate basic structure-transport relations at the single-molecule limit.

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

PubMed

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.

Single-Cell and Single-Molecule Analysis of Gene Expression Regulation.

PubMed

Vera, Maria; Biswas, Jeetayu; Senecal, Adrien; Singer, Robert H; Park, Hye Yoon

2016-11-23

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.

Memory and aging effects of molecular nanomagnet Mn12 benzoate

NASA Astrophysics Data System (ADS)

Palakkal, Jasnamol P.; Sankar, Cheriyedath Raj; Varma, Manoj R.

2018-05-01

The single-molecule nanomagnet Mn12-benzoate was synthesized by an exchange of acetate groups present in the Mn12-acetate by benzoate ligands. The hysteresis loop recorded at 1.85 K exhibit clear step-like feature implying the quantum tunneling effect of the synthesized single-molecule magnet. The thermomagnetic measurements with various protocols identified a blocking temperature at Tb=2.8 K and spin-glass-like memory effect of a pause at an intermittent temperature below Tb. Spin glass property of Mn12 benzoate is further confirmed by a zero field cooled aging test below Tb and obtained stretching parameter β=0.622(1) in the range reported for many spin glass systems. The relaxation rate S shows an inflexion point near the characteristic relaxation time τr=215 s.

Molecular electronics with single molecules in solid-state devices.

PubMed

Moth-Poulsen, Kasper; Bjørnholm, Thomas

2009-09-01

The ultimate aim of molecular electronics is to understand and master single-molecule devices. Based on the latest results on electron transport in single molecules in solid-state devices, we focus here on new insights into the influence of metal electrodes on the energy spectrum of the molecule, and on how the electron transport properties of the molecule depend on the strength of the electronic coupling between it and the electrodes. A variety of phenomena are observed depending on whether this coupling is weak, intermediate or strong.

Single molecule views of Nature's nano-machines

NASA Astrophysics Data System (ADS)

Ha, Taekjip

2006-03-01

We are interested in the perturbational analysis of biological molecules to better understand their mechanisms. Our readout is the fluorescence signal from individual biomolecules, mainly in the form of single molecule fluorescence resonance energy transfer (FRET). We are pioneering approaches to perturb and control biomolecular conformations using external force (combination of single molecule FRET and optical trap) or other biological motifs (DNA hybridization, G-quadruplex, aptamers,.). In this talk, I will present our latest results on mapping the conformational energy landscape of the Holliday junction through simultaneous fluorescence and force measurements. In addition, a new nanomechanical device called single molecule nano-metronome will be discussed with an outlook toward controlling protein conformations using nucleic acids motifs.

Symmetry of Carrier-Envelope Phase Difference Effects in Strong-Field, Few-Cycle Ionization of Atoms and Molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martiny, Christian Per Juul; Madsen, Lars Bojer

2006-09-01

In few-cycle pulses, the exact value of the carrier-envelope phase difference (CEPD) has a pronounced influence on the ionization dynamics of atoms and molecules. We show that, for atoms in circularly polarized light, a change in the CEPD is mapped uniquely to an overall rotation of the system, and results for arbitrary CEPD are obtained by rotation of the results from a single calculation with fixed CEPD. For molecules, this is true only for linear molecules aligned parallel with the propagation direction of the field. The effects of CEPD are classified as geometric or nongeometric. The observations are exemplified bymore » strong-field calculations on hydrogen.« less

SINGLE STRAND-CONTAINING REPLICATING MOLECULES OF CIRCULAR MITOCHONDRIAL DNA

PubMed Central

Wolstenholme, David R.; Koike, Katsuro; Cochran-Fouts, Patricia

1973-01-01

Mitochondrial DNAs (mtDNAs) from Chang rat solid hepatomas and Novikoff rat ascites hepatomas were examined in the electron microscope after preparation by the aqueous and by the formamide protein monolayer techniques. MtDNAs from both tumors were found to include double-forked circular molecules with a form and size suggesting they were replicative intermediates. These molecules were of two classes. In molecules of one class, all three segments were apparently totally double stranded. Molecules of the second class were distinguished by the fact that one of the segments spanning the region between the forks in which replication had occurred (the daughter segments) was either totally single stranded, or contained a single-stranded region associated with one of the forks. Daughter segments of both totally double-stranded and single strand-containing replicating molecules varied in length from about 3 to about 80% of the circular contour length of the molecule. Similar classes of replicating molecules were found in mtDNA from regenerating rat liver and chick embryos, indicating them to be normal intermediates in the replication of mtDNA All of the mtDNAs examined included partially single-stranded simple (nonforked) circular molecules. A possible scheme for the replication of mtDNA is presented, based on the different molecular forms observed PMID:4345165

Single-Molecule Electronic Measurements with Metal Electrodes

ERIC Educational Resources Information Center

Lindsay, Stuart

2005-01-01

A review of concepts like tunneling through a metal-molecule-metal-junction, contrast with electrochemical and optical-charge injection, strong-coupling limit, calculations of tunnel transport, electron transfer through Redox-active molecules is presented. This is followed by a discussion of experimental approaches for single-molecule measurements.

Three-dimensional single-molecule localization with nanometer accuracy using Metal-Induced Energy Transfer (MIET) imaging

NASA Astrophysics Data System (ADS)

Karedla, Narain; Chizhik, Anna M.; Stein, Simon C.; Ruhlandt, Daja; Gregor, Ingo; Chizhik, Alexey I.; Enderlein, Jörg

2018-05-01

Our paper presents the first theoretical and experimental study using single-molecule Metal-Induced Energy Transfer (smMIET) for localizing single fluorescent molecules in three dimensions. Metal-Induced Energy Transfer describes the resonant energy transfer from the excited state of a fluorescent emitter to surface plasmons in a metal nanostructure. This energy transfer is strongly distance-dependent and can be used to localize an emitter along one dimension. We have used Metal-Induced Energy Transfer in the past for localizing fluorescent emitters with nanometer accuracy along the optical axis of a microscope. The combination of smMIET with single-molecule localization based super-resolution microscopy that provides nanometer lateral localization accuracy offers the prospect of achieving isotropic nanometer localization accuracy in all three spatial dimensions. We give a thorough theoretical explanation and analysis of smMIET, describe its experimental requirements, also in its combination with lateral single-molecule localization techniques, and present first proof-of-principle experiments using dye molecules immobilized on top of a silica spacer, and of dye molecules embedded in thin polymer films.

Watching cellular machinery in action, one molecule at a time.

PubMed

Monachino, Enrico; Spenkelink, Lisanne M; van Oijen, Antoine M

2017-01-02

Single-molecule manipulation and imaging techniques have become important elements of the biologist's toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components. © 2017 Monachino et al.

Detailed analysis of complex single molecule FRET data with the software MASH

NASA Astrophysics Data System (ADS)

Hadzic, Mélodie C. A. S.; Kowerko, Danny; Börner, Richard; Zelger-Paulus, Susann; Sigel, Roland K. O.

2016-04-01

The processing and analysis of surface-immobilized single molecule FRET (Förster resonance energy transfer) data follows systematic steps (e.g. single molecule localization, clearance of different sources of noise, selection of the conformational and kinetic model, etc.) that require a solid knowledge in optics, photophysics, signal processing and statistics. The present proceeding aims at standardizing and facilitating procedures for single molecule detection by guiding the reader through an optimization protocol for a particular experimental data set. Relevant features were determined from single molecule movies (SMM) imaging Cy3- and Cy5-labeled Sc.ai5γ group II intron molecules synthetically recreated, to test the performances of four different detection algorithms. Up to 120 different parameterizations per method were routinely evaluated to finally establish an optimum detection procedure. The present protocol is adaptable to any movie displaying surface-immobilized molecules, and can be easily reproduced with our home-written software MASH (multifunctional analysis software for heterogeneous data) and script routines (both available in the download section of www.chem.uzh.ch/rna).

Single-molecule pull-down (SiMPull) for new-age biochemistry: methodology and biochemical applications of single-molecule pull-down (SiMPull) for probing biomolecular interactions in crude cell extracts.

PubMed

Aggarwal, Vasudha; Ha, Taekjip

2014-11-01

Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools. © 2014 WILEY Periodicals, Inc.

A reversible single-molecule switch based on activated antiaromaticity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Xiaodong; Zang, Yaping; Zhu, Liangliang

Single-molecule electronic devices provide researchers with an unprecedented ability to relate novel physical phenomena to molecular chemical structures. Typically, conjugated aromatic molecular backbones are relied upon to create electronic devices, where the aromaticity of the building blocks is used to enhance conductivity. We capitalize on the classical physical organic chemistry concept of Hückel antiaromaticity by demonstrating a single-molecule switch that exhibits low conductance in the neutral state and, upon electrochemical oxidation, reversibly switches to an antiaromatic high-conducting structure. We form single-molecule devices using the scanning tunneling microscope–based break-junction technique and observe an on/off ratio of ~70 for a thiophenylidene derivativemore » that switches to an antiaromatic state with 6-4-6-p electrons. Through supporting nuclear magnetic resonance measurements, we show that the doubly oxidized core has antiaromatic character and we use density functional theory calculations to rationalize the origin of the high-conductance state for the oxidized single-molecule junction. Together, our work demonstrates how the concept of antiaromaticity can be exploited to create single-molecule devices that are highly conducting.« less

A reversible single-molecule switch based on activated antiaromaticity

DOE PAGES

Yin, Xiaodong; Zang, Yaping; Zhu, Liangliang; ...

2017-10-27

Single-molecule electronic devices provide researchers with an unprecedented ability to relate novel physical phenomena to molecular chemical structures. Typically, conjugated aromatic molecular backbones are relied upon to create electronic devices, where the aromaticity of the building blocks is used to enhance conductivity. We capitalize on the classical physical organic chemistry concept of Hückel antiaromaticity by demonstrating a single-molecule switch that exhibits low conductance in the neutral state and, upon electrochemical oxidation, reversibly switches to an antiaromatic high-conducting structure. We form single-molecule devices using the scanning tunneling microscope–based break-junction technique and observe an on/off ratio of ~70 for a thiophenylidene derivativemore » that switches to an antiaromatic state with 6-4-6-p electrons. Through supporting nuclear magnetic resonance measurements, we show that the doubly oxidized core has antiaromatic character and we use density functional theory calculations to rationalize the origin of the high-conductance state for the oxidized single-molecule junction. Together, our work demonstrates how the concept of antiaromaticity can be exploited to create single-molecule devices that are highly conducting.« less

Integrated single-walled carbon nanotube/microfluidic devices for the study of the sensing mechanism of nanotube sensors.

PubMed

Fu, Qiang; Liu, Jie

2005-07-21

A method to fabricate integrated single-walled carbon nanotube/microfluidic devices was developed. This simple process could be used to directly prepare nanotube thin film transistors within the microfluidic channel and to register SWNT devices with the microfludic channel without the need of an additional alignment step. The microfluidic device was designed to have several inlets that deliver multiple liquid flows to a single main channel. The location and width of each flow in the main channel could be controlled by the relative flow rates. This capability enabled us to study the effect of the location and the coverage area of the liquid flow that contained charged molecules on the conduction of the nanotube devices, providing important information on the sensing mechanism of carbon nanotube sensors. The results showed that in a sensor based on a nanotube thin film field effect transistor, the sensing signal came from target molecules absorbed on or around the nanotubes. The effect from adsorption on metal electrodes was weak.

Quantum rotor model for a Bose-Einstein condensate of dipolar molecules.

PubMed

Armaitis, J; Duine, R A; Stoof, H T C

2013-11-22

We show that a Bose-Einstein condensate of heteronuclear molecules in the regime of small and static electric fields is described by a quantum rotor model for the macroscopic electric dipole moment of the molecular gas cloud. We solve this model exactly and find the symmetric, i.e., rotationally invariant, and dipolar phases expected from the single-molecule problem, but also an axial and planar nematic phase due to many-body effects. Investigation of the wave function of the macroscopic dipole moment also reveals squeezing of the probability distribution for the angular momentum of the molecules.

MPAI (mass probes aided ionization) method for total analysis of biomolecules by mass spectrometry.

PubMed

Honda, Aki; Hayashi, Shinichiro; Hifumi, Hiroki; Honma, Yuya; Tanji, Noriyuki; Iwasawa, Naoko; Suzuki, Yoshio; Suzuki, Koji

2007-01-01

We have designed and synthesized various mass probes, which enable us to effectively ionize various molecules to be detected with mass spectrometry. We call the ionization method using mass probes the "MPAI (mass probes aided ionization)" method. We aim at the sensitive detection of various biological molecules, and also the detection of bio-molecules by a single mass spectrometry serially without changing the mechanical settings. Here, we review mass probes for small molecules with various functional groups and mass probes for proteins. Further, we introduce newly developed mass probes for proteins for highly sensitive detection.

Procedure for normalization of cDNA libraries

DOEpatents

Bonaldo, Maria DeFatima; Soares, Marcelo Bento

1997-01-01

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

NASA Astrophysics Data System (ADS)

Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

2009-02-01

Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics.

PubMed

Földes-Papp, Zeno; Baumann, Gerd; Demel, Ulrike; Tilz, Gernot P

2004-04-01

Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bai, Rui; Yi, Shaoqiong; Zhang, Xuejie

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 inducedmore » 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.« less

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

PubMed

Geng, Tao; Mathies, Richard A

2015-01-01

The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

NASA Astrophysics Data System (ADS)

Reed, Jason; Hsueh, Carlin; Mishra, Bud; Gimzewski, James K.

2008-09-01

We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing.

Rapid and efficient detection of single chromophore molecules in aqueous solution

NASA Astrophysics Data System (ADS)

Li, Li-Qiang; Davis, Lloyd M.

1995-06-01

The first experiments on the detection of single fluorescent molecules in a flowing stream of an aqueous solution with high total efficiency are reported. A capillary injection system for sample delivery causes all the dye molecules to pass in a diffusion-broadened stream within a fast-moving sheath flow, through the center of the tightly focused laser excitation beam. Single-molecule detection with a transit time of approximately 1 ms is accomplished with a high-quantum-efficiency single-photon avalanche diode and a low dead-time time-gating circuit for discrimination of Raman-scattered light from the solvent.

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

PubMed

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

2018-04-11

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.

Single molecule junction conductance and binding geometry

NASA Astrophysics Data System (ADS)

Kamenetska, Maria

This Thesis addresses the fundamental problem of controlling transport through a metal-organic interface by studying electronic and mechanical properties of single organic molecule-metal junctions. Using a Scanning Tunneling Microscope (STM) we image, probe energy-level alignment and perform STM-based break junction (BJ) measurements on molecules bound to a gold surface. Using Scanning Tunneling Microscope-based break-junction (STM-BJ) techniques, we explore the effect of binding geometry on single-molecule conductance by varying the structure of the molecules, metal-molecule binding chemistry and by applying sub-nanometer manipulation control to the junction. These experiments are performed both in ambient conditions and in ultra high vacuum (UHV) at cryogenic temperatures. First, using STM imaging and scanning tunneling spectroscopy (STS) measurements we explore binding configurations and electronic properties of an amine-terminated benzene derivative on gold. We find that details of metal-molecule binding affect energy-level alignment at the interface. Next, using the STM-BJ technique, we form and rupture metal-molecule-metal junctions ˜104 times to obtain conductance-vs-extension curves and extract most likely conductance values for each molecule. With these measurements, we demonstrated that the control of junction conductance is possible through a choice of metal-molecule binding chemistry and sub-nanometer positioning. First, we show that molecules terminated with amines, sulfides and phosphines bind selectively on gold and therefore demonstrate constant conductance levels even as the junction is elongated and the metal-molecule attachment point is modified. Such well-defined conductance is also obtained with paracyclophane molecules which bind to gold directly through the pi system. Next, we are able to create metal-molecule-metal junctions with more than one reproducible conductance signatures that can be accessed by changing junction geometry. In the case of pyridine-linked molecules, conductance can be reliably switched between two distinct conductance states using sub-nanometer mechanical manipulation. Using a methyl sulfide linker attached to an oligoene backbone, we are able to create a 3-nm-long molecular potentiometer, whose resistance can be tuned exponentially with Angstom-scale modulations in metal-molecule configuration. These experiments points to a new paradigm for attaining reproducible electrical characteristics of metal-organic devices which involves controlling linker-metal chemistry rather than fabricating identically structured metal-molecule interfaces. By choosing a linker group which is either insensitive to or responds reproducibly to changes in metal-molecule configuration, one can design single molecule devices with functionality more complex than a simple resistor. These ambient temperature experiments were combined with UHV conductance measurements performed in a commercial STM on amine-terminated benzene derivatives which conduct through a non-resonant tunneling mechanism, at temperatures varying from 5 to 300 Kelvin. Our results indicate that while amine-gold binding remains selective irrespective of environment, conductance is not temperature independent, in contrast to what is expected for a tunneling mechanism. Furthermore, using temperature-dependent measurements in ambient conditions we find that HOMO-conducting amines and LUMO-conducting pyridines show opposite dependence of conductance on temperature. These results indicate that energy-level alignment between the molecule and the electrodes changes as a result of varying electrode structure at different temperatures. We find that temperature can serve as a knob with which to tune transport properties of single molecule-metal junctions.

Assembly and diploid architecture of an individual human genome via single-molecule technologies

PubMed Central

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

2015-01-01

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. PMID:26121404

Assembly and diploid architecture of an individual human genome via single-molecule technologies.

PubMed

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.

Detection of kinetic change points in piece-wise linear single molecule motion

NASA Astrophysics Data System (ADS)

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.

Tracking the ultrafast motion of a single molecule by femtosecond orbital imaging

NASA Astrophysics Data System (ADS)

Cocker, Tyler L.; Peller, Dominik; Yu, Ping; Repp, Jascha; Huber, Rupert

2016-11-01

Watching a single molecule move on its intrinsic timescale has been one of the central goals of modern nanoscience, and calls for measurements that combine ultrafast temporal resolution with atomic spatial resolution. Steady-state experiments access the requisite spatial scales, as illustrated by direct imaging of individual molecular orbitals using scanning tunnelling microscopy or the acquisition of tip-enhanced Raman and luminescence spectra with sub-molecular resolution. But tracking the intrinsic dynamics of a single molecule directly in the time domain faces the challenge that interactions with the molecule must be confined to a femtosecond time window. For individual nanoparticles, such ultrafast temporal confinement has been demonstrated by combining scanning tunnelling microscopy with so-called lightwave electronics, which uses the oscillating carrier wave of tailored light pulses to directly manipulate electronic motion on timescales faster even than a single cycle of light. Here we build on ultrafast terahertz scanning tunnelling microscopy to access a state-selective tunnelling regime, where the peak of a terahertz electric-field waveform transiently opens an otherwise forbidden tunnelling channel through a single molecular state. It thereby removes a single electron from an individual pentacene molecule’s highest occupied molecular orbital within a time window shorter than one oscillation cycle of the terahertz wave. We exploit this effect to record approximately 100-femtosecond snapshot images of the orbital structure with sub-ångström spatial resolution, and to reveal, through pump/probe measurements, coherent molecular vibrations at terahertz frequencies directly in the time domain. We anticipate that the combination of lightwave electronics and the atomic resolution of our approach will open the door to visualizing ultrafast photochemistry and the operation of molecular electronics on the single-orbital scale.

Visualizing biological reaction intermediates with DNA curtains

NASA Astrophysics Data System (ADS)

Zhao, Yiling; Jiang, Yanzhou; Qi, Zhi

2017-04-01

Single-molecule approaches have tremendous potential analyzing dynamic biological reaction with heterogeneity that cannot be effectively accessed via traditional ensemble-level biochemical approaches. The approach of deoxyribonucleic acid (DNA) curtains developed by Dr Eric Greene and his research team at Columbia University is a high-throughput single-molecule technique that utilizes fluorescent imaging to visualize protein-DNA interactions directly and allows the acquisition of statistically relevant information from hundreds or even thousands of individual reactions. This review aims to summarize the past, present, and future of DNA curtains, with an emphasis on its applications to solve important biological questions.

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong

2016-01-08

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively boundmore » the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.« less

The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

DOE PAGES

Hu, Xiaohu; Hong, Liang; Smith, Micholas Dean; ...

2015-11-23

Here, internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behavior with effective relaxation times existing over many decades in time, from ps up to ~10 2s (refs 1-4). Here, using molecular dynamics simulations, we show that, on timescales from 10 –12 to 10 –5s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of themore » energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behavior persists up to timescales approaching the in vivo lifespan of individual protein molecules.« less

Non-sticky translocation of bio-molecules through Tween 20-coated solid-state nanopores in a wide pH range

NASA Astrophysics Data System (ADS)

Li, Xiaoqing; Hu, Rui; Li, Ji; Tong, Xin; Diao, J. J.; Yu, Dapeng; Zhao, Qing

2016-10-01

Nanopore-based sensing technology is considered high-throughput and low-cost for single molecule detection, but solid-state nanopores have suffered from pore clogging issues. A simple Tween 20 coating method is applied to ensure long-term (several hours) non-sticky translocation of various types of bio-molecules through SiN nanopores in a wide pH range (4.0-13.0). We also emphasize the importance of choosing appropriate concentration of Tween 20 coating buffer for desired effect. By coating nanopores with a Tween 20 layer, we are able to differentiate between single-stranded DNA and double-stranded DNA, to identify drift-dominated domain for single-stranded DNA, to estimate BSA volume and to observe the shape of individual nucleosome translocation event without non-specific adsorption. The wide pH endurance from 4.0 to 13.0 and the broad types of detection analytes including nucleic acids, proteins, and biological complexes highlight the great application potential of Tween 20-coated solid-state nanopores.

Biochemical and single-molecule analyses of the RNA silencing suppressing activity of CrPV-1A.

PubMed

Watanabe, Mariko; Iwakawa, Hiro-Oki; Tadakuma, Hisashi; Tomari, Yukihide

2017-10-13

Viruses often encode viral silencing suppressors (VSSs) to counteract the hosts' RNA silencing activity. The cricket paralysis virus 1A protein (CrPV-1A) is a unique VSS that binds to a specific Argonaute protein (Ago)-the core of the RNA-induced silencing complex (RISC)-in insects to suppress its target cleavage reaction. However, the precise molecular mechanism of CrPV-1A action remains unclear. Here we utilized biochemical and single-molecule imaging approaches to analyze the effect of CrPV-1A during target recognition and cleavage by Drosophila Ago2-RISC. Our results suggest that CrPV-1A obstructs the initial target searching by Ago2-RISC via base pairing in the seed region. The combination of biochemistry and single-molecule imaging may help to pave the way for mechanistic understanding of VSSs with diverse functions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Detection of the barium daughter in 136Xe -->136Ba + 2e- by in situ single-molecule fluorescence imaging

NASA Astrophysics Data System (ADS)

Nygren, David

2015-10-01

To proceed toward effective ``discovery class'' ton-scale detectors in the search for neutrino-less double beta decay, a robust technique for rejection of all radioactivity-induced backgrounds is urgently needed. An efficient technique for detection of the barium daughter in the decay 136Xe -->136Ba + 2e- would provide a long-sought pathway toward this goal. Single-molecule fluorescent imaging appears to offer a new way to detect the barium daughter atom, which emerges naturally in an ionized state in pure xenon. A doubly charged barium ion can initiate a chelation process with a non-fluorescent precursor molecule, leading to a highly fluorescent complex. Repeated photo-excitation of the complex can reveal both presence and location of a single ionized atom with high precision and selectivity. Detection within the active volume of a xenon gas Time Projection Chamber operating at high pressure would be automatic, and with a capability for redundant confirmation.

Chain-Length-Dependent Exciton Dynamics in Linear Oligothiophenes Probed Using Ensemble and Single-Molecule Spectroscopy.

PubMed

Kim, Tae-Woo; Kim, Woojae; Park, Kyu Hyung; Kim, Pyosang; Cho, Jae-Won; Shimizu, Hideyuki; Iyoda, Masahiko; Kim, Dongho

2016-02-04

Exciton dynamics in π-conjugated molecular systems is highly susceptible to conformational disorder. Using time-resolved and single-molecule spectroscopic techniques, the effect of chain length on the exciton dynamics in a series of linear oligothiophenes, for which the conformational disorder increased with increasing chain length, was investigated. As a result, extraordinary features of the exciton dynamics in longer-chain oligothiophene were revealed. Ultrafast fluorescence depolarization processes were observed due to exciton self-trapping in longer and bent chains. Increase in exciton delocalization during dynamic planarization processes was also observed in the linear oligothiophenes via time-resolved fluorescence spectra but was restricted in L-10T because of its considerable conformational disorder. Exciton delocalization was also unexpectedly observed in a bent chain using single-molecule fluorescence spectroscopy. Such delocalization modulates the fluorescence spectral shape by attenuating the 0-0 peak intensity. Collectively, these results provide significant insights into the exciton dynamics in conjugated polymers.

Single-molecule studies of the neuronal SNARE fusion machinery.

PubMed

Brunger, Axel T; Weninger, Keith; Bowen, Mark; Chu, Steven

2009-01-01

SNAREs are essential components of the machinery for Ca(2+)-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca(2+) sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

Tunneling effects in the kinetics of helium and hydrogen isotopes desorption from single-walled carbon nanotube bundles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Danilchenko, B. A., E-mail: danil@iop.kiev.ua; Yaskovets, I. I.; Uvarova, I. Y.

2014-04-28

The kinetics of desorption both helium isotopes and molecules of hydrogen and deuterium from open-ended or γ-irradiated single-walled carbon nanotube bundles was investigated in temperature range of 10–300 K. The gases desorption rates obey the Arrhenius law at high temperatures, deviate from it with temperature reduction and become constant at low temperatures. These results indicate the quantum nature of gas outflow from carbon nanotube bundles. We had deduced the crossover temperature below which the quantum corrections to the effective activation energy of desorption become significant. This temperature follows linear dependence against the inverse mass of gas molecule and is consistent withmore » theoretical prediction.« less

Efficient spin-filtering, magnetoresistance and negative differential resistance effects of a one-dimensional single-molecule magnet Mn(dmit)2-based device with graphene nanoribbon electrodes

NASA Astrophysics Data System (ADS)

Liu, N.; Liu, J. B.; Yao, K. L.

2017-12-01

We present first-principle spin-dependent quantum transport calculations in a molecular device constructed by one single-molecule magnet Mn(dmit)2 and two graphene nanoribbon electrodes. Our results show that the device could generate perfect spin-filtering performance in a certain bias range both in the parallel configuration (PC) and the antiparallel configuration (APC). At the same time, a magnetoresistance effect, up to a high value of 103%, can be realized. Moreover, visible negative differential resistance phenomenon is obtained for the spin-up current of the PC. These results suggest that our one-dimensional molecular device is a promising candidate for multi-functional spintronics devices.

Robust nonparametric quantification of clustering density of molecules in single-molecule localization microscopy

PubMed Central

Jiang, Shenghang; Park, Seongjin; Challapalli, Sai Divya; Fei, Jingyi; Wang, Yong

2017-01-01

We report a robust nonparametric descriptor, J′(r), for quantifying the density of clustering molecules in single-molecule localization microscopy. J′(r), based on nearest neighbor distribution functions, does not require any parameter as an input for analyzing point patterns. We show that J′(r) displays a valley shape in the presence of clusters of molecules, and the characteristics of the valley reliably report the clustering features in the data. Most importantly, the position of the J′(r) valley (rJm′) depends exclusively on the density of clustering molecules (ρc). Therefore, it is ideal for direct estimation of the clustering density of molecules in single-molecule localization microscopy. As an example, this descriptor was applied to estimate the clustering density of ptsG mRNA in E. coli bacteria. PMID:28636661

Reversible on-surface wiring of resistive circuits.

PubMed

Inkpen, Michael S; Leroux, Yann R; Hapiot, Philippe; Campos, Luis M; Venkataraman, Latha

2017-06-01

Whilst most studies in single-molecule electronics involve components first synthesized ex situ , there is also great potential in exploiting chemical transformations to prepare devices in situ . Here, as a first step towards this goal, we conduct reversible reactions on monolayers to make and break covalent bonds between alkanes of different lengths, then measure the conductance of these molecules connected between electrodes using the scanning tunneling microscopy-based break junction (STM-BJ) method. In doing so, we develop the critical methodology required for assembling and disassembling surface-bound single-molecule circuits. We identify effective reaction conditions for surface-bound reagents, and importantly demonstrate that the electronic characteristics of wires created in situ agree with those created ex situ . Finally, we show that the STM-BJ technique is unique in its ability to definitively probe surface reaction yields both on a local (∼50 nm 2 ) and pseudo-global (≥10 mm 2 ) level. This investigation thus highlights a route to the construction and integration of more complex, and ultimately functional, surface-based single-molecule circuitry, as well as advancing a methodology that facilitates studies beyond the reach of traditional ex situ synthetic approaches.

Model Checking Temporal Logic Formulas Using Sticker Automata

PubMed Central

Feng, Changwei; Wu, Huanmei

2017-01-01

As an important complex problem, the temporal logic model checking problem is still far from being fully resolved under the circumstance of DNA computing, especially Computation Tree Logic (CTL), Interval Temporal Logic (ITL), and Projection Temporal Logic (PTL), because there is still a lack of approaches for DNA model checking. To address this challenge, a model checking method is proposed for checking the basic formulas in the above three temporal logic types with DNA molecules. First, one-type single-stranded DNA molecules are employed to encode the Finite State Automaton (FSA) model of the given basic formula so that a sticker automaton is obtained. On the other hand, other single-stranded DNA molecules are employed to encode the given system model so that the input strings of the sticker automaton are obtained. Next, a series of biochemical reactions are conducted between the above two types of single-stranded DNA molecules. It can then be decided whether the system satisfies the formula or not. As a result, we have developed a DNA-based approach for checking all the basic formulas of CTL, ITL, and PTL. The simulated results demonstrate the effectiveness of the new method. PMID:29119114

Exploring the chemical enhancement for surface-enhanced Raman scattering with Au bowtie nanoantennas

PubMed Central

Fromm, David P.; Kinkhabwala, Anika; Schuck, P. James; Moerner, W. E.; Sundaramurthy, Arvind; Kino, Gordon S.

2006-01-01

Single metallic bowtie nanoantennas provide a controllable environment for surface-enhanced Raman scattering (SERS) of adsorbed molecules. Bowties have experimentally measured electromagnetic enhancements, enabling estimation of chemical enhancement for both the bulk and the few-molecule regime. Strong fluctuations of selected Raman lines imply that a small number of p-mercaptoaniline molecules on a single bowtie show chemical enhancement >107, much larger than previously believed, likely due to charge transfer between the Au surface and the molecule. This chemical sensitivity of SERS has significant implications for ultra-sensitive detection of single molecules. PMID:16483189

Unraveling helicase mechanisms one molecule at a time

PubMed Central

Rasnik, Ivan; Myong, Sua; Ha, Taekjip

2006-01-01

Recent years have seen an increasing number of biological applications of single molecule techniques, evolving from a proof of principle type to the more sophisticated studies. Here we compare the capabilities and limitations of different single molecule techniques in studying the activities of helicases. Helicases share a common catalytic activity but present a high variability in kinetic and phenomenological behavior, making their studies ideal in exemplifying the use of the new single molecule techniques to answer biological questions. Unexpected phenomena have also been observed from individual molecules suggesting extended or alternative functionality of helicases in vivo. PMID:16935883

Noise characteristics of single-walled carbon nanotube network transistors.

PubMed

Kim, Un Jeong; Kim, Kang Hyun; Kim, Kyu Tae; Min, Yo-Sep; Park, Wanjun

2008-07-16

The noise characteristics of randomly networked single-walled carbon nanotubes grown directly by plasma enhanced chemical vapor deposition (PECVD) are studied with field effect transistors (FETs). Due to the geometrical complexity of nanotube networks in the channel area and the large number of tube-tube/tube-metal junctions, the inverse frequency, 1/f, dependence of the noise shows a similar level to that of a single single-walled carbon nanotube transistor. Detailed analysis is performed with the parameters of number of mobile carriers and mobility in the different environment. This shows that the change in the number of mobile carriers resulting in the mobility change due to adsorption and desorption of gas molecules (mostly oxygen molecules) to the tube surface is a key factor in the 1/f noise level for carbon nanotube network transistors.

Serial single molecule electron diffraction imaging: diffraction background of superfluid helium droplets

NASA Astrophysics Data System (ADS)

Zhang, Jie; He, Yunteng; Lei, Lei; Alghamdi, Maha; Oswalt, Andrew; Kong, Wei

2017-08-01

In an effort to solve the crystallization problem in crystallography, we have been engaged in developing a method termed "serial single molecule electron diffraction imaging" (SS-EDI). The unique features of SS-EDI are superfluid helium droplet cooling and field-induced orientation: together the two features constitute a molecular goniometer. Unfortunately, the helium atoms surrounding the sample molecule also contribute to a diffraction background. In this report, we analyze the properties of a superfluid helium droplet beam and its doping statistics, and demonstrate the feasibility of overcoming the background issue by using the velocity slip phenomenon of a pulsed droplet beam. Electron diffraction profiles and pair correlation functions of ferrocene-monomer-doped droplets and iodine-nanocluster-doped droplets are presented. The timing of the pulsed electron gun and the effective doping efficiency under different dopant pressures can both be controlled for size selection. This work clears any doubt of the effectiveness of superfluid helium droplets in SS-EDI, thereby advancing the effort in demonstrating the "proof-of-concept" one step further.

Field-Effect Control of Graphene-Fullerene Thermoelectric Nanodevices.

PubMed

Gehring, Pascal; Harzheim, Achim; Spièce, Jean; Sheng, Yuewen; Rogers, Gregory; Evangeli, Charalambos; Mishra, Aadarsh; Robinson, Benjamin J; Porfyrakis, Kyriakos; Warner, Jamie H; Kolosov, Oleg V; Briggs, G Andrew D; Mol, Jan A

2017-11-08

Although it was demonstrated that discrete molecular levels determine the sign and magnitude of the thermoelectric effect in single-molecule junctions, full electrostatic control of these levels has not been achieved to date. Here, we show that graphene nanogaps combined with gold microheaters serve as a testbed for studying single-molecule thermoelectricity. Reduced screening of the gate electric field compared to conventional metal electrodes allows control of the position of the dominant transport orbital by hundreds of meV. We find that the power factor of graphene-fullerene junctions can be tuned over several orders of magnitude to a value close to the theoretical limit of an isolated Breit-Wigner resonance. Furthermore, our data suggest that the power factor of an isolated level is only given by the tunnel coupling to the leads and temperature. These results open up new avenues for exploring thermoelectricity and charge transport in individual molecules and highlight the importance of level alignment and coupling to the electrodes for optimum energy conversion in organic thermoelectric materials.

The symmetry of single-molecule conduction.

PubMed

Solomon, Gemma C; Gagliardi, Alessio; Pecchia, Alessandro; Frauenheim, Thomas; Di Carlo, Aldo; Reimers, Jeffrey R; Hush, Noel S

2006-11-14

We introduce the conductance point group which defines the symmetry of single-molecule conduction within the nonequilibrium Green's function formalism. It is shown, either rigorously or to within a very good approximation, to correspond to a molecular-conductance point group defined purely in terms of the properties of the conducting molecule. This enables single-molecule conductivity to be described in terms of key qualitative chemical descriptors that are independent of the nature of the molecule-conductor interfaces. We apply this to demonstrate how symmetry controls the conduction through 1,4-benzenedithiol chemisorbed to gold electrodes as an example system, listing also the molecular-conductance point groups for a range of molecules commonly used in molecular electronics research.

Single molecules can operate as primitive biological sensors, switches and oscillators.

PubMed

Hernansaiz-Ballesteros, Rosa D; Cardelli, Luca; Csikász-Nagy, Attila

2018-06-18

Switch-like and oscillatory dynamical systems are widely observed in biology. We investigate the simplest biological switch that is composed of a single molecule that can be autocatalytically converted between two opposing activity forms. We test how this simple network can keep its switching behaviour under perturbations in the system. We show that this molecule can work as a robust bistable system, even for alterations in the reactions that drive the switching between various conformations. We propose that this single molecule system could work as a primitive biological sensor and show by steady state analysis of a mathematical model of the system that it could switch between possible states for changes in environmental signals. Particularly, we show that a single molecule phosphorylation-dephosphorylation switch could work as a nucleotide or energy sensor. We also notice that a given set of reductions in the reaction network can lead to the emergence of oscillatory behaviour. We propose that evolution could have converted this switch into a single molecule oscillator, which could have been used as a primitive timekeeper. We discuss how the structure of the simplest known circadian clock regulatory system, found in cyanobacteria, resembles the proposed single molecule oscillator. Besides, we speculate if such minimal systems could have existed in an RNA world.

Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations

PubMed Central

Serag, Maged F.; Abadi, Maram; Habuchi, Satoshi

2014-01-01

Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields. PMID:25283876

A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase.

PubMed

Liebherr, Raphaela B; Renner, Max; Gorris, Hans H

2014-04-23

The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.

Imaging and Force Recognition of Single Molecular Behaviors Using Atomic Force Microscopy

PubMed Central

Li, Mi; Dang, Dan; Liu, Lianqing; Xi, Ning; Wang, Yuechao

2017-01-01

The advent of atomic force microscopy (AFM) has provided a powerful tool for investigating the behaviors of single native biological molecules under physiological conditions. AFM can not only image the conformational changes of single biological molecules at work with sub-nanometer resolution, but also sense the specific interactions of individual molecular pair with piconewton force sensitivity. In the past decade, the performance of AFM has been greatly improved, which makes it widely used in biology to address diverse biomedical issues. Characterizing the behaviors of single molecules by AFM provides considerable novel insights into the underlying mechanisms guiding life activities, contributing much to cell and molecular biology. In this article, we review the recent developments of AFM studies in single-molecule assay. The related techniques involved in AFM single-molecule assay were firstly presented, and then the progress in several aspects (including molecular imaging, molecular mechanics, molecular recognition, and molecular activities on cell surface) was summarized. The challenges and future directions were also discussed. PMID:28117741

Arrhenius plot for a reaction catalyzed by a single molecule of β-galactosidase.

PubMed

Craig, Douglas B; Chase, Linden N

2012-02-21

The activity of a single enzyme molecule of Escherichia coli β-galactosidase was measured using a capillary electrophoresis continuous flow assay. As the enzyme molecule traversed the capillary the incubation temperature was increased from 27 to 37 °C, providing a continuous record of the change in rate with temperature. This data was used to develop a single enzyme molecule Arrhenius plot, from which the activation energy of the reaction was determined to be 31 kJ mol(-1).

Lucky Imaging: Improved Localization Accuracy for Single Molecule Imaging

PubMed Central

Cronin, Bríd; de Wet, Ben; Wallace, Mark I.

2009-01-01

We apply the astronomical data-analysis technique, Lucky imaging, to improve resolution in single molecule fluorescence microscopy. We show that by selectively discarding data points from individual single-molecule trajectories, imaging resolution can be improved by a factor of 1.6 for individual fluorophores and up to 5.6 for more complex images. The method is illustrated using images of fluorescent dye molecules and quantum dots, and the in vivo imaging of fluorescently labeled linker for activation of T cells. PMID:19348772

Manipulating molecular quantum states with classical metal atom inputs: demonstration of a single molecule NOR logic gate.

PubMed

Soe, We-Hyo; Manzano, Carlos; Renaud, Nicolas; de Mendoza, Paula; De Sarkar, Abir; Ample, Francisco; Hliwa, Mohamed; Echavarren, Antonio M; Chandrasekhar, Natarajan; Joachim, Christian

2011-02-22

Quantum states of a trinaphthylene molecule were manipulated by putting its naphthyl branches in contact with single Au atoms. One Au atom carries 1-bit of classical information input that is converted into quantum information throughout the molecule. The Au-trinaphthylene electronic interactions give rise to measurable energy shifts of the molecular electronic states demonstrating a NOR logic gate functionality. The NOR truth table of the single molecule logic gate was characterized by means of scanning tunnelling spectroscopy.

Targeting neurotransmitter receptors with nanoparticles in vivo allows single-molecule tracking in acute brain slices

NASA Astrophysics Data System (ADS)

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.

Single molecule characterization of DNA binding and strand displacement reactions on lithographic DNA origami microarrays.

PubMed

Scheible, Max B; Pardatscher, Günther; Kuzyk, Anton; Simmel, Friedrich C

2014-03-12

The combination of molecular self-assembly based on the DNA origami technique with lithographic patterning enables the creation of hierarchically ordered nanosystems, in which single molecules are positioned at precise locations on multiple length scales. Based on a hybrid assembly protocol utilizing DNA self-assembly and electron-beam lithography on transparent glass substrates, we here demonstrate a DNA origami microarray, which is compatible with the requirements of single molecule fluorescence and super-resolution microscopy. The spatial arrangement allows for a simple and reliable identification of single molecule events and facilitates automated read-out and data analysis. As a specific application, we utilize the microarray to characterize the performance of DNA strand displacement reactions localized on the DNA origami structures. We find considerable variability within the array, which results both from structural variations and stochastic reaction dynamics prevalent at the single molecule level.

Single molecule sequencing of the M13 virus genome without amplification

PubMed Central

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X.; Yan, Qin; Deem, Michael W.; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias. PMID:29253901

Single molecule sequencing of the M13 virus genome without amplification.

PubMed

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X; Yan, Qin; Deem, Michael W; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

In situ temperature monitoring in single-molecule FRET experiments

NASA Astrophysics Data System (ADS)

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.

Torsional mechanics of DNA are regulated by small-molecule intercalation.

PubMed

Celedon, Alfredo; Wirtz, Denis; Sun, Sean

2010-12-23

Whether the bend and twist mechanics of DNA molecules are coupled is unclear. Here, we report the direct measurement of the resistive torque of single DNA molecules to study the effect of ethidium bromide (EtBr) intercalation and pulling force on DNA twist mechanics. DNA molecules were overwound and unwound using recently developed magnetic tweezers where the molecular resistive torque was obtained from Brownian angular fluctuations. The effect of EtBr intercalation on the twist stiffness was found to be significantly different from the effect on the bend persistence length. The twist stiffness of DNA was dramatically reduced at low intercalator concentration (<10 nM); however, it did not decrease further when the intercalator concentration was increased by 3 orders of magnitude. We also determined the dependence of EtBr intercalation on the torque applied to DNA. We propose a model for the elasticity of DNA base pairs with intercalated EtBr molecules to explain the abrupt decrease of twist stiffness at low EtBr concentration. These results indicate that the bend and twist stiffnesses of DNA are independent and can be differently affected by small-molecule binding.

Procedure for normalization of cDNA libraries

DOEpatents

Bonaldo, M.D.; Soares, M.B.

1997-12-30

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library. 1 fig.

Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy

PubMed Central

Michalet, Xavier; Ingargiola, Antonino; Colyer, Ryan A.; Scalia, Giuseppe; Weiss, Shimon; Maccagnani, Piera; Gulinatti, Angelo; Rech, Ivan; Ghioni, Massimo

2014-01-01

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements. PMID:25309114

Development of new photon-counting detectors for single-molecule fluorescence microscopy.

PubMed

Michalet, X; Colyer, R A; Scalia, G; Ingargiola, A; Lin, R; Millaud, J E; Weiss, S; Siegmund, Oswald H W; Tremsin, Anton S; Vallerga, John V; Cheng, A; Levi, M; Aharoni, D; Arisaka, K; Villa, F; Guerrieri, F; Panzeri, F; Rech, I; Gulinatti, A; Zappa, F; Ghioni, M; Cova, S

2013-02-05

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Development of new photon-counting detectors for single-molecule fluorescence microscopy

PubMed Central

Michalet, X.; Colyer, R. A.; Scalia, G.; Ingargiola, A.; Lin, R.; Millaud, J. E.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Cheng, A.; Levi, M.; Aharoni, D.; Arisaka, K.; Villa, F.; Guerrieri, F.; Panzeri, F.; Rech, I.; Gulinatti, A.; Zappa, F.; Ghioni, M.; Cova, S.

2013-01-01

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level. PMID:23267185

Precision analysis for standard deviation measurements of immobile single fluorescent molecule images.

PubMed

DeSantis, Michael C; DeCenzo, Shawn H; Li, Je-Luen; Wang, Y M

2010-03-29

Standard deviation measurements of intensity profiles of stationary single fluorescent molecules are useful for studying axial localization, molecular orientation, and a fluorescence imaging system's spatial resolution. Here we report on the analysis of the precision of standard deviation measurements of intensity profiles of single fluorescent molecules imaged using an EMCCD camera.We have developed an analytical expression for the standard deviation measurement error of a single image which is a function of the total number of detected photons, the background photon noise, and the camera pixel size. The theoretical results agree well with the experimental, simulation, and numerical integration results. Using this expression, we show that single-molecule standard deviation measurements offer nanometer precision for a large range of experimental parameters.

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

DOEpatents

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.

Single DNA molecule detection using nanopipettes and nanoparticles.

PubMed

Karhanek, Miloslav; Kemp, Jennifer T; Pourmand, Nader; Davis, Ronald W; Webb, Chris D

2005-02-01

Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

2017-07-18

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

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.

Single molecule tracking of Ace1p in Saccharomyces cerevisiae defines a characteristic residence time for non-specific interactions of transcription factors with chromatin

PubMed Central

Ball, David A.; Mehta, Gunjan D.; Salomon-Kent, Ronit; Mazza, Davide; Morisaki, Tatsuya; Mueller, Florian; McNally, James G.; Karpova, Tatiana S.

2016-01-01

In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12–0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast. PMID:27566148

Study of gas adsorption on as-produced and modified carbon nanotubes

NASA Astrophysics Data System (ADS)

Rawat, Dinesh Singh

Volumetric adsorption isotherm measurements were used to study the adsorption characteristics of Ethane (C2H6) and Butane (C 4H10) on as-produced single-walled carbon nanotubes. The binding energy of the adsorbed alkane molecule was found to increase with increasing carbon chain length. Two adsorption substeps were obtained for each alkane molecule. However, the size of the high pressure substep was found to be gradually smearing with the increase in size of the adsorbed molecule. This phenomenon is interpreted as size entropy effect for linear molecules. This interpretation was also verified by determining the specific surface area of the substrate using linear molecules of different sizes. Kinetics measurements of alkane adsorption on SWNTs were also conducted and their dependence on the molecular length was determined. Similar adsorption measurements were performed for Argon (Ar) on as-produced single-walled carbon nanotubes and nanotubes that were structurally modified using acid treatment. Enhancement of the sorptive capacity and the presence of two distinct kinetics of gas adsorption verified partial opening of single walled carbon nanotubes as a result of chemical treatment. Mutiwalled carbon nanotubes were exposed to oxygen plasma treatment for varying time periods. Afterwards, adsorption measurements of Methane (CH 4) were conducted on untreated and oxygen plasma treated tubes. The presence of an additional substep, after exposing multiwalled carbon nanotubes to oxygen plasma for varying time periods, suggested progressive cleaning of nanotube surface.

Measurement of a linear free energy relationship one molecule at a time

PubMed Central

Rao, B. V.; Kwon, K.-Y.; Liu, A.; Bartels, L.

2004-01-01

A systematic study of the dehydrogenation of substituted thiophenols by controlled charge injection from the tip of a scanning tunneling microscope (STM) reveals a pronounced dependence of the reaction yield on the position and the chemical nature of the substituent. We evaluate the dehydrogenation rate of para-halo-substituted species within a linear free energy relationship, namely the Hammett equation. The resultant ρ value of 1.4 can faithfully predict the reaction rates of molecules that are meta-halo-substituted or para-methyl-substituted. The positive sign of ρ suggests a negatively charged transition state at the core of the STM-induced process, and the magnitude of the ρ value indicates that the presence of the substrate does not preclude substantial substituent effects. The applicability of the Hammett equation to single-molecule chemistry offers facile prediction of the rate of STM-based single-molecule chemistry in a field, which so far has been addressed by focusing on involved quantum-mechanical modeling of its underlying processes. PMID:15601774

Measurement of a linear free energy relationship one molecule at a time.

PubMed

Rao, B V; Kwon, K-Y; Liu, A; Bartels, L

2004-12-28

A systematic study of the dehydrogenation of substituted thiophenols by controlled charge injection from the tip of a scanning tunneling microscope (STM) reveals a pronounced dependence of the reaction yield on the position and the chemical nature of the substituent. We evaluate the dehydrogenation rate of para-halo-substituted species within a linear free energy relationship, namely the Hammett equation. The resultant rho value of 1.4 can faithfully predict the reaction rates of molecules that are meta-halo-substituted or para-methyl-substituted. The positive sign of rho suggests a negatively charged transition state at the core of the STM-induced process, and the magnitude of the rho value indicates that the presence of the substrate does not preclude substantial substituent effects. The applicability of the Hammett equation to single-molecule chemistry offers facile prediction of the rate of STM-based single-molecule chemistry in a field, which so far has been addressed by focusing on involved quantum-mechanical modeling of its underlying processes.

Effect of different substitution position on the switching behavior in single-molecule device with carbon nanotube electrodes

NASA Astrophysics Data System (ADS)

Yang, Jingjuan; Han, Xiaoxiao; Yuan, Peipei; Bian, Baoan; Wang, Yixiang

2018-01-01

We investigate the electronic transport properties of dihydroazulene (DHA) and vinylheptafulvene (VHF) molecule sandwiched between two carbon nanotubes using density functional theory and non-equilibrium Green's function. The device displays significantly switching behavior between DHA and VHF isomerizations. It is found the different substitution position of F in the molecule influences the switching ratio of device, which is analyzed by transmission spectra and molecular projected self-consistent Hamiltonian. The observed negative differential resistance effect is explained by transmission spectra and transmission eigenstates of transmission peak in the bias window. The observed reverse of current in VHF form in which two H atoms on the right side of the benzene ring of the molecule are replaced by F is explained by transmission spectra and molecule-electrode coupling with the varied bias. The results suggest that the reasonable substitution position of molecule may improve the switching ratio, displaying a potential application in future molecular circuit.

Current rectification in a single molecule diode: the role of electrode coupling.

PubMed

Sherif, Siya; Rubio-Bollinger, Gabino; Pinilla-Cienfuegos, Elena; Coronado, Eugenio; Cuevas, Juan Carlos; Agraït, Nicolás

2015-07-24

We demonstrate large rectification ratios (> 100) in single-molecule junctions based on a metal-oxide cluster (polyoxometalate), using a scanning tunneling microscope (STM) both at ambient conditions and at low temperature. These rectification ratios are the largest ever observed in a single-molecule junction, and in addition these junctions sustain current densities larger than 10(5) A cm(-2). By following the variation of the I-V characteristics with tip-molecule separation we demonstrate unambiguously that rectification is due to asymmetric coupling to the electrodes of a molecule with an asymmetric level structure. This mechanism can be implemented in other type of molecular junctions using both organic and inorganic molecules and provides a simple strategy for the rational design of molecular diodes.

Current rectification in a single molecule diode: the role of electrode coupling

NASA Astrophysics Data System (ADS)

Sherif, Siya; Rubio-Bollinger, Gabino; Pinilla-Cienfuegos, Elena; Coronado, Eugenio; Cuevas, Juan Carlos; Agraït, Nicolás

2015-07-01

We demonstrate large rectification ratios (\\gt 100) in single-molecule junctions based on a metal-oxide cluster (polyoxometalate), using a scanning tunneling microscope (STM) both at ambient conditions and at low temperature. These rectification ratios are the largest ever observed in a single-molecule junction, and in addition these junctions sustain current densities larger than 105 A cm-2. By following the variation of the I-V characteristics with tip-molecule separation we demonstrate unambiguously that rectification is due to asymmetric coupling to the electrodes of a molecule with an asymmetric level structure. This mechanism can be implemented in other type of molecular junctions using both organic and inorganic molecules and provides a simple strategy for the rational design of molecular diodes.

Distinguishing Lead and Molecule States in Graphene-Based Single-Electron Transistors

PubMed Central

2017-01-01

Graphene provides a two-dimensional platform for contacting individual molecules, which enables transport spectroscopy of molecular orbital, spin, and vibrational states. Here we report single-electron tunneling through a molecule that has been anchored to two graphene leads. Quantum interference within the graphene leads gives rise to an energy-dependent transmission and fluctuations in the sequential tunnel-rates. The lead states are electrostatically tuned by a global back-gate, resulting in a distinct pattern of varying intensity in the measured conductance maps. This pattern could potentially obscure transport features that are intrinsic to the molecule under investigation. Using ensemble averaged magneto-conductance measurements, lead and molecule states are disentangled, enabling spectroscopic investigation of the single molecule. PMID:28423272

Quantum design rules for single molecule logic gates.

PubMed

Renaud, N; Hliwa, M; Joachim, C

2011-08-28

Recent publications have demonstrated how to implement a NOR logic gate with a single molecule using its interaction with two surface atoms as logical inputs [W. Soe et al., ACS Nano, 2011, 5, 1436]. We demonstrate here how this NOR logic gate belongs to the general family of quantum logic gates where the Boolean truth table results from a full control of the quantum trajectory of the electron transfer process through the molecule by very local and classical inputs practiced on the molecule. A new molecule OR gate is proposed for the logical inputs to be also single metal atoms, one per logical input.

Single-molecule fluorescence microscopy review: shedding new light on old problems

PubMed Central

Shashkova, Sviatlana

2017-01-01

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. PMID:28694303

Conformation-based signal transfer and processing at the single-molecule level

NASA Astrophysics Data System (ADS)

Li, Chao; Wang, Zhongping; Lu, Yan; Liu, Xiaoqing; Wang, Li

2017-11-01

Building electronic components made of individual molecules is a promising strategy for the miniaturization and integration of electronic devices. However, the practical realization of molecular devices and circuits for signal transmission and processing at room temperature has proven challenging. Here, we present room-temperature intermolecular signal transfer and processing using SnCl2Pc molecules on a Cu(100) surface. The in-plane orientations of the molecules are effectively coupled via intermolecular interaction and serve as the information carrier. In the coupled molecular arrays, the signal can be transferred from one molecule to another in the in-plane direction along predesigned routes and processed to realize logical operations. These phenomena enable the use of molecules displaying intrinsic bistable states as complex molecular devices and circuits with novel functions.

Nanomanipulation of Single RNA Molecules by Optical Tweezers

PubMed Central

Stephenson, William; Wan, Gorby; Tenenbaum, Scott A.; Li, Pan T. X.

2014-01-01

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed. PMID:25177917

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions.

PubMed

Zheng, Haocheng; Goldner, Lori S; Leuba, Sanford H

2007-03-01

Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

Single-molecule studies of multi-protein machines

NASA Astrophysics Data System (ADS)

van Oijen, Antoine

2010-03-01

Advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. In a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. Our group is developing the single-molecule tools that will make it possible to study biochemical pathways of arbitrary complexity at the single-molecule level. I will discuss results of single-molecule experiments on the replisome, the molecular machinery that is responsible for replication of DNA. We stretch individual DNA molecules and use their elastic properties to obtain dynamic information on the proteins that unwind the double helix and copy its genetic information. Furthermore, we visualize fluorescently labeled components of the replisome and thus obtain information on stochiometry and exchange kinetics. This simultaneous observation of catalytic activity and composition allows us to gain deeper insight into the structure-function relationship of the replisome.

A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force

NASA Astrophysics Data System (ADS)

Bano, Fouzia; Banerji, Suneale; Howarth, Mark; Jackson, David G.; Richter, Ralf P.

2016-09-01

Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Despite the recognized significance of mechanical stimuli in cellular communication, however, only few single molecule methods are currently available to study how monovalent and multivalent GAG·protein bonds respond to directed mechanical forces. Here, we have devised such a method, by combining purpose-designed surfaces that afford immobilization of GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy (SMFS). We apply the method to study the interaction of the GAG polymer hyaluronan (HA) with CD44, its receptor in vascular endothelium. Individual bonds between HA and CD44 are remarkably resistant to rupture under force in comparison to their low binding affinity. Multiple bonds along a single HA chain rupture sequentially and independently under load. We also demonstrate how strong non-covalent bonds, which are versatile for controlled protein and GAG immobilization, can be effectively used as molecular anchors in SMFS. We thus establish a versatile method for analyzing the nanomechanics of GAG·protein interactions at the level of single GAG chains, which provides new molecular-level insight into the role of mechanical forces in the assembly and function of GAG-rich extracellular matrices.

Force and Conductance Spectroscopy of Single Molecule Junctions

NASA Astrophysics Data System (ADS)

Frei, Michael

Investigation of mechanical properties of single molecule junctions is crucial to develop an understanding and enable control of single molecular junctions. This work presents an experimental and analytical approach that enables the statistical evaluation of force and simultaneous conductance data of metallic atomic point contacts and molecular junctions. A conductive atomic force microscope based break junction technique is developed to form single molecular junctions and collect conductance and force data simultaneously. Improvements of the optical components have been achieved through the use of a super-luminescent diode, enabling tremendous increases in force resolution. An experimental procedure to collect data for various molecular junctions has been developed and includes deposition, calibration, and analysis methods. For the statistical analysis of force, novel approaches based on two dimensional histograms and a direct force identification method are presented. The two dimensional method allows for an unbiased evaluation of force events that are identified using corresponding conductance signatures. This is not always possible however, and in these situations, the force based identification of junction rearrangement events is an attractive alternative method. This combined experimental and analytical approach is then applied to three studies: First, the impact of molecular backbones to the mechanical behavior of single molecule junctions is investigated and it is found that junctions formed with identical linkers but different backbone structure result in junctions with varying breaking forces. All molecules used show a clear molecular signature and force data can be evaluated using the 2D method. Second, the effects of the linker group used to attach molecules to gold electrodes are investigated. A study of four alkane molecules with different linkers finds a drastic difference in the evolution of donor-acceptor and covalently bonded molecules respectively. In fact, the covalent bond is found to significantly distort the metal electrode rearrangement such that junction rearrangement events can no longer be identified with a clean and well defined conductance signature. For this case, the force based identification process is used. Third, results for break junction measurements with different metals are presented. It is found that silver and palladium junctions rupture with forces different from those of gold contacts. In the case of silver experiments in ambient conditions, we can also identify oxygen impurities in the silver contact formation process, leading to force and conductance measurements of silver-oxygen structures. For the future, this work provides an experimental and analytical foundation that will enable insights into single molecule systems not previously accessible.

Electric-field-induced spin switch of endohedral dodecahedrane heterodimers H@C20Hn-C20Hn@M (M= Cu, Ag and Au, n = 15, 18, and 19): a theoretical study.

PubMed

Hou, Jianhua; Yang, Zhixiong; Li, Zhiru; Chai, Haoyu; Zhao, Ruiqi

2017-08-01

We designed nine endohedral dodecahedrane heterodimers H@C 20 H n -C 20 H n @M (M = Cu, Ag, and Au, n = 15, 18, and 19) that may act as single-molecule spin switches, and we predicted theoretically that the ground states of the dimmers shift from low-spin states (S = 0) to the high-spin states (S = 1) under an external electric field applied parallel or perpendicular to the molecular symmetry axes, consisting well with the analyses of Stark effect. Molecular orbitals analyses provide an intuitive insight into the spin crossover behavior. This study expands the application of endohedral chemistry and provides new molecules for designing single-molecule spin switch.

Nanopore arrays in a silicon membrane for parallel single-molecule detection: fabrication

NASA Astrophysics Data System (ADS)

Schmidt, Torsten; Zhang, Miao; Sychugov, Ilya; Roxhed, Niclas; Linnros, Jan

2015-08-01

Solid state nanopores enable translocation and detection of single bio-molecules such as DNA in buffer solutions. Here, sub-10 nm nanopore arrays in silicon membranes were fabricated by using electron-beam lithography to define etch pits and by using a subsequent electrochemical etching step. This approach effectively decouples positioning of the pores and the control of their size, where the pore size essentially results from the anodizing current and time in the etching cell. Nanopores with diameters as small as 7 nm, fully penetrating 300 nm thick membranes, were obtained. The presented fabrication scheme to form large arrays of nanopores is attractive for parallel bio-molecule sensing and DNA sequencing using optical techniques. In particular the signal-to-noise ratio is improved compared to other alternatives such as nitride membranes suffering from a high-luminescence background.

Nanopore arrays in a silicon membrane for parallel single-molecule detection: fabrication.

PubMed

Schmidt, Torsten; Zhang, Miao; Sychugov, Ilya; Roxhed, Niclas; Linnros, Jan

2015-08-07

Solid state nanopores enable translocation and detection of single bio-molecules such as DNA in buffer solutions. Here, sub-10 nm nanopore arrays in silicon membranes were fabricated by using electron-beam lithography to define etch pits and by using a subsequent electrochemical etching step. This approach effectively decouples positioning of the pores and the control of their size, where the pore size essentially results from the anodizing current and time in the etching cell. Nanopores with diameters as small as 7 nm, fully penetrating 300 nm thick membranes, were obtained. The presented fabrication scheme to form large arrays of nanopores is attractive for parallel bio-molecule sensing and DNA sequencing using optical techniques. In particular the signal-to-noise ratio is improved compared to other alternatives such as nitride membranes suffering from a high-luminescence background.

Single cell and single molecule techniques for the analysis of the epigenome

NASA Astrophysics Data System (ADS)

Wallin, Christopher Benjamin

Epigenetic regulation is a critical biological process for the health and development of a cell. Epigenetic regulation is facilitated by covalent modifications to the underlying DNA and chromatin proteins. A fundamental understanding of these epigenetic modifications and their associated interactions at the molecular scale is necessary to explain phenomena including cellular identity, stem cell plasticity, and neoplastic transformation. It is widely known that abnormal epigenetic profiles have been linked to many diseases, most notably cancer. While the field of epigenetics has progressed rapidly with conventional techniques, significant advances remain to be made with respect to combinatoric analysis of epigenetic marks and single cell epigenetics. Therefore, in this dissertation, I will discuss our development of devices and methodologies to address these pertinent issues. First, we designed a preparatory polydimethylsiloxane (PDMS) microdevice for the extraction, purification, and stretching of human chromosomal DNA and chromatin from small cell populations down to a single cell. The valveless device captures cells by size exclusion within the micropillars, entraps the DNA or chromatin in the micropillars after cell lysis, purifies away the cellular debris, and fluorescently labels the DNA and/or chromatin all within a single reaction chamber. With the device, we achieve nearly 100% extraction efficiency of the DNA. The device is also used for in-channel immunostaining of chromatin followed by downstream single molecule chromatin analysis in nanochannels (SCAN). Second, using multi-color, time-correlated single molecule measurements in nanochannels, simultaneous coincidence detection of 2 epigenetic marks is demonstrated. Coincidence detection of 3 epigenetic marks is also established using a pulsed interleaved excitation scheme. With these two promising results, genome-wide quantification of epigenetic marks was pursued. Unfortunately, quantitative SCAN never materialized. Reasons for this, including poor signal to background, are explained in detail. Third, development of mobility-SCAN, an analytical technique for measuring and analyzing single molecules based on their fluorescent signature and their electrophoretic mobility in nanochannels is described. We use the technique to differentiate biomolecules from complex mixtures and derive parameters such as diffusion coefficients and effective charges. Finally, the device is used to detect binding interactions of various complexes similar to affinity capillary electrophoresis, but on a single molecule level. Fourth, we conclude by briefly discussing SCAN-sort, a technique to sort individual chromatin molecules based on their fluorescent emissions for further downstream analysis such as DNA sequencing. We demonstrate a 2-fold enrichment of chromatin from sorting and discuss possible system modifications for better performance in the future.

Atomic-Scale Control of Electron Transport through Single Molecules

NASA Astrophysics Data System (ADS)

Wang, Y. F.; Kröger, J.; Berndt, R.; Vázquez, H.; Brandbyge, M.; Paulsson, M.

2010-04-01

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 of the surface electrode. Nonequilibrium Green’s function calculations reproduce the trend of the conductance and visualize the current flow through the junction, which is guided through molecule-electrode chemical bonds.

Unfolding single RNA molecules: bridging the gap between equilibrium and non-equilibrium statistical thermodynamics.

PubMed

Bustamante, Carlos

2005-11-01

During the last 15 years, scientists have developed methods that permit the direct mechanical manipulation of individual molecules. Using this approach, they have begun to investigate the effect of force and torque in chemical and biochemical reactions. These studies span from the study of the mechanical properties of macromolecules, to the characterization of molecular motors, to the mechanical unfolding of individual proteins and RNA. Here I present a review of some of our most recent results using mechanical force to unfold individual molecules of RNA. These studies make it possible to follow in real time the trajectory of each molecule as it unfolds and characterize the various intermediates of the reaction. Moreover, if the process takes place reversibly it is possible to extract both kinetic and thermodynamic information from these experiments at the same time that we characterize the forces that maintain the three-dimensional structure of the molecule in solution. These studies bring us closer to the biological unfolding processes in the cell as they simulate in vitro, the mechanical unfolding of RNAs carried out in the cell by helicases. If the unfolding process occurs irreversibly, I show here that single-molecule experiments can still provide equilibrium, thermodynamic information from non-equilibrium data by using recently discovered fluctuation theorems. Such theorems represent a bridge between equilibrium and non-equilibrium statistical mechanics. In fact, first derived in 1997, the first experimental demonstration of the validity of fluctuation theorems was obtained by unfolding mechanically a single molecule of RNA. It is perhaps a sign of the times that important physical results are these days used to extract information about biological systems and that biological systems are being used to test and confirm fundamental new laws in physics.

Probing Enzyme-Surface Interactions via Protein Engineering and Single-Molecule Techniques

DTIC Science & Technology

2017-06-26

SECURITY CLASSIFICATION OF: The overall objective of this research was to exploit protein engineering and fluorescence single-molecule methods to... Engineering and Single-Molecule Techniques The views, opinions and/or findings contained in this report are those of the author(s) and should not...Status: Technology Transfer: Report Date: 1 FINAL REPORT Project Title: Probing Enzyme-Surface Interactions via Protein Engineering and

A model for competition for ribosomes in the cell

PubMed Central

Raveh, Alon; Margaliot, Michael; Sontag, Eduardo D.; Tuller, Tamir

2016-01-01

A single mammalian cell includes an order of 104–105 mRNA molecules and as many as 105–106 ribosomes. Large-scale simultaneous mRNA translation induces correlations between the mRNA molecules, as they all compete for the finite pool of available ribosomes. This has important implications for the cell's functioning and evolution. Developing a better understanding of the intricate correlations between these simultaneous processes, rather than focusing on the translation of a single isolated transcript, should help in gaining a better understanding of mRNA translation regulation and the way elongation rates affect organismal fitness. A model of simultaneous translation is specifically important when dealing with highly expressed genes, as these consume more resources. In addition, such a model can lead to more accurate predictions that are needed in the interconnection of translational modules in synthetic biology. We develop and analyse a general dynamical model for large-scale simultaneous mRNA translation and competition for ribosomes. This is based on combining several ribosome flow models (RFMs) interconnected via a pool of free ribosomes. We use this model to explore the interactions between the various mRNA molecules and ribosomes at steady state. We show that the compound system always converges to a steady state and that it always entrains or phase locks to periodically time-varying transition rates in any of the mRNA molecules. We then study the effect of changing the transition rates in one mRNA molecule on the steady-state translation rates of the other mRNAs that results from the competition for ribosomes. We show that increasing any of the codon translation rates in a specific mRNA molecule yields a local effect, an increase in the translation rate of this mRNA, and also a global effect, the translation rates in the other mRNA molecules all increase or all decrease. These results suggest that the effect of codon decoding rates of endogenous and heterologous mRNAs on protein production is more complicated than previously thought. In addition, we show that increasing the length of an mRNA molecule decreases the production rate of all the mRNAs. PMID:26962028

Spontaneous encapsulation behavior of ionic liquid into carbon nanotube

NASA Astrophysics Data System (ADS)

Jiang, Yanyan; Zhang, Kun; Li, Hui; He, Yezeng; Song, Xigui

2012-10-01

Molecular dynamics simulations and density functional theory have been performed to investigate the spontaneous encapsulation of 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) into single-walled carbon nanotubes (SWCNTs). This phenomenon can be attributed to the van der Waals attractive force, hydrogen bonds and especially the π-π stacking effect. The [Bmim][Cl] molecules enter SWCNTs with larger diameters more rapidly, showing an interesting dependence on tube size. A high temperature is not beneficial to, and may even disrupt, the encapsulation of the [Bmim][Cl] molecules. It is also worth noting that the graphene nanoribbon entering the SWCNT would have an extremely different effect on this encapsulation process from when they wrap around the outer surface. Furthermore, the [Bmim][Cl] molecules can assist water transport in the SWCNT by expelling water molecules from the SWCNT. The proposed discoveries eventually provide a powerful way to fabricate nanoscale materials and devices and tune their properties.Molecular dynamics simulations and density functional theory have been performed to investigate the spontaneous encapsulation of 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) into single-walled carbon nanotubes (SWCNTs). This phenomenon can be attributed to the van der Waals attractive force, hydrogen bonds and especially the π-π stacking effect. The [Bmim][Cl] molecules enter SWCNTs with larger diameters more rapidly, showing an interesting dependence on tube size. A high temperature is not beneficial to, and may even disrupt, the encapsulation of the [Bmim][Cl] molecules. It is also worth noting that the graphene nanoribbon entering the SWCNT would have an extremely different effect on this encapsulation process from when they wrap around the outer surface. Furthermore, the [Bmim][Cl] molecules can assist water transport in the SWCNT by expelling water molecules from the SWCNT. The proposed discoveries eventually provide a powerful way to fabricate nanoscale materials and devices and tune their properties. Electronic supplementary information (ESI) available: (1) Movie: the encapsulation process of the ILs into the SWCNT (16,16). (2) More calculation details, including the data of the morphology vs. the encapsulation process from both ends and different tube diameters. See DOI: 10.1039/c2nr31432k

The nature of the force-induced conformation transition of dsDNA studied by using single molecule force spectroscopy.

PubMed

Liu, Ningning; Bu, Tianjia; Song, Yu; Zhang, Wei; Li, Jinjing; Zhang, Wenke; Shen, Jiacong; Li, Hongbin

2010-06-15

Single-stranded DNA binding proteins (SSB) interact with single-stranded DNA (ssDNA) specifically. Taking advantage of this character, we have employed Bacillus subtilis SSB protein to investigate the nature of force-induced conformation transition of double-stranded DNA (dsDNA) by using AFM-based single molecule force spectroscopy (SMFS) technique. Our results show that, when a dsDNA is stretched beyond its contour length, the dsDNA is partially melted, producing some ssDNA segments which can be captured by SSB proteins. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. Furthermore, the effect of proflavine, a DNA intercalator, on the SSB-DNA interactions has been investigated, and the results indicate that the proflavine-saturated dsDNA can be stabilized to the extent that the dsDNA will no longer melt into ssDNA under the mechanical force even up to 150 pN, and no SSB-DNA interactions are detectable.

Direct Observation of Quantum Coherence in Single-Molecule Magnets

NASA Astrophysics Data System (ADS)

Schlegel, C.; van Slageren, J.; Manoli, M.; Brechin, E. K.; Dressel, M.

2008-10-01

Direct evidence of quantum coherence in a single-molecule magnet in a frozen solution is reported with coherence times as long as T2=630±30ns. We can strongly increase the coherence time by modifying the matrix in which the single-molecule magnets are embedded. The electron spins are coupled to the proton nuclear spins of both the molecule itself and, interestingly, also to those of the solvent. The clear observation of Rabi oscillations indicates that we can manipulate the spin coherently, an essential prerequisite for performing quantum computations.

Nanopores and nucleic acids: prospects for ultrarapid sequencing

NASA Technical Reports Server (NTRS)

Deamer, D. W.; Akeson, M.

2000-01-01

DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.

Single-molecule tracking study of the permeability and transverse width of individual cylindrical microdomains in solvent-swollen polystyrene- block-poly(ethylene oxide) films

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sapkota, Dol Raj; Tran-Ba, Khanh-Hoa; Elwell-Cuddy, Trevor

Understanding the properties of solvent-swollen block copolymer (BCP) microdomains is important for better solvent-based control of microdomain morphology, orientation, and permeability. In this study, single-molecule tracking (SMT) was explored to assess the permeability and transverse width of individual cylindrical microdomains in solvent-swollen polystyrene- block-poly(ethylene oxide) (PS-b-PEO) films. PS-b-PEO films comprising shear-elongated cylindrical PEO microdomains were prepared by sandwiching its benzene or tetrahydrofuran (THF) solution between two glass substrates. In this paper, SMT measurements were performed at different drying times to investigate the effects of solvent evaporation on the microdomain properties. SMT data showed one-dimensional (1D) motions of single fluorescent moleculesmore » (sulforhodamine B) based on their diffusion within the cylindrical microdomains. Microdomain permeability and transverse width were assessed from the single-molecule diffusion coefficients (D SMT) and transverse variance of the 1D trajectories (σ δ 2), respectively. The D SMT and σ δ 2 values from individual 1D trajectories were widely distributed with no evidence of correlation on a single molecule basis, possibly because the individual microdomains in a film were swollen to different extents. On average, microdomain permeability (D) and effective radius (r) gradually decreased within the first 3 days of drying due to solvent evaporation, and changed negligibly thereafter. PS-b-PEO films prepared from THF solutions exhibited larger changes in D and r as compared with those from benzene solutions due to the better swelling of the PEO microdomains by THF. Importantly, changes in D were more prominent than those in r, suggesting that the permeability of the PEO microdomains is very susceptible to the presence of solvent. Finally, these results reveal the unique capability of SMT to assess the properties of individual cylindrical microdomains in a solvent-swollen BCP film.« less

Single-molecule tracking study of the permeability and transverse width of individual cylindrical microdomains in solvent-swollen polystyrene- block-poly(ethylene oxide) films

DOE PAGES

Sapkota, Dol Raj; Tran-Ba, Khanh-Hoa; Elwell-Cuddy, Trevor; ...

2016-11-04

Understanding the properties of solvent-swollen block copolymer (BCP) microdomains is important for better solvent-based control of microdomain morphology, orientation, and permeability. In this study, single-molecule tracking (SMT) was explored to assess the permeability and transverse width of individual cylindrical microdomains in solvent-swollen polystyrene- block-poly(ethylene oxide) (PS-b-PEO) films. PS-b-PEO films comprising shear-elongated cylindrical PEO microdomains were prepared by sandwiching its benzene or tetrahydrofuran (THF) solution between two glass substrates. In this paper, SMT measurements were performed at different drying times to investigate the effects of solvent evaporation on the microdomain properties. SMT data showed one-dimensional (1D) motions of single fluorescent moleculesmore » (sulforhodamine B) based on their diffusion within the cylindrical microdomains. Microdomain permeability and transverse width were assessed from the single-molecule diffusion coefficients (D SMT) and transverse variance of the 1D trajectories (σ δ 2), respectively. The D SMT and σ δ 2 values from individual 1D trajectories were widely distributed with no evidence of correlation on a single molecule basis, possibly because the individual microdomains in a film were swollen to different extents. On average, microdomain permeability (D) and effective radius (r) gradually decreased within the first 3 days of drying due to solvent evaporation, and changed negligibly thereafter. PS-b-PEO films prepared from THF solutions exhibited larger changes in D and r as compared with those from benzene solutions due to the better swelling of the PEO microdomains by THF. Importantly, changes in D were more prominent than those in r, suggesting that the permeability of the PEO microdomains is very susceptible to the presence of solvent. Finally, these results reveal the unique capability of SMT to assess the properties of individual cylindrical microdomains in a solvent-swollen BCP film.« less

Spectrally resolved single-molecule electrometry

NASA Astrophysics Data System (ADS)

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.

Apigenin Impacts the Growth of the Gut Microbiota and Alters the Gene Expression of Enterococcus.

PubMed

Wang, Minqian; Firrman, Jenni; Zhang, Liqing; Arango-Argoty, Gustavo; Tomasula, Peggy; Liu, LinShu; Xiao, Weidong; Yam, Kit

2017-08-03

Apigenin is a major dietary flavonoid with many bioactivities, widely distributed in plants. Apigenin reaches the colon region intact and interacts there with the human gut microbiota, however there is little research on how apigenin affects the gut bacteria. This study investigated the effect of pure apigenin on human gut bacteria, at both the single strain and community levels. The effect of apigenin on the single gut bacteria strains Bacteroides galacturonicus , Bifidobacterium catenulatum , Lactobacillus rhamnosus GG, and Enterococcus caccae , was examined by measuring their anaerobic growth profiles. The effect of apigenin on a gut microbiota community was studied by culturing a fecal inoculum under in vitro conditions simulating the human ascending colon. 16S rRNA gene sequencing and GC-MS analysis quantified changes in the community structure. Single molecule RNA sequencing was used to reveal the response of Enterococcus caccae to apigenin. Enterococcus caccae was effectively inhibited by apigenin when cultured alone, however, the genus Enterococcus was enhanced when tested in a community setting. Single molecule RNA sequencing found that Enterococcus caccae responded to apigenin by up-regulating genes involved in DNA repair, stress response, cell wall synthesis, and protein folding. Taken together, these results demonstrate that apigenin affects both the growth and gene expression of Enterococcus caccae .

Biology and polymer physics at the single-molecule level.

PubMed

Chu, Steven

2003-04-15

The ability to look at individual molecules has given us new insights into molecular processes. Examples of our recent work are given to illustrate how behaviour that may otherwise be hidden from view can be clearly seen in single-molecule experiments.

From single-molecule spectroscopy to super-resolution imaging of the neuron: a review

PubMed Central

Laine, Romain F; Kaminski Schierle, Gabriele S; van de Linde, Sebastian; Kaminski, Clemens F

2016-01-01

Abstract For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods, ca. 10 years ago, which overcome the limitations imposed by optical diffraction. Today, single molecule super-resolution imaging is routinely used in the study of macromolecular function and structure in the cell. Concomitantly, computational methods have been developed that provide information on numbers and positions of molecules at the nanometer-scale. In this overview, we outline the technical developments that have led to the emergence of localization microscopy techniques from single-molecule spectroscopy. We then provide a comprehensive review on the application of the technique in the field of neuroscience research. PMID:28809165

A Comparison of Single-Molecule Emission in Aluminum and Gold Zero-Mode Waveguides

DOE PAGES

Martin, William Elliott; Srijanto, Bernadeta R.; Collier, C. Patrick; ...

2016-08-06

We characterized the effect of gold and aluminum zero-mode waveguides (ZMWs) on the brightness of immobilized single emitters by probing fluorophores that absorb in the green and red regions of the visible spectrum. Aluminum ZMWs enhance the emission of Atto565 fluorophores upon green excitation, but they do not enhance the emission of Atto647N fluorophores upon red excitation. Gold ZMWs increase emission of both fluorophores with Atto647N showing enhancement that is threefold higher than that observed for Atto565. Our work indicates that 200 nm gold ZMWs are better suited for single-molecule fluorescence studies in the red region of the visible spectrum,more » while aluminum appears more suited for the green region of the visible spectrum.« less

A Comparison of Single-Molecule Emission in Aluminum and Gold Zero-Mode Waveguides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, William Elliott; Srijanto, Bernadeta R.; Collier, C. Patrick

We characterized the effect of gold and aluminum zero-mode waveguides (ZMWs) on the brightness of immobilized single emitters by probing fluorophores that absorb in the green and red regions of the visible spectrum. Aluminum ZMWs enhance the emission of Atto565 fluorophores upon green excitation, but they do not enhance the emission of Atto647N fluorophores upon red excitation. Gold ZMWs increase emission of both fluorophores with Atto647N showing enhancement that is threefold higher than that observed for Atto565. Our work indicates that 200 nm gold ZMWs are better suited for single-molecule fluorescence studies in the red region of the visible spectrum,more » while aluminum appears more suited for the green region of the visible spectrum.« less

Conductance of Single Molecule Junctions: Dependence on Structure and Conformation

NASA Astrophysics Data System (ADS)

Venkataraman, Latha

2007-03-01

We recently demonstrated that the conductance of single molecule junctions formed by breaking Au point contacts in an environment of molecules with amine linkages can be measured reliably and reproducibly^1. We have now studied junctions formed by approximately 30 different amine terminated molecules, allowing systematic study of the correlation between molecular properties and single molecule junction conductance. This talk will focus on the relation between molecular conductance and molecule conformation for the simple case of a biphenyl, two benzene rings linked together by a single C-C bond. Our results from a series of seven biphenyl derivatives show that the molecular junction conductance depends on the twist angle. Specifically, we find that the planar molecule has the highest conductance, and the conductance for the series decreases with increasing twist angle, consistent with a cosine squared relation predicted theoretically^2. 1. L. Venkataraman, J.E. Klare, I.W. Tam, C. Nuckolls, M.S Hybertsen and M. Steigerwald, Nano Letters, vol. 5, pp. 458-462, 2006. 2. L. Venkataraman, J.E. Klare, C. Nuckolls, M.S Hybertsen and M. Steigerwald, Nature, vol. 442, pp. 904-907, 2006.

Site-Selection in Single-Molecule Junction for Highly Reproducible Molecular Electronics.

PubMed

Kaneko, Satoshi; Murai, Daigo; Marqués-González, Santiago; Nakamura, Hisao; Komoto, Yuki; Fujii, Shintaro; Nishino, Tomoaki; Ikeda, Katsuyoshi; Tsukagoshi, Kazuhito; Kiguchi, Manabu

2016-02-03

Adsorption sites of molecules critically determine the electric/photonic properties and the stability of heterogeneous molecule-metal interfaces. Then, selectivity of adsorption site is essential for development of the fields including organic electronics, catalysis, and biology. However, due to current technical limitations, site-selectivity, i.e., precise determination of the molecular adsorption site, remains a major challenge because of difficulty in precise selection of meaningful one among the sites. We have succeeded the single site-selection at a single-molecule junction by performing newly developed hybrid technique: simultaneous characterization of surface enhanced Raman scattering (SERS) and current-voltage (I-V) measurements. The I-V response of 1,4-benzenedithiol junctions reveals the existence of three metastable states arising from different adsorption sites. Notably, correlated SERS measurements show selectivity toward one of the adsorption sites: "bridge sites". This site-selectivity represents an essential step toward the reliable integration of individual molecules on metallic surfaces. Furthermore, the hybrid spectro-electric technique reveals the dependence of the SERS intensity on the strength of the molecule-metal interaction, showing the interdependence between the optical and electronic properties in single-molecule junctions.

Surface enhanced single-molecule magnetism involving 4f spin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yachao, E-mail: yachao.zhang@pku.edu.cn

2016-03-28

We study the magnetic anisotropy energy (MAE) of the isolated and deposited Eu(C{sub 8}H{sub 8}){sub 2} by first-principles calculations considering the van der Waals correction and the strong correlation effects. We find that both the molecular spin moment and the easy-axis magnetic anisotropy are enhanced upon deposition on Cu(111). We propose a mechanism in terms of the weakened spin polarization of the π-2p orbitals and the induced anisotropic occupations of the 4f orbitals. Our findings pave the way for raising the MAE of 4f-element single-molecule magnets by tailoring the molecule–surface contacts.

A theoretical study of symmetry-breaking organic overlayers on single- and bi-layer graphene

NASA Astrophysics Data System (ADS)

Morales-Cifuentes, Josue; Einstein, T. L.

2013-03-01

An ``overlayer'' of molecules that breaks the AB symmetry of graphene can produce (modify) a band gap in single- (bi-) layer graphene.[2] Since the triangular shaped trimesic acid (TMA) molecule forms two familiar symmetry breaking configurations, we are motivated to model TMA physisorption on graphene surfaces in conjunction with experiments by Groce et al. at UMD. Using VASP, with ab initio van der Waals density functionals (vdW-DF), we simulate adsorption of TMA onto a graphene surface in several symmetry-breaking arrangements in order to predict/understand the effect of TMA adsorption on experimental observables. Supported by NSF-MRSEC Grant DMR 05-20471.

A Simple Bioconjugate Attachment Protocol for Use in Single Molecule Force Spectroscopy Experiments Based on Mixed Self-Assembled Monolayers

PubMed Central

Attwood, Simon J.; Simpson, Anna M. C.; Stone, Rachael; Hamaia, SamirW.; Roy, Debdulal; Farndale, RichardW.; Ouberai, Myriam; Welland, Mark E.

2012-01-01

Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin. PMID:23202965

Single-molecule dilution and multiple displacement amplification for molecular haplotyping.

PubMed

Paul, Philip; Apgar, Josh

2005-04-01

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

Nanohole optical tweezers in heterogeneous mixture analysis

NASA Astrophysics Data System (ADS)

Hacohen, Noa; Ip, Candice J. X.; Laxminarayana, Gurunatha K.; DeWolf, Timothy S.; Gordon, Reuven

2017-08-01

Nanohole optical trapping is a tool that has been shown to analyze proteins at the single molecule level using pure samples. The next step is to detect and study single molecules with dirty samples. We demonstrate that using our double nanohole optical tweezing configuration, single particles in an egg white solution can be classified when trapped. Different sized molecules provide different signal variations in their trapped state, allowing the proteins to be statistically characterized. Root mean squared variation and trap stiffness are methods used on trapped signals to distinguish between the different proteins. This method to isolate and determine single molecules in heterogeneous samples provides huge potential to become a reliable tool for use within biomedical and scientific communities.

Development of a reference material of a single DNA molecule for the quality control of PCR testing.

PubMed

Mano, Junichi; Hatano, Shuko; Futo, Satoshi; Yoshii, Junji; Nakae, Hiroki; Naito, Shigehiro; Takabatake, Reona; Kitta, Kazumi

2014-09-02

We developed a reference material of a single DNA molecule with a specific nucleotide sequence. The double-strand linear DNA which has PCR target sequences at the both ends was prepared as a reference DNA molecule, and we named the PCR targets on each side as confirmation sequence and standard sequence. The highly diluted solution of the reference molecule was dispensed into 96 wells of a plastic PCR plate to make the average number of molecules in a well below one. Subsequently, the presence or absence of the reference molecule in each well was checked by real-time PCR targeting for the confirmation sequence. After an enzymatic treatment of the reaction mixture in the positive wells for the digestion of PCR products, the resultant solution was used as the reference material of a single DNA molecule with the standard sequence. PCR analyses revealed that the prepared samples included only one reference molecule with high probability. The single-molecule reference material developed in this study will be useful for the absolute evaluation of a detection limit of PCR-based testing methods, the quality control of PCR analyses, performance evaluations of PCR reagents and instruments, and the preparation of an accurate calibration curve for real-time PCR quantitation.

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine.

PubMed

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W; Cai, Jiye

2014-11-07

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In this review, we attempt to summarize the characteristics of these advanced techniques for use in the in situ single molecule imaging of cell membranes. We believe that this work will help to promote the technological and methodological developments of super-resolution techniques for the single molecule imaging of cell membranes and help researchers better understand which technique is most suitable for their future exploring of membrane biomolecules; ultimately promoting further developments in cell biology, immunology and medicine.

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

NASA Astrophysics Data System (ADS)

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

2014-10-01

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In this review, we attempt to summarize the characteristics of these advanced techniques for use in the in situ single molecule imaging of cell membranes. We believe that this work will help to promote the technological and methodological developments of super-resolution techniques for the single molecule imaging of cell membranes and help researchers better understand which technique is most suitable for their future exploring of membrane biomolecules; ultimately promoting further developments in cell biology, immunology and medicine.

DNA Origami Directed Au Nanostar Dimers for Single-Molecule Surface-Enhanced Raman Scattering.

PubMed

Tanwar, Swati; Haldar, Krishna Kanta; Sen, Tapasi

2017-12-06

We demonstrate the synthesis of Au nanostar dimers with tunable interparticle gap and controlled stoichiometry assembled on DNA origami. Au nanostars with uniform and sharp tips were immobilized on rectangular DNA origami dimerized structures to create nanoantennas containing monomeric and dimeric Au nanostars. Single Texas red (TR) dye was specifically attached in the junction of the dimerized origami to act as a Raman reporter molecule. The SERS enhancement factors of single TR dye molecules located in the conjunction region in dimer structures having interparticle gaps of 7 and 13 nm are 2 × 10 10 and 8 × 10 9 , respectively, which are strong enough for single analyte detection. The highly enhanced electromagnetic field generated by the plasmon coupling between sharp tips and cores of two Au nanostars in the wide conjunction region allows the accommodation and specific detection of large biomolecules. Such DNA-directed assembled nanoantennas with controlled interparticle separation distance and stoichiometry, and well-defined geometry, can be used as excellent substrates in single-molecule SERS spectroscopy and will have potential applications as a reproducible platform in single-molecule sensing.

Single-Molecule Interfacial Electron Transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, H. Peter

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 andmore » 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 TiO 2 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 TiO 2 nanoparticle systems that strongly involves with and regulated by molecule-surface interactions. We have studied interfacial electron transfer on TiO 2 nanoparticle surfaces by using ultrafast single-molecule spectroscopy and electrochemical AFM metal tip scanning microscopy, focusing on understanding the interfacial electron transfer dynamics at specific nanoscale electron transfer sites with high-spatially and temporally resolved topographic-and-spectroscopic characterization at individual molecule basis, characterizing single-molecule rate processes, reaction driving force, and molecule-substrate electronic coupling. One of the most significant characteristics of our new approach is that we are able to interrogate the complex interfacial electron transfer dynamics by actively pin-point energetic manipulation of the surface interaction and electronic couplings, beyond the conventional excitation and observation.« less

Direct observation and control of hydrogen-bond dynamics using low-temperature scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Kumagai, Takashi

2015-08-01

Hydrogen(H)-bond dynamics are involved in many elementary processes in chemistry and biology. Because of its fundamental importance, a variety of experimental and theoretical approaches have been employed to study the dynamics in gas, liquid, solid phases, and their interfaces. This review describes the recent progress of direct observation and control of H-bond dynamics in several model systems on a metal surface by using low-temperature scanning tunneling microscopy (STM). General aspects of H-bond dynamics and the experimental methods are briefly described in chapter 1 and 2. In the subsequent four chapters, I present direct observation of an H-bond exchange reaction within a single water dimer (chapter 3), a symmetric H bond (chapter 4) and H-atom relay reactions (chapter 5) within water-hydroxyl complexes, and an intramolecular H-atom transfer reaction (tautomerization) within a single porphycene molecule (chapter 6). These results provide novel microscopic insights into H-bond dynamics at the single-molecule level, and highlight significant impact on the process from quantum effects, namely tunneling and zero-point vibration, resulting from the small mass of H atom. Additionally, local environmental effect on H-bond dynamics is also examined by using atom/molecule manipulation with the STM.

Measurement and control of detailed electronic properties in a single molecule break junction.

PubMed

Wang, Kun; Hamill, Joseph; Zhou, Jianfeng; Guo, Cunlan; Xu, Bingqian

2014-01-01

The lack of detailed experimental controls has been one of the major obstacles hindering progress in molecular electronics. While large fluctuations have been occurring in the experimental data, specific details, related mechanisms, and data analysis techniques are in high demand to promote our physical understanding at the single-molecule level. A series of modulations we recently developed, based on traditional scanning probe microscopy break junctions (SPMBJs), have helped to discover significant properties in detail which are hidden in the contact interfaces of a single-molecule break junction (SMBJ). For example, in the past we have shown that the correlated force and conductance changes under the saw tooth modulation and stretch-hold mode of PZT movement revealed inherent differences in the contact geometries of a molecular junction. In this paper, using a bias-modulated SPMBJ and utilizing emerging data analysis techniques, we report on the measurement of the altered alignment of the HOMO of benzene molecules with changing the anchoring group which coupled the molecule to metal electrodes. Further calculations based on Landauer fitting and transition voltage spectroscopy (TVS) demonstrated the effects of modulated bias on the location of the frontier molecular orbitals. Understanding the alignment of the molecular orbitals with the Fermi level of the electrodes is essential for understanding the behaviour of SMBJs and for the future design of more complex devices. With these modulations and analysis techniques, fruitful information has been found about the nature of the metal-molecule junction, providing us insightful clues towards the next step for in-depth study.

A Single-Level Tunnel Model to Account for Electrical Transport through Single Molecule- and Self-Assembled Monolayer-based Junctions

PubMed Central

Garrigues, Alvar R.; Yuan, Li; Wang, Lejia; Mucciolo, Eduardo R.; Thompon, Damien; del Barco, Enrique; Nijhuis, Christian A.

2016-01-01

We present a theoretical analysis aimed at understanding electrical conduction in molecular tunnel junctions. We focus on discussing the validity of coherent versus incoherent theoretical formulations for single-level tunneling to explain experimental results obtained under a wide range of experimental conditions, including measurements in individual molecules connecting the leads of electromigrated single-electron transistors and junctions of self-assembled monolayers (SAM) of molecules sandwiched between two macroscopic contacts. We show that the restriction of transport through a single level in solid state junctions (no solvent) makes coherent and incoherent tunneling formalisms indistinguishable when only one level participates in transport. Similar to Marcus relaxation processes in wet electrochemistry, the thermal broadening of the Fermi distribution describing the electronic occupation energies in the electrodes accounts for the exponential dependence of the tunneling current on temperature. We demonstrate that a single-level tunnel model satisfactorily explains experimental results obtained in three different molecular junctions (both single-molecule and SAM-based) formed by ferrocene-based molecules. Among other things, we use the model to map the electrostatic potential profile in EGaIn-based SAM junctions in which the ferrocene unit is placed at different positions within the molecule, and we find that electrical screening gives rise to a strongly non-linear profile across the junction. PMID:27216489

A wireless centrifuge force microscope (CFM) enables multiplexed single-molecule experiments in a commercial centrifuge.

PubMed

Hoang, Tony; Patel, Dhruv S; Halvorsen, Ken

2016-08-01

The centrifuge force microscope (CFM) was recently introduced as a platform for massively parallel single-molecule manipulation and analysis. Here we developed a low-cost and self-contained CFM module that works directly within a commercial centrifuge, greatly improving accessibility and ease of use. Our instrument incorporates research grade video microscopy, a power source, a computer, and wireless transmission capability to simultaneously monitor many individually tethered microspheres. We validated the instrument by performing single-molecule force shearing of short DNA duplexes. For a 7 bp duplex, we observed over 1000 dissociation events due to force dependent shearing from 2 pN to 12 pN with dissociation times in the range of 10-100 s. We extended the measurement to a 10 bp duplex, applying a 12 pN force clamp and directly observing single-molecule dissociation over an 85 min experiment. Our new CFM module facilitates simple and inexpensive experiments that dramatically improve access to single-molecule analysis.

Quantitative Connection Between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryo-EM and smFRET Investigations of the Ribosome

PubMed Central

Frank, Joachim; Gonzalez, Ruben L.

2015-01-01

At equilibrium, thermodynamic and kinetic information can be extracted from biomolecular energy landscapes by many techniques. However, while static, ensemble techniques yield thermodynamic data, often only dynamic, single-molecule techniques can yield the kinetic data that describes transition-state energy barriers. Here we present a generalized framework based upon dwell-time distributions that can be used to connect such static, ensemble techniques with dynamic, single-molecule techniques, and thus characterize energy landscapes to greater resolutions. We demonstrate the utility of this framework by applying it to cryogenic electron microscopy and single-molecule fluorescence resonance energy transfer studies of the bacterial ribosomal pretranslocation complex. Among other benefits, application of this framework to these data explains why two transient, intermediate conformations of the pretranslocation complex, which are observed in a cryogenic electron microscopy study, may not be observed in several single-molecule fluorescence resonance energy transfer studies. PMID:25785884

Super-resolution and super-localization microscopy: A novel tool for imaging chemical and biological processes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Bin

2015-01-01

Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes.This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the singlemore » molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM).« less

Single-Molecule Chemistry with Surface- and Tip-Enhanced Raman Spectroscopy.

PubMed

Zrimsek, Alyssa B; Chiang, Naihao; Mattei, Michael; Zaleski, Stephanie; McAnally, Michael O; Chapman, Craig T; Henry, Anne-Isabelle; Schatz, George C; Van Duyne, Richard P

2017-06-14

Single-molecule (SM) surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) have emerged as analytical techniques for characterizing molecular systems in nanoscale environments. SERS and TERS use plasmonically enhanced Raman scattering to characterize the chemical information on single molecules. Additionally, TERS can image single molecules with subnanometer spatial resolution. In this review, we cover the development and history of SERS and TERS, including the concept of SERS hot spots and the plasmonic nanostructures necessary for SM detection, the past and current methodologies for verifying SMSERS, and investigations into understanding the signal heterogeneities observed with SMSERS. Moving on to TERS, we cover tip fabrication and the physical origins of the subnanometer spatial resolution. Then, we highlight recent advances of SMSERS and TERS in fields such as electrochemistry, catalysis, and SM electronics, which all benefit from the vibrational characterization of single molecules. SMSERS and TERS provide new insights on molecular behavior that would otherwise be obscured in an ensemble-averaged measurement.

Single-Molecule Electrical Random Resequencing of DNA and RNA

NASA Astrophysics Data System (ADS)

Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

2012-07-01

Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

Quantitative Connection between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome.

PubMed

Thompson, Colin D Kinz; Sharma, Ajeet K; Frank, Joachim; Gonzalez, Ruben L; Chowdhury, Debashish

2015-08-27

At equilibrium, thermodynamic and kinetic information can be extracted from biomolecular energy landscapes by many techniques. However, while static, ensemble techniques yield thermodynamic data, often only dynamic, single-molecule techniques can yield the kinetic data that describe transition-state energy barriers. Here we present a generalized framework based upon dwell-time distributions that can be used to connect such static, ensemble techniques with dynamic, single-molecule techniques, and thus characterize energy landscapes to greater resolutions. We demonstrate the utility of this framework by applying it to cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) studies of the bacterial ribosomal pre-translocation complex. Among other benefits, application of this framework to these data explains why two transient, intermediate conformations of the pre-translocation complex, which are observed in a cryo-EM study, may not be observed in several smFRET studies.

Binding configurations and intramolecular strain in single-molecule devices.

PubMed

Rascón-Ramos, Habid; Artés, Juan Manuel; Li, Yuanhui; Hihath, Joshua

2015-05-01

The development of molecular-scale electronic devices has made considerable progress over the past decade, and single-molecule transistors, diodes and wires have all been demonstrated. Despite this remarkable progress, the agreement between theoretically predicted conductance values and those measured experimentally remains limited. One of the primary reasons for these discrepancies lies in the difficulty to experimentally determine the contact geometry and binding configuration of a single-molecule junction. In this Article, we apply a small-amplitude, high-frequency, sinusoidal mechanical signal to a series of single-molecule devices during junction formation and breakdown. By measuring the current response at this frequency, it is possible to determine the most probable binding and contact configurations for the molecular junction at room temperature in solution, and to obtain information about how an applied strain is distributed within the molecular junction. These results provide insight into the complex configuration of single-molecule devices, and are in excellent agreement with previous predictions from theoretical models.

Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules.

PubMed

Sung, Jongmin; Nag, Suman; Mortensen, Kim I; Vestergaard, Christian L; Sutton, Shirley; Ruppel, Kathleen; Flyvbjerg, Henrik; Spudich, James A

2015-08-04

Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using 'harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.

Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules

PubMed Central

Sung, Jongmin; Nag, Suman; Mortensen, Kim I.; Vestergaard, Christian L.; Sutton, Shirley; Ruppel, Kathleen; Flyvbjerg, Henrik; Spudich, James A.

2015-01-01

Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using ‘harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load. PMID:26239258

Detection of gas molecules on single Mn adatom adsorbed graphyne: a DFT-D study

NASA Astrophysics Data System (ADS)

Lu, Zhansheng; Lv, Peng; Ma, Dongwei; Yang, Xinwei; Li, Shuo; Yang, Zongxian

2018-02-01

As one of the prominent applications in intelligent systems, gas sensing technology has attracted great interest in both industry and academia. In the current study, the pristine graphyne (GY) without and with a single Mn atom is investigated to detect the gas molecules (CO, CH4, CO2, NH3, NO and O2). The pristine GY is promising to detect O2 molecules because of its chemical adsorption on GY with large electron transfer. The great stability of the Mn/GY is found, and the Mn atom prefers to anchor at the alkyne ring as a single atom. Upon single Mn atom anchoring, the sensitivity and selectivity of GY based gas sensors is significantly improved for various molecules, except CH4. The recovery time of the Mn/GY after detecting the gas molecules may help to appraise the detection efficiency for the Mn/GY. The current study will help to understand the mechanism of detecting the gas molecules, and extend the potentially fascinating applications of GY-based materials.

Measurement and statistical analysis of single-molecule current-voltage characteristics, transition voltage spectroscopy, and tunneling barrier height.

PubMed

Guo, Shaoyin; Hihath, Joshua; Díez-Pérez, Ismael; Tao, Nongjian

2011-11-30

We report on the measurement and statistical study of thousands of current-voltage characteristics and transition voltage spectra (TVS) of single-molecule junctions with different contact geometries that are rapidly acquired using a new break junction method at room temperature. This capability allows one to obtain current-voltage, conductance voltage, and transition voltage histograms, thus adding a new dimension to the previous conductance histogram analysis at a fixed low-bias voltage for single molecules. This method confirms the low-bias conductance values of alkanedithiols and biphenyldithiol reported in literature. However, at high biases the current shows large nonlinearity and asymmetry, and TVS allows for the determination of a critically important parameter, the tunneling barrier height or energy level alignment between the molecule and the electrodes of single-molecule junctions. The energy level alignment is found to depend on the molecule and also on the contact geometry, revealing the role of contact geometry in both the contact resistance and energy level alignment of a molecular junction. Detailed statistical analysis further reveals that, despite the dependence of the energy level alignment on contact geometry, the variation in single-molecule conductance is primarily due to contact resistance rather than variations in the energy level alignment.

Unimolecular Logic Gate with Classical Input by Single Gold Atoms.

PubMed

Skidin, Dmitry; Faizy, Omid; Krüger, Justus; Eisenhut, Frank; Jancarik, Andrej; Nguyen, Khanh-Hung; Cuniberti, Gianaurelio; Gourdon, Andre; Moresco, Francesca; Joachim, Christian

2018-02-27

By a combination of solution and on-surface chemistry, we synthesized an asymmetric starphene molecule with two long anthracenyl input branches and a short naphthyl output branch on the Au(111) surface. Starting from this molecule, we could demonstrate the working principle of a single molecule NAND logic gate by selectively contacting single gold atoms by atomic manipulation to the longer branches of the molecule. The logical input "1" ("0") is defined by the interaction (noninteraction) of a gold atom with one of the input branches. The output is measured by scanning tunneling spectroscopy following the shift in energy of the electronic tunneling resonances at the end of the short branch of the molecule.

Single Fluorescent Molecules as Nano-Illuminators for Biological Structure and Function

NASA Astrophysics Data System (ADS)

Moerner, W. E.

2011-03-01

Since the first optical detection and spectroscopy of a single molecule in a solid (Phys. Rev. Lett. {62}, 2535 (1989)), much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. For example, the shape of single filaments in a living cell can be extracted simply by allowing a single molecule to move through the filament (PNAS {103}, 10929 (2006)). The addition of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (super-resolution) and a new array of acronyms (PALM, STORM, F-PALM etc.) and advances have appeared. We have used the native blinking and switching of a common yellow-emitting variant of green fluorescent protein (EYFP) reported more than a decade ago (Nature {388}, 355 (1997)) to achieve sub-40 nm super-resolution imaging of several protein structures in the bacterium Caulobacter crescentus: the quasi-helix of the actin-like protein MreB (Nat. Meth. {5}, 947 (2008)), the cellular distribution of the DNA binding protein HU (submitted), and the recently discovered division spindle composed of ParA filaments (Nat. Cell Biol. {12}, 791 (2010)). Even with these advances, better emitters would provide more photons and improved resolution, and a new photoactivatable small-molecule emitter has recently been synthesized and targeted to specific structures in living cells to provide super-resolution images (JACS {132}, 15099 (2010)). Finally, a new optical method for extracting three-dimensional position information based on a double-helix point spread function enables quantitative tracking of single mRNA particles in living yeast cells with 15 ms time resolution and 25-50 nm spatial precision (PNAS {107}, 17864 (2010)). These examples illustrate the power of single-molecule optical imaging in extracting new structural and functional information in living cells.

Role of solvent environments in single molecule conductance used insulator-modified mechanically controlled break junctions

NASA Astrophysics Data System (ADS)

Muthusubramanian, Nandini; Maity, Chandan; Galan Garcia, Elena; Eelkema, Rienk; Grozema, Ferdinand; van der Zant, Herre; Kavli Institute of Nanoscience Collaboration; Department of Chemical Engineering Collaboration

We present a method for studying the effects of polar solvents on charge transport through organic/biological single molecules by developing solvent-compatible mechanically controlled break junctions of gold coated with a thin layer of aluminium oxide using plasma enhanced atomic layer deposition (ALD). The optimal oxide thickness was experimentally determined to be 15 nm deposited at ALD operating temperature of 300°C which yielded atomically sharp electrodes and reproducible single-barrier tunnelling behaviour across a wide conductance range between 1 G0 and 10-7 G0. The insulator protected MCBJ devices were found to be effective in various solvents such as deionized water, phosphate buffered saline, methanol, acetonitrile and dichlorobenzene. The yield of molecular junctions using such insulated electrodes was tested by developing a chemical protocol for synthesizing an amphipathic form of oligo-phenylene ethynylene (OPE3-PEO) with thioacetate anchoring groups. This work has further applications in studying effects of solvation, dipole orientation and other thermodynamic interactions on charge transport. Eu Marie Curie Initial Training Network (ITN). MOLECULAR-SCALE ELECTRONICS: ``MOLESCO'' Project Number 606728.

Electron scattering effects at physisorbed hydrogen molecules on break-junction electrodes and nanowires formation in hydrogen environment

NASA Astrophysics Data System (ADS)

van der Maas, M.; Vasnyov, S.; Hendriksen, B. L. M.; Shklyarevskii, O. I.; Speller, S.

2012-06-01

Physisorption of hydrogen molecules on the surface of gold and other coinage metals has been studied using distance tunneling spectroscopy. We have observed that the distance dependence of the tunnel current (resistance) displays a strong N-shaped deviation from exponential behavior. Such deviations are difficult to explain within the Tersoff-Hamann approximation. We suggest the scattering of tunneling electrons by H2 molecules as an origin for the observed effect. We have found that this phenomenon is also common for strongly adsorbed organic molecules with a single anchoring group. Pulling Au, Cu and Pt nanowires at 22 K in hydrogen environment shows that the break-junction electrodes are still connected through hydrogen-metal monoatomic chains down to very low conductance values of 10-4-10-6 G0.

Inter-dot strain field effect on the optoelectronic properties of realistic InP lateral quantum-dot molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barettin, Daniele, E-mail: Daniele.Barettin@uniroma2.it; Auf der Maur, Matthias; De Angelis, Roberta

2015-03-07

We report on numerical simulations of InP surface lateral quantum-dot molecules on In{sub 0.48}Ga{sub 0.52 }P buffer, using a model strictly derived by experimental results by extrapolation of the molecules shape from atomic force microscopy images. Our study has been inspired by the comparison of a photoluminescence spectrum of a high-density InP surface quantum dot sample with a numerical ensemble average given by a weighted sum of simulated single quantum-dot spectra. A lack of experimental optical response from the smaller dots of the sample is found to be due to strong inter-dot strain fields, which influence the optoelectronic properties of lateralmore » quantum-dot molecules. Continuum electromechanical, k{sup →}·p{sup →} bandstructure, and optical calculations are presented for two different molecules, the first composed of two dots of nearly identical dimensions (homonuclear), the second of two dots with rather different sizes (heteronuclear). We show that in the homonuclear molecule the hydrostatic strain raises a potential barrier for the electrons in the connection zone between the dots, while conversely the holes do not experience any barrier, which considerably increases the coupling. Results for the heteronuclear molecule show instead that its dots do not appear as two separate and distinguishable structures, but as a single large dot, and no optical emission is observed in the range of higher energies where the smaller dot is supposed to emit. We believe that in samples of such a high density the smaller dots result as practically incorporated into bigger molecular structures, an effect strongly enforced by the inter-dot strain fields, and consequently it is not possible to experimentally obtain a separate optical emission from the smaller dots.« less

Inter-dot strain field effect on the optoelectronic properties of realistic InP lateral quantum-dot molecules

NASA Astrophysics Data System (ADS)

Barettin, Daniele; Auf der Maur, Matthias; De Angelis, Roberta; Prosposito, Paolo; Casalboni, Mauro; Pecchia, Alessandro

2015-03-01

We report on numerical simulations of InP surface lateral quantum-dot molecules on In0.48Ga0.52P buffer, using a model strictly derived by experimental results by extrapolation of the molecules shape from atomic force microscopy images. Our study has been inspired by the comparison of a photoluminescence spectrum of a high-density InP surface quantum dot sample with a numerical ensemble average given by a weighted sum of simulated single quantum-dot spectra. A lack of experimental optical response from the smaller dots of the sample is found to be due to strong inter-dot strain fields, which influence the optoelectronic properties of lateral quantum-dot molecules. Continuum electromechanical, k →.p → bandstructure, and optical calculations are presented for two different molecules, the first composed of two dots of nearly identical dimensions (homonuclear), the second of two dots with rather different sizes (heteronuclear). We show that in the homonuclear molecule the hydrostatic strain raises a potential barrier for the electrons in the connection zone between the dots, while conversely the holes do not experience any barrier, which considerably increases the coupling. Results for the heteronuclear molecule show instead that its dots do not appear as two separate and distinguishable structures, but as a single large dot, and no optical emission is observed in the range of higher energies where the smaller dot is supposed to emit. We believe that in samples of such a high density the smaller dots result as practically incorporated into bigger molecular structures, an effect strongly enforced by the inter-dot strain fields, and consequently it is not possible to experimentally obtain a separate optical emission from the smaller dots.

Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques.

PubMed

van 't Hoff, Marcel; Reuter, Marcel; Dryden, David T F; Oheim, Martin

2009-09-21

Bacteriophage lambda-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single lambda-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and lambda-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching approximately 1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of lambda-DNA shown here.

DNA Molecules Adsorbed on Rippled Supported Cationic Lipid Membranes -- A new way to stretch DNAs

NASA Astrophysics Data System (ADS)

Golubovic, Leonardo

2005-03-01

We discuss a novel approach to control to shapes of DNA molecules. We elucidate the recent experimental work of M. Hochrein, L. Golubovic and J. Raedler, on the conformational behavior of DNA molecules adsorbed on lipid membranes that are supported on grooved micro-structured surfaces. We explain the striking ability of the edges formed on these supported membranes to adsorb and completely orient (stretch) very long DNA molecules. Here we explain the experimentally observed DNA stretching effect in terms of the surface curvature dependent electrostatic potential seen by the adsorbed DNA molecules. On the curved, rippled membrane, we show that the DNA molecules undergo localization transitions causing them to stretch by binding to the ripple edges of the supported membrane. In the future, this stretching will allow to directly image, by the common fluorescence microscopy, fundamental biological processes of the interactions between DNA and single protein molecules.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

Theoretical electrical conductivity of hydrogen-bonded benzamide-derived molecules and single DNA bases.

PubMed

Chen, Xiang

2013-09-01

A benzamide molecule is used as a "reader" molecule to form hydrogen bonds with five single DNA bases, i.e., four normal single DNA bases A,T,C,G and one for 5methylC. The whole molecule is then attached to the gold surface so that a meta-molecule junction is formed. We calculate the transmission function and conductance for the five metal-molecule systems, with the implementation of density functional theory-based non-equilibrium Green function method. Our results show that each DNA base exhibits a unique conductance and most of them are on the pS level. The distinguishable conductance of each DNA base provides a way for the fast sequencing of DNA. We also investigate the dependence of conductivity of such a metal-molecule system on the hydrogen bond length between the "reader" molecule and DNA base, which shows that conductance follows an exponential decay as the hydrogen bond length increases, i.e., the conductivity is highly sensitive to the change in hydrogen bond length.

DFT study of adsorption of picric acid molecule on the surface of single-walled ZnO nanotube; as potential new chemical sensor

NASA Astrophysics Data System (ADS)

Farmanzadeh, Davood; Tabari, Leila

2015-01-01

Using density functional theory (DFT), we have investigated the adsorption of picric acid (PA) molecule on the surface of (8,0) single-walled ZnO nanotube (ZnONT). The results show that the PA molecule can be chemisorbed on the surface of ZnONT with adsorption energies of -82.01 and -75.26 kJ/mol in gas and aqueous phase, respectively. Frontier molecular orbital analysis show that HOMO/LUMO gap of ZnONT reduces from 1.66 and 1.75 eV in the pristine nanotube to 0.83 and 0.72 eV in PA-adsorbed form in gas and aqueous phase, respectively. It suggests that the process can affect the electronic properties of the studied nanotube which would lead to its conductance change upon the adsorption of PA molecule. The modifying effect on the electrical conductance of ZnONT underlies the working mechanism of gas sensors for detecting the PA molecules. Analyses of the adsorption behavior of the electrically charged ZnONT toward PA molecule in the gas phase show that the PA molecule can be strongly adsorbed on the negatively charged ZnONT surface with significant adsorption energy (-135.1 kJ/mol). However, from the HOMO/LUMO gap changes, it can be concluded that the positive ZnONT might sensitively detect the PA molecule in comparison to the negative tube. These results can provide helpful information for experimental investigation to develop novel nanotube-based sensors.

In Situ Oxidation Synthesis of p-Type Composite with Narrow-Bandgap Small Organic Molecule Coating on Single-Walled Carbon Nanotube: Flexible Film and Thermoelectric Performance.

PubMed

Gao, Caiyan; Chen, Guangming

2018-03-01

Although composites of organic polymers or n-type small molecule/carbon nanotube (CNT) have achieved significant advances in thermoelectric (TE) applications, p-type TE composites of small organic molecules as thick surface coating layers on the surfaces of inorganic nanoparticles still remain a great challenge. Taking advantage of in situ oxidation reaction of thieno[3,4-b]pyrazine (TP) into TP di-N-oxide (TPNO) on single-walled CNT (SWCNT) surface, a novel synthesis strategy is proposed to achieve flexible films of TE composites with narrow-bandgap (1.19 eV) small molecule coating on SWCNT surface. The TE performance can be effectively enhanced and conveniently tuned by poly(sodium-p-styrenesulfonate) content, TPNO/SWCNT mass ratio, and posttreatment by various polar solvents. The maximum of the composite power factor at room temperature is 29.4 ± 1.0 µW m -1 K -2 . The work presents a way to achieve flexible films of p-type small organic molecule/inorganic composites with clear surface coating morphology for TE application. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells.

PubMed

Avogaro, Laura; Querido, Emmanuelle; Dalachi, Myriam; Jantsch, Michael F; Chartrand, Pascal; Cusanelli, Emilio

2018-04-16

Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

Kinetics of molecular transitions with dynamic disorder in single-molecule pulling experiments

NASA Astrophysics Data System (ADS)

Zheng, Yue; Li, Ping; Zhao, Nanrong; Hou, Zhonghuai

2013-05-01

Macromolecular transitions are subject to large fluctuations of rate constant, termed as dynamic disorder. The individual or intrinsic transition rates and activation free energies can be extracted from single-molecule pulling experiments. Here we present a theoretical framework based on a generalized Langevin equation with fractional Gaussian noise and power-law memory kernel to study the kinetics of macromolecular transitions to address the effects of dynamic disorder on barrier-crossing kinetics under external pulling force. By using the Kramers' rate theory, we have calculated the fluctuating rate constant of molecular transition, as well as the experimentally accessible quantities such as the force-dependent mean lifetime, the rupture force distribution, and the speed-dependent mean rupture force. Particular attention is paid to the discrepancies between the kinetics with and without dynamic disorder. We demonstrate that these discrepancies show strong and nontrivial dependence on the external force or the pulling speed, as well as the barrier height of the potential of mean force. Our results suggest that dynamic disorder is an important factor that should be taken into account properly in accurate interpretations of single-molecule pulling experiments.

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules

PubMed Central

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A.M.

2017-01-01

Abstract Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. PMID:28077562

Single-Molecule Analysis for RISC Assembly and Target Cleavage.

PubMed

Sasaki, Hiroshi M; Tadakuma, Hisashi; Tomari, Yukihide

2018-01-01

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as.

PubMed

Kocks, Christine; Boltengagen, Anastasiya; Piwecka, Monika; Rybak-Wolf, Agnieszka; Rajewsky, Nikolaus

2018-01-01

Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus.

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

PubMed Central

Lee, Wonbae; Gillies, John P.; Jose, Davis; Israels, Brett A.; von Hippel, Peter H.; Marcus, Andrew H.

2016-01-01

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed. PMID:27694621

An information-theoretic approach to designing the plane spacing for multifocal plane microscopy

PubMed Central

Tahmasbi, Amir; Ram, Sripad; Chao, Jerry; Abraham, Anish V.; Ward, E. Sally; Ober, Raimund J.

2015-01-01

Multifocal plane microscopy (MUM) is a 3D imaging modality which enables the localization and tracking of single molecules at high spatial and temporal resolution by simultaneously imaging distinct focal planes within the sample. MUM overcomes the depth discrimination problem of conventional microscopy and allows high accuracy localization of a single molecule in 3D along the z-axis. An important question in the design of MUM experiments concerns the appropriate number of focal planes and their spacings to achieve the best possible 3D localization accuracy along the z-axis. Ideally, it is desired to obtain a 3D localization accuracy that is uniform over a large depth and has small numerical values, which guarantee that the single molecule is continuously detectable. Here, we address this concern by developing a plane spacing design strategy based on the Fisher information. In particular, we analyze the Fisher information matrix for the 3D localization problem along the z-axis and propose spacing scenarios termed the strong coupling and the weak coupling spacings, which provide appropriate 3D localization accuracies. Using these spacing scenarios, we investigate the detectability of the single molecule along the z-axis and study the effect of changing the number of focal planes on the 3D localization accuracy. We further review a software module we recently introduced, the MUMDesignTool, that helps to design the plane spacings for a MUM setup. PMID:26113764

Real-time Molecular Study of Bystander Effects of Low dose Low LET radiation Using Living Cell Imaging and Nanoparticale Optics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Natarajan, Mohan; Xu, Nancy R; Mohan, Sumathy

2013-06-03

In this study two novel approaches are proposed to investigate precisely the low dose low LET radiation damage and its effect on bystander cells in real time. First, a flow shear model system, which would provide us a near in vivo situation where endothelial cells in the presence of extra cellular matrix experiencing continuous flow shear stress, will be used. Endothelial cells on matri-gel (simulated extra cellular matrix) will be subjected to physiological flow shear (that occurs in normal blood vessels). Second, a unique tool (Single nano particle/single live cell/single molecule microscopy and spectroscopy; Figure A) will be used tomore » track the molecular trafficking by single live cell imaging. Single molecule chemical microscopy allows one to single out and study rare events that otherwise might be lost in assembled average measurement, and monitor many target single molecules simultaneously in real-time. Multi color single novel metal nanoparticle probes allow one to prepare multicolor probes (Figure B) to monitor many single components (events) simultaneously and perform multi-complex analysis in real-time. These nano-particles resist to photo bleaching and hence serve as probes for unlimited timeframe of analysis. Single live cell microscopy allows one to image many single cells simultaneously in real-time. With the combination of these unique tools, we will be able to study under near-physiological conditions the cellular and sub-cellular responses (even subtle changes at one molecule level) to low and very low doses of low LET radiation in real time (milli-second or nano-second) at sub-10 nanometer spatial resolution. This would allow us to precisely identify, at least in part, the molecular mediators that are responsible of radiation damage in the irradiated cells and the mediators that are responsible for initiating the signaling in the neighboring cells. Endothelial cells subjected to flow shear (2 dynes/cm2 or 16 dynes/cm2) and exposed to 0.1, 1 and 10 cGy on coverslips will be examined for (a) low LET radiation-induced alterations of cellular function and its physiological relevance in real time; and (b) radiation damage triggered bystander effect on the neighboring unirradiated cells. First, to determine the low LET radiation induced alteration of cellular function we will examine: (i) the real time transformation of single membrane transporters in single living cells; (ii) the pump efficiency of membrane efflux pump of live cells in real time at the molecular level; (iii) the kinetics of single-ligand receptor interaction on single live cell surface (Figure C); and (iv) alteration in chromosome replication in living cell. Second, to study the radiation triggered bystander responses, we will examine one of the key signaling pathway i.e. TNF- alpha/NF-kappa B mediated signaling. TNF-alpha specific nano particle sensors (green) will be developed to detect the releasing dynamics, transport mechanisms and ligand-receptor binding on live cell surface in real time. A second sensor (blue) will be developed to simultaneously monitor the track of NF-kB inside the cell. The proposed nano-particle optics approach would complement our DOE funded study on biochemical mechanisms of TNF-alpha- NF-kappa B-mediated bystander effect.« less

Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World.

PubMed

Ray, Sujay; Widom, Julia R; Walter, Nils G

2018-04-25

The emergence of single-molecule (SM) fluorescence techniques has opened up a vast new toolbox for exploring the molecular basis of life. The ability to monitor individual biomolecules in real time enables complex, dynamic folding pathways to be interrogated without the averaging effect of ensemble measurements. In parallel, modern biology has been revolutionized by our emerging understanding of the many functions of RNA. In this comprehensive review, we survey SM fluorescence approaches and discuss how the application of these tools to RNA and RNA-containing macromolecular complexes in vitro has yielded significant insights into the underlying biology. Topics covered include the three-dimensional folding landscapes of a plethora of isolated RNA molecules, their assembly and interactions in RNA-protein complexes, and the relation of these properties to their biological functions. In all of these examples, the use of SM fluorescence methods has revealed critical information beyond the reach of ensemble averages.

Chemical bond imaging using higher eigenmodes of tuning fork sensors in atomic force microscopy

NASA Astrophysics Data System (ADS)

Ebeling, Daniel; Zhong, Qigang; Ahles, Sebastian; Chi, Lifeng; Wegner, Hermann A.; Schirmeisen, André

2017-05-01

We demonstrate the ability of resolving the chemical structure of single organic molecules using non-contact atomic force microscopy with higher normal eigenmodes of quartz tuning fork sensors. In order to achieve submolecular resolution, CO-functionalized tips at low temperatures are used. The tuning fork sensors are operated in ultrahigh vacuum in the frequency modulation mode by exciting either their first or second eigenmode. Despite the high effective spring constant of the second eigenmode (on the order of several tens of kN/m), the force sensitivity is sufficiently high to achieve atomic resolution above the organic molecules. This is observed for two different tuning fork sensors with different tip geometries (small tip vs. large tip). These results represent an important step towards resolving the chemical structure of single molecules with multifrequency atomic force microscopy techniques where two or more eigenmodes are driven simultaneously.

Core-mass nonadiabatic corrections to molecules: H2, H2+, and isotopologues.

PubMed

Diniz, Leonardo G; Alijah, Alexander; Mohallem, José Rachid

2012-10-28

For high-precision calculations of rovibrational states of light molecules, it is essential to include non-adiabatic corrections. In the absence of crossings of potential energy surfaces, they can be incorporated in a single surface picture through coordinate-dependent vibrational and rotational reduced masses. We present a compact method for their evaluation and relate in particular the vibrational mass to a well defined nuclear core mass derived from a Mulliken analysis of the electronic density. For the rotational mass we propose a simple, but very effective parametrization. The use of these masses in the nuclear Schrödinger equation yields numerical data for the corrections of a much higher quality than can be obtained with optimized constant masses, typically better than 0.1 cm(-1). We demonstrate the method for H(2), H(2)(+), and singly deuterated isotopologues. Isotopic asymmetry does not present any particular difficulty. Generalization to polyatomic molecules is straightforward.

Polymer physics experiments with single DNA molecules

NASA Astrophysics Data System (ADS)

Smith, Douglas E.

1999-11-01

Bacteriophage DNA molecules were taken as a model flexible polymer chain for the experimental study of polymer dynamics at the single molecule level. Video fluorescence microscopy was used to directly observe the conformational dynamics of fluorescently labeled molecules, optical tweezers were used to manipulate individual molecules, and micro-fabricated flow cells were used to apply controlled hydrodynamic strain to molecules. These techniques constitute a powerful new experimental approach in the study of basic polymer physics questions. I have used these techniques to study the diffusion and relaxation of isolated and entangled polymer molecules and the hydrodynamic deformation of polymers in elongational and shear flows. These studies revealed a rich, and previously unobserved, ``molecular individualism'' in the dynamical behavior of single molecules. Individual measurements on ensembles of identical molecules allowed the average conformation to be determined as well as the underlying probability distributions for molecular conformation. Scaling laws, that predict the dependence of properties on chain length and concentration, were also tested. The basic assumptions of the reptation model were directly confirmed by visualizing the dynamics of entangled chains.

High-resolution single-molecule recognition imaging of the molecular details of ricin-aptamer interaction

USDA-ARS?s Scientific Manuscript database

The molecular details of DNA aptamer-ricin interactions were investigated. The toxic protein ricin molecules were immobilized on Au(111) surface using N-hydroxysuccinimide (NHS) ester to specifically react with lysine residues located on the ricin B chains. A single ricin molecule was visualized in ...

Fast temporal fluctuations in single-molecule junctions.

PubMed

Ochs, Roif; Secker, Daniel; Elbing, Mark; Mayor, Marcel; Weber, Heiko B

2006-01-01

The noise within the electrical current through single-molecule junctions is studied cryogenic temperature. The organic sample molecules were contacted with the mechanically controlled break-junction technique. The noise spectra refer to a where only few Lorentzian fluctuators occur in the conductance. The frequency dependence shows qualitative variations from sample to sample.

PhotoGate microscopy: tracking single molecules in a cytoplasm (Conference Presentation)

NASA Astrophysics Data System (ADS)

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.

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

PubMed Central

Ardui, Simon; Ameur, Adam; Vermeesch, Joris R; Hestand, Matthew S

2018-01-01

Abstract Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing. PMID:29401301

Detection of Single Molecules Illuminated by a Light-Emitting Diode

PubMed Central

Gerhardt, Ilja; Mai, Lijian; Lamas-Linares, Antía; Kurtsiefer, Christian

2011-01-01

Optical detection and spectroscopy of single molecules has become an indispensable tool in biological imaging and sensing. Its success is based on fluorescence of organic dye molecules under carefully engineered laser illumination. In this paper we demonstrate optical detection of single molecules on a wide-field microscope with an illumination based on a commercially available, green light-emitting diode. The results are directly compared with laser illumination in the same experimental configuration. The setup and the limiting factors, such as light transfer to the sample, spectral filtering and the resulting signal-to-noise ratio are discussed. A theoretical and an experimental approach to estimate these parameters are presented. The results can be adapted to other single emitter and illumination schemes. PMID:22346610

Magnetic blocking in a linear iron(I) complex.

PubMed

Zadrozny, Joseph M; Xiao, Dianne J; Atanasov, Mihail; Long, Gary J; Grandjean, Fernande; Neese, Frank; Long, Jeffrey R

2013-07-01

Single-molecule magnets that contain one spin centre may represent the smallest possible unit for spin-based computational devices. Such applications, however, require the realization of molecules with a substantial energy barrier for spin inversion, achieved through a large axial magnetic anisotropy. Recently, significant progress has been made in this regard by using lanthanide centres such as terbium(III) and dysprosium(III), whose anisotropy can lead to extremely high relaxation barriers. We contend that similar effects should be achievable with transition metals by maintaining a low coordination number to restrict the magnitude of the d-orbital ligand-field splitting energy (which tends to hinder the development of large anisotropies). Herein we report the first two-coordinate complex of iron(I), [Fe(C(SiMe3)3)2](-), for which alternating current magnetic susceptibility measurements reveal slow magnetic relaxation below 29 K in a zero applied direct-current field. This S =  complex exhibits an effective spin-reversal barrier of Ueff = 226(4) cm(-1), the largest yet observed for a single-molecule magnet based on a transition metal, and displays magnetic blocking below 4.5 K.

Superresolution Imaging using Single-Molecule Localization

PubMed Central

Patterson, George; Davidson, Michael; Manley, Suliana; Lippincott-Schwartz, Jennifer

2013-01-01

Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (∼10–20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales. PMID:20055680

Single-particle studies of band alignment effects on electron transfer dynamics from semiconductor hetero-nanostructures to single-walled carbon nanotubes.

PubMed

Yuan, Chi-Tsu; Wang, Yong-Gang; Huang, Kuo-Yen; Chen, Ting-Yu; Yu, Pyng; Tang, Jau; Sitt, Amit; Banin, Uri; Millo, Oded

2012-01-24

We utilize single-molecule spectroscopy combined with time-correlated single-photon counting to probe the electron transfer (ET) rates from various types of semiconductor hetero-nanocrystals, having either type-I or type-II band alignment, to single-walled carbon nanotubes. A significantly larger ET rate was observed for type-II ZnSe/CdS dot-in-rod nanostructures as compared to type-I spherical CdSe/ZnS core/shell quantum dots and to CdSe/CdS dot-in-rod structures. Furthermore, such rapid ET dynamics can compete with both Auger and radiative recombination processes, with significance for effective photovoltaic operation. © 2011 American Chemical Society

Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

PubMed Central

Balogh, Zoltán; Makk, Péter

2015-01-01

Summary We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference. PMID:26199840

Alternative types of molecule-decorated atomic chains in Au-CO-Au single-molecule junctions.

PubMed

Balogh, Zoltán; Makk, Péter; Halbritter, András

2015-01-01

We investigate the formation and evolution of Au-CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference.

Ultrasensitive Laser Spectroscopy in Solids: Single-Molecule Detection

DTIC Science & Technology

1989-10-25

spite of detection intensity constraints necessary to avoid power broadening, the optical absorption spectrum of single molecules of pentacene In p...molecule detection, or SMD) would provide a useful tool for the study of local host-absorber interactions where tihe absorbing ,ontor is essentially at...modulation techniques 7. 8 for the model system composed of pentacene substitutional impurities in p-terphenyl crystals at 1.5K. The pontacene molecules can

Resolving metal-molecule interfaces at single-molecule junctions

NASA Astrophysics Data System (ADS)

Komoto, Yuki; Fujii, Shintaro; Nakamura, Hisao; Tada, Tomofumi; Nishino, Tomoaki; Kiguchi, Manabu

2016-05-01

Electronic and structural detail at the electrode-molecule interface have a significant influence on charge transport across molecular junctions. Despite the decisive role of the metal-molecule interface, a complete electronic and structural characterization of the interface remains a challenge. This is in no small part due to current experimental limitations. Here, we present a comprehensive approach to obtain a detailed description of the metal-molecule interface in single-molecule junctions, based on current-voltage (I-V) measurements. Contrary to conventional 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 energy alignment between the conduction orbital and the Fermi level of the electrode. This exhaustive statistical approach was employed to study single-molecule junctions of 1,4-benzenediamine (BDA), 1,4-butanediamine (C4DA), and 1,4-benzenedithiol (BDT). A single interfacial configuration was observed for both BDA and C4DA junctions, while three different interfacial arrangements were resolved for BDT. This multiplicity is due to different molecular adsorption sites on the Au surface namely on-top, hollow, and bridge. Furthermore, C4DA junctions present a fluctuating I-V curve arising from the greater conformational freedom of the saturated alkyl chain, in sharp contrast with the rigid aromatic backbone of both BDA and BDT.

Photoinduced electron transfer of oxazine 1/TiO2 nanoparticles at single molecule level by using confocal fluorescence microscopy.

PubMed

Chen, Yi-Ju; Tzeng, Hsin-Yu; Fan, Hsiu-Fang; Chen, Ming-Shiang; Huang, Jer-Shing; Lin, King-Chuen

2010-06-01

Kinetics of photoinduced electron transfer (ET) from oxazine 1 dye to TiO(2) nanoparticles (NPs) surface is studied at a single molecule level by using confocal fluorescence microscopy. Upon irradiation with a pulsed laser at 630 nm, the fluorescence lifetimes sampled among 100 different dye molecules are determined to yield an average lifetime of 2.9 +/- 0.3 ns, which is close to the value of 3.0 +/- 0.6 ns measured on the bare coverslip. The lifetime proximity suggests that most interfacial electron transfer (IFET) processes for the current system are inefficient, probably caused by physisorption between dye and the TiO(2) film. However, there might exist some molecules which are quenched before fluorescing and fail to be detected. With the aid of autocorrelation analysis under a three-level energy system, the IFET kinetics of single dye molecules in the conduction band of TiO(2) NPs is evaluated to be (1.0 +/- 0.1) x 10(4) s(-1) averaged over 100 single molecules and the back ET rate constant is 4.7 +/- 0.9 s(-1). When a thicker TiO(2) film is substituted, the resultant kinetic data do not make a significant difference. The trend of IFET efficacy agrees with the method of fluorescence lifetime measurements. The obtained forward ET rate constants are about ten times smaller than the photovoltage response measured in an assembled dye-sensitized solar cell. The discrepancy is discussed. The inhomogeneous and fluctuation characters for the IFET process are attributed to microenvironment variation for each single molecule. The obtained ET rates are much slower than the fluorescence relaxation. Such a small ET quantum yield is yet feasibly detectable at a single molecule level.

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

NASA Astrophysics Data System (ADS)

Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary; Benkovic, Stephen J.; Hammes, Gordon G.

2002-10-01

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Measurement of the conductance properties of single organic molecules using gold nanoparticles

NASA Astrophysics Data System (ADS)

Gordin, Yoav

In this work we describe the development and application of a new method for the electrical conductance measurement of single molecules. The issue of reliable theoretical modeling of molecular electronic transport is still very much in debate. The experimental methods used in the field are difficult to realize and interpret; most have very low yield, preventing proper statistical analysis and many have problems in the researchers' ability to characterize the system properly. We address this issue by using self assembly of gold nanoparticle-molecule-gold nanoparticle objects called dimers. This method allows fabrication of molecular junctions with greater ease; moreover it allows individual characterization of the various elements of the junction, removing much of the uncertainties that exist in this kind of measurements. We make use of home grown gold nanoparticles with a few tens of nanometer diameter to form the hybrid dimers. The dimers are large enough to connect between electrodes fabricated using electron beam lithography and to measure the electric properties of the molecule. We have invested significant effort in the characterization of the system, ensuring that the dimers are indeed bridged by the molecules, and that the chances that more than a single molecule exists in a dimer are negligibly small. We have made measurements on single gold nanoparticles, to characterize their properties separately from those of the molecule. These measurements have allowed us to observe single electron transistor (SET) behavior, resulting from the requirement that electrons charge the nanoparticle during transport. We have shown that the energy associated with this charging scales with nanoparticle size as expected. We have performed measurements on single organic molecules, showing that there is a very strong influence of molecular conjugation (the way electronic orbitals are spread along the molecular backbone) on its conductance. The molecules with broken conjugation conduct more than an order of magnitude less than those that are fully conjugated. A distinct feature of the conjugated molecule is the appearance of pronounced peaks in its conductance at certain voltage values. We have shown that these peaks can be gated randomly by the electrostatic environment, but the peak spectrum is reproducible among the different samples of the same molecular species that we studied. To properly study and understand the peak structure we developed the ability to add gate dependent measurements to our system. Unfortunately the backdrop of this was a drastic reduction in the yield of good samples for measurement. We focused on four different conjugated molecules to attempt to understand the effect of the molecular structure on the properties of the peak spectra. We have been able to measure three of these molecules, and obtained SET diamond plots reminiscent of those seen for the single particles. The molecular diamonds have a larger energy gap than that found in single particles, as can be expected from their smaller size. We do not yet have enough data on this issue to make any definite statements on the influence of the molecular structure on the peak structure. Another topic investigated in this work is the physics of the two gold nanoparticles, giving rise to double quantum dot (DQD) phenomena. This physics is observed in dimers that do not exhibit "molecular" (high energy) features, or at low voltages before the appearance of the molecular peaks. We have used these phenomena to fully characterize the properties of our system and understand better the role the molecule plays in transport at low bias (below the voltage of the first peak). I begin this thesis with an introduction to the field of molecular electronics; I briefly review the theoretical approaches and the experimental methods used. I then describe in detail the dimer method, whose development took up a major part of this work, relaying in detail the relevant issues and considerations. I relate briefly some early measurements done on single nanoparticles to verify our understanding of transport in the system. Moving to the description of single molecule conductance measurements I start by describing measurements demonstrating the importance of conjugation to molecular conductance. I discuss in detail the peaks appearing in the conductance measurements of the conjugated molecules describing their sample to sample variability, temporal behavior and temperature dependence. I describe the importance of gating for a comprehensive understanding of the peak spectrum and present some preliminary measurements of three different molecular species that we have been able to gate. In the final chapter of this work I describe the double quantum dot phenomena observed in the dimer system; these phenomena are not immediately relevant to the issue of single molecule conductance, but serve as a tool to characterize our system fully while providing assurance that the current goes through both nanoparticles. (Abstract shortened by UMI.)

Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

PubMed

Kim, Kyung Lock; Park, Kyeng Min; Murray, James; Kim, Kimoon; Ryu, Sung Ho

2018-05-23

Combinatorial post-translational modifications (PTMs), which can serve as dynamic "molecular barcodes", have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

Optimal Background Estimators in Single-Molecule FRET Microscopy.

PubMed

Preus, Søren; Hildebrandt, Lasse L; Birkedal, Victoria

2016-09-20

Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Förster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry

PubMed Central

2018-01-01

Combinatorial post-translational modifications (PTMs), which can serve as dynamic “molecular barcodes”, have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

From Molecules to Cells to Organisms: Understanding Health and Disease with Multidimensional Single-Cell Methods

NASA Astrophysics Data System (ADS)

Candia, Julián

2013-03-01

The multidimensional nature of many single-cell measurements (e.g. multiple markers measured simultaneously using Fluorescence-Activated Cell Sorting (FACS) technologies) offers unprecedented opportunities to unravel emergent phenomena that are governed by the cooperative action of multiple elements across different scales, from molecules and proteins to cells and organisms. We will discuss an integrated analysis framework to investigate multicolor FACS data from different perspectives: Singular Value Decomposition to achieve an effective dimensional reduction in the data representation, machine learning techniques to separate different patient classes and improve diagnosis, as well as a novel cell-similarity network analysis method to identify cell subpopulations in an unbiased manner. Besides FACS data, this framework is versatile: in this vein, we will demonstrate an application to the multidimensional single-cell shape analysis of healthy and prematurely aged cells.

Probing Biomolecular Structures and Dynamics of Single Molecules Using In-Gel Alternating-Laser Excitation

PubMed Central

Santoso, Yusdi; Kapanidis, Achillefs N.

2009-01-01

Gel electrophoresis is a standard biochemical technique used for separating biomolecules on the basis of size and charge. Despite the use of gels in early single-molecule experiments, gel electrophoresis has not been widely adopted for single-molecule fluorescence spectroscopy. We present a novel method that combines gel electrophoresis and single-molecule fluorescence spectroscopy to simultaneously purify and analyze biomolecules in a gel matrix. Our method, in-gel ALEX, uses non-denaturing gels to purify biomolecular complexes of interest from free components, aggregates, and non-specific complexes. The gel matrix also slows down translational diffusion of molecules, giving rise to long, high-resolution time traces without surface immobilization, which allow extended observations of conformational dynamics in a biologically friendly environment. We demonstrated the compatibility of this method with different types of single molecule spectroscopy techniques, including confocal detection and fluorescence-correlation spectroscopy. We demonstrated that in-gel ALEX can be used to study conformational dynamics at the millisecond timescale; by studying a DNA hairpin in gels, we directly observed fluorescence fluctuations due to conformational interconversion between folded and unfolded states. Our method is amenable to the addition of small molecules that can alter the equilibrium and dynamic properties of the system. In-gel ALEX will be a versatile tool for studying structures and dynamics of complex biomolecules and their assemblies. PMID:19863108

Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.

PubMed Central

Peck, K; Stryer, L; Glazer, A N; Mathies, R A

1989-01-01

A theory for single-molecule fluorescence detection is developed and then used to analyze data from subpicomolar solutions of B-phycoerythrin (PE). The distribution of detected counts is the convolution of a Poissonian continuous background with bursts arising from the passage of individual fluorophores through the focused laser beam. The autocorrelation function reveals single-molecule events and provides a criterion for optimizing experimental parameters. The transit time of fluorescent molecules through the 120-fl imaged volume was 800 microseconds. The optimal laser power (32 mW at 514.5 nm) gave an incident intensity of 1.8 x 10(23) photons.cm-2.s-1, corresponding to a mean time of 1.1 ns between absorptions. The mean incremental count rate was 1.5 per 100 microseconds for PE monomers and 3.0 for PE dimers above a background count rate of 1.0. The distribution of counts and the autocorrelation function for 200 fM monomer and 100 fM dimer demonstrate that single-molecule detection was achieved. At this concentration, the mean occupancy was 0.014 monomer molecules in the probed volume. A hard-wired version of this detection system was used to measure the concentration of PE down to 1 fM. This single-molecule counter is 3 orders of magnitude more sensitive than conventional fluorescence detection systems. PMID:2726766

Modified atomic force microscope applied to the measurement of elastic modulus for a single peptide molecule

NASA Astrophysics Data System (ADS)

Ptak, Arkadiusz; Takeda, Seiji; Nakamura, Chikashi; Miyake, Jun; Kageshima, Masami; Jarvis, Suzanne P.; Tokumoto, Hiroshi

2001-09-01

A modified atomic force microscopy (AFM) system, based on a force modulation technique, has been used to find an approximate value for the elastic modulus of a single peptide molecule directly from a mechanical test. For this purpose a self-assembled monolayer built from two kinds of peptides, reactive (able to anchor to the AFM tip) and nonreactive, was synthesized. In a typical experiment a single C3K30C (C=cysteine, K=lysine) peptide molecule was stretched between a Au(111) substrate and the gold-coated tip of an AFM cantilever to which it was attached via gold-sulfur bonds. The amplitude of the cantilever oscillations, due to an external force applied via a magnetic particle to the cantilever, was recorded by a lock-in amplifier and recalculated into stiffness of the stretched molecule. A longitudinal Young's modulus for the α-helix of a single peptide molecule and for the elongated state of this molecule has been estimated. The obtained values; 1.2±0.3 and 50±15 GPa, for the peptide α-helix and elongated peptide backbone, respectively, seem to be reasonable comparing them to the Young's modulus of protein crystals and linear organic polymers. We believe this research opens up a means by which scientists can perform quantitative studies of the elastic properties of single molecule, especially of biologically important polymers like peptides or DNA.

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level*

PubMed Central

Nagy, Attila; Takagi, Yasuharu; Billington, Neil; Sun, Sara A.; Hong, Davin K. T.; Homsher, Earl; Wang, Aibing; Sellers, James R.

2013-01-01

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ∼14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼20–25%) motor. The ADP release step (∼0.35 s−1) of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s−1). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (∼0.4 s−1). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ∼6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments. PMID:23148220

"Anticlumping" and Other Combinatorial Effects on Clumped Isotopes: Implications for Tracing Biogeochemical Cycling

NASA Astrophysics Data System (ADS)

Yeung, L.

2015-12-01

I present a mode of isotopic ordering that has purely combinatorial origins. It can be important when identical rare isotopes are paired by coincidence (e.g., they are neighbors on the same molecule), or when extrinsic factors govern the isotopic composition of the two atoms that share a chemical bond. By itself, combinatorial isotope pairing yields products with isotopes either randomly distributed or with a deficit relative to a random distribution of isotopes. These systematics arise because of an unconventional coupling between the formation of singly- and multiply-substituted isotopic moieties. In a random distribution, rare isotopes are symmetrically distributed: Single isotopic substitutions (e.g., H‒D and D‒H in H2) occur with equal probability, and double isotopic substitutions (e.g., D2) occur according to random chance. The absence of symmetry in a bond-making complex can yield unequal numbers of singly-substituted molecules (e.g., more H‒D than D‒H in H2), which is recorded in the product molecule as a deficit in doubly-substituted moieties and an "anticlumped" isotope distribution (i.e., Δn < 0). Enzymatic isotope pairing reactions, which can have site-specific isotopic fractionation factors and atom reservoirs, should express this class of combinatorial isotope effect. Chemical-kinetic isotope effects, which are related to the bond-forming transition state, arise independently and express second-order combinatorial effects. In general, both combinatorial and chemical factors are important for calculating and interpreting clumped-isotope signatures of individual reactions. In many reactions relevant to geochemical oxygen, carbon, and nitrogen cycling, combinatorial isotope pairing likely plays a strong role in the clumped isotope distribution of the products. These isotopic signatures, manifest as either directly bound isotope clumps or as features of a molecule's isotopic anatomy, could be exploited as tracers of biogeochemistry that can relate molecular mechanisms to signals observable at environmentally relevant spatial scales.

From genes to protein mechanics on a chip.

PubMed

Otten, Marcus; Ott, Wolfgang; Jobst, Markus A; Milles, Lukas F; Verdorfer, Tobias; Pippig, Diana A; Nash, Michael A; Gaub, Hermann E

2014-11-01

Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

The adsorption, stability and properties of Mn6Cr Single-Molecule-Magnets studied by means of nc-AFM, STM, XAS and Spin-resolved Photoelectron Spectroscopy

NASA Astrophysics Data System (ADS)

Heinzmann, U.; Gryzia, A.; Helmstedt, A.; Dohmeier, N.; Predatsch, H.; Brechling, A.; Müller, N.; Sacher, M.; Hoeke, V.; Krickemeyer, E.; Glaser, T.; Bouvron, S.; Fonin, M.; Neumann, M.

2012-11-01

The ionic single-molecule-magnet [MnIII6CrIII]3 with corresponding three counterions has been deposited on different surfaces and studied with respect to its structure and its electronic and magnetic properties. This is the first time that spin polarization of photoelectrons ejected by means of circularly polarized synchrotron radiation has been measured in a single-molecule-magnet.

Single molecules and single nanoparticles as windows to the nanoscale

NASA Astrophysics Data System (ADS)

Caldarola, Martín; Orrit, Michel

2018-05-01

Since the first optical detection of single molecules, they have been used as nanometersized optical sensors to explore the physical properties of materials and light-matter interaction at the nanoscale. Understanding nanoscale properties of materials is fundamental for the development of new technology that requires precise control of atoms and molecules when the quantum nature of matter cannot be ignored. In the following lines, we illustrate this journey into nanoscience with some experiments from our group.

Detecting Intramolecular Conformational Dynamics of Single Molecules in Short Distance Range with Sub-Nanometer Sensitivity

PubMed Central

Zhou, Ruobo; Kunzelmann, Simone; Webb, Martin R.; Ha, Taekjip

2011-01-01

Single molecule detection is useful for characterizing nanoscale objects such as biological macromolecules, nano-particles and nano-devices with nano-meter spatial resolution. Fluorescence resonance energy transfer (FRET) is widely used as a single-molecule assay to monitor intramolecular dynamics in the distance range of 3–8 nm. Here we demonstrate that self-quenching of two rhodamine derivatives can be used to detect small conformational dynamics corresponding to sub-nanometer distance changes in a FRET-insensitive short range at the single molecule level. A ParM protein mutant labeled with two rhodamines works as a single molecule ADP sensor which has 20 times brighter fluorescence signal in the ADP bound state than the unbound state. Single molecule time trajectories show discrete transitions between fluorescence on and off states that can be directly ascribed to ADP binding and dissociation events. The conformational changes observed with 20:1 contrast are only 0.5 nm in magnitude and are between crystallographic distances of 1.6 nm and 2.1 nm, demonstrating exquisite sensitivity to short distance scale changes. The systems also allowed us to gain information on the photophysics of self-quenching induced by rhodamine stacking: (1) photobleaching of either of the two rhodamines eliminates quenching of the other rhodamine fluorophore and (2) photobleaching from the highly quenched, stacked state is only two-fold slower than from the unstacked state. PMID:22023515

Single molecule dynamics at a mechanically controllable break junction in solution at room temperature.

PubMed

Konishi, Tatsuya; Kiguchi, Manabu; Takase, Mai; Nagasawa, Fumika; Nabika, Hideki; Ikeda, Katsuyoshi; Uosaki, Kohei; Ueno, Kosei; Misawa, Hiroaki; Murakoshi, Kei

2013-01-23

The in situ observation of geometrical and electronic structural dynamics of a single molecule junction is critically important in order to further progress in molecular electronics. Observations of single molecular junctions are difficult, however, because of sensitivity limits. Here, we report surface-enhanced Raman scattering (SERS) of a single 4,4'-bipyridine molecule under conditions of in situ current flow in a nanogap, by using nano-fabricated, mechanically controllable break junction (MCBJ) electrodes. When adsorbed at room temperature on metal nanoelectrodes in solution to form a single molecule junction, statistical analysis showed that nontotally symmetric b(1) and b(2) modes of 4,4'-bipyridine were strongly enhanced relative to observations of the same modes in solid or aqueous solutions. Significant changes in SERS intensity, energy (wavenumber), and selectivity of Raman vibrational bands that are coincident with current fluctuations provide information on distinct states of electronic and geometrical structure of the single molecule junction, even under large thermal fluctuations occurring at room temperature. We observed the dynamics of 4,4'-bipyridine motion between vertical and tilting configurations in the Au nanogap via b(1) and b(2) mode switching. A slight increase in the tilting angle of the molecule was also observed by noting the increase in the energies of Raman modes and the decrease in conductance of the molecular junction.

Tracking single mRNA molecules in live cells

NASA Astrophysics Data System (ADS)

Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

2016-06-01

mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

Detectors for single-molecule fluorescence imaging and spectroscopy

PubMed Central

MICHALET, X.; SIEGMUND, O.H.W.; VALLERGA, J.V.; JELINSKY, P.; MILLAUD, J.E.; WEISS, S.

2010-01-01

Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy. PMID:20157633

Coherent interaction of single molecules and plasmonic nanowires

NASA Astrophysics Data System (ADS)

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.

Isolated single-molecule magnets on native gold.

PubMed

Zobbi, Laura; Mannini, Matteo; Pacchioni, Mirko; Chastanet, Guillaume; Bonacchi, Daniele; Zanardi, Chiara; Biagi, Roberto; Del Pennino, Umberto; Gatteschi, Dante; Cornia, Andrea; Sessoli, Roberta

2005-03-28

The incorporation of thioether groups in the structure of a Mn12 single-molecule magnet, [Mn12(O12)(L)16(H2O)4] with L = 4-(methylthio)benzoate, is a successful route to the deposition of well-separated clusters on native gold surfaces and to the addressing of individual molecules by scanning tunnelling microscopy.

Single Molecule Sensing by Nanopores and Nanopore Devices

PubMed Central

Gu, Li-Qun; Shim, Ji Wook

2010-01-01

Molecular-scale pore structures, called nanopores, can be assembled by protein ion channels through genetic engineering or be artificially fabricated on solid substrates using fashion nanotechnology. When target molecules interact with the functionalized lumen of a nanopore, they characteristically block the ion pathway. The resulting conductance changes allow for identification of single molecules and quantification of target species in the mixture. In this review, we first overview nanopore-based sensory techniques that have been created for the detection of myriad biomedical targets, from metal ions, drug compounds, and cellular second messengers to proteins and DNA. Then we introduce our recent discoveries in nanopore single molecule detection: (1) using the protein nanopore to study folding/unfolding of the G-quadruplex aptamer; (2) creating a portable and durable biochip that is integrated with a single-protein pore sensor (this chip is compared with recently developed protein pore sensors based on stabilized bilayers on glass nanopore membranes and droplet interface bilayer); and (3) creating a glass nanopore-terminated probe for single-molecule DNA detection, chiral enantiomer discrimination, and identification of the bioterrorist agent ricin with an aptamer-encoded nanopore. PMID:20174694

Dual Lifetimes for Complexes between Glutathione-S-transferase (hGSTA1-1) and Product-like Ligands Detected by Single-Molecule Fluorescence Imaging.

PubMed

Pettersson, John R; Lanni, Frederick; Rule, Gordon S

2017-08-08

Single-molecule fluorescence techniques were used to characterize the binding of products and inhibitors to human glutathione S-transferase A1-1 (hGSTA1-1). The identification of at least two different bound states for the wild-type enzyme suggests that there are at least two conformations of the protein, consistent with the model that ligand binding promotes closure of the carboxy-terminal helix over the active site. Ligand induced changes in ensemble fluorescence energy transfer support this proposed structural change. The more predominant state in the ensemble of single molecules shows a significantly faster off-rate, suggesting that the carboxy-terminal helix is delocalized in this state, permitting faster exit of the bound ligand. A point mutation (I219A), which is known to interfere with the association of the carboxy-terminal helix with the enzyme, shows increased rates of interconversion between the open and closed state. Kinematic traces of fluorescence from single molecules show that a single molecule readily samples a number of different conformations, each with a characteristic off-rate.

Quantifying the assembly of multicomponent molecular machines by single-molecule total internal reflection fluorescence microscopy

PubMed Central

Boehm, Elizabeth M.; Subramanyam, Shyamal; Ghoneim, Mohamed; Washington, M. Todd; Spies, Maria

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, quaternary, etc.) complexes using multi-color 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 has become today. PMID:27793278

Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging

PubMed Central

Nishimura, Hirohito; Ritchie, Ken; Kasai, Rinshi S.; Goto, Miki; Morone, Nobuhiro; Sugimura, Hiroyuki; Tanaka, Koichiro; Sase, Ichiro; Yoshimura, Akihiko; Nakano, Yoshitaro; Fujiwara, Takahiro K.

2013-01-01

Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1–2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed. PMID:24043702

Single molecule microscopy in 3D cell cultures and tissues.

PubMed

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.

Cell biochemistry studied by single-molecule imaging.

PubMed

Mashanov, G I; Nenasheva, T A; Peckham, M; Molloy, J E

2006-11-01

Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

Low-bias negative differential conductance controlled by electrode separation

NASA Astrophysics Data System (ADS)

Yi, Xiao-Hua; Liu, Ran; Bi, Jun-Jie; Jiao, Yang; Wang, Chuan-Kui; Li, Zong-Liang

2016-12-01

The electronic transport properties of a single thiolated arylethynylene molecule with 9,10-dihydroanthracene core, denoted as TADHA, is studied by using non-equilibrium Green’s function formalism combined with ab initio calculations. The numerical results show that the TADHA molecule exhibits excellent negative differential conductance (NDC) behavior at lower bias regime as probed experimentally. The NDC behavior of TADHA molecule originates from the Stark effect of the applied bias voltage, by which the highest occupied molecular orbital (HOMO) and the HOMO-1 are pulled apart and become localized. The NDC behavior of TADHA molecular system is tunable by changing the electrode distance. Shortening the electrode separation can enhance the NDC effect which is attributed to the possible increase of coupling between the two branches of TADHA molecule. Project supported by the National Natural Science Foundation of China (Grant Nos. 11374195 and 11405098) and the Natural Science Foundation of Shandong Province, China (Grant No. ZR2013FM006).

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

PubMed

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, 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine perchlorate, used to interrogate the available free volume in their vicinity, were loaded into the mesoporous silica membranes at subnanomolar concentrations. The mesoporous silica films were prepared using a nonionic ethylene oxide-propylene oxide-ethylene oxide triblock copolymer surfactant, Pluronic P123, on single crystal silicon substrates using dip coating of a silica sol. Membranes were prepared resulting in an average pore diameter of approximately 5 nm as measured by helium, nitrogen permeance, and porosimetry. Fluorescent images and time transient experiments were recorded using a custom built single-molecule scanning confocal microscope at differing temperatures (10, 20, 30, 40, and 50 degrees C). Time-dependent polarization anisotropy was used to obtain the enthalpy of adsorption and Henry's law constant of the probe molecule.

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

NASA Astrophysics Data System (ADS)

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

2008-04-01

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, 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine perchlorate, used to interrogate the available free volume in their vicinity, were loaded into the mesoporous silica membranes at subnanomolar concentrations. The mesoporous silica films were prepared using a nonionic ethylene oxide-propylene oxide-ethylene oxide triblock copolymer surfactant, Pluronic P123, on single crystal silicon substrates using dip coating of a silica sol. Membranes were prepared resulting in an average pore diameter of approximately 5nm as measured by helium, nitrogen permeance, and porosimetry. Fluorescent images and time transient experiments were recorded using a custom built single-molecule scanning confocal microscope at differing temperatures (10, 20, 30, 40, and 50°C). Time-dependent polarization anisotropy was used to obtain the enthalpy of adsorption and Henry's law constant of the probe molecule.

Fano Description of Single-Hydrocarbon Fluorescence Excited by a Scanning Tunneling Microscope.

PubMed

Kröger, Jörg; Doppagne, Benjamin; Scheurer, Fabrice; Schull, Guillaume

2018-06-13

The detection of fluorescence with submolecular resolution enables the exploration of spatially varying photon yields and vibronic properties at the single-molecule level. By placing individual polycyclic aromatic hydrocarbon molecules into the plasmon cavity formed by the tip of a scanning tunneling microscope and a NaCl-covered Ag(111) surface, molecular light emission spectra are obtained that unravel vibrational progression. In addition, light spectra unveil a signature of the molecule even when the tunneling current is injected well separated from the molecular emitter. This signature exhibits a distance-dependent Fano profile that reflects the subtle interplay between inelastic tunneling electrons, the molecular exciton and localized plasmons in at-distance as well as on-molecule fluorescence. The presented findings open the path to luminescence of a different class of molecules than investigated before and contribute to the understanding of single-molecule luminescence at surfaces in a unified picture.

Multiplexed single-molecule force spectroscopy using a centrifuge.

PubMed

Yang, Darren; Ward, Andrew; Halvorsen, Ken; Wong, Wesley P

2016-03-17

We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.

Multiplexed single-molecule force spectroscopy using a centrifuge

PubMed Central

Yang, Darren; Ward, Andrew; Halvorsen, Ken; Wong, Wesley P.

2016-01-01

We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise. PMID:26984516

Thermal deposition of intact tetrairon(III) single-molecule magnets in high-vacuum conditions.

PubMed

Margheriti, Ludovica; Mannini, Matteo; Sorace, Lorenzo; Gorini, Lapo; Gatteschi, Dante; Caneschi, Andrea; Chiappe, Daniele; Moroni, Riccardo; de Mongeot, Francesco Buatier; Cornia, Andrea; Piras, Federica M; Magnani, Agnese; Sessoli, Roberta

2009-06-01

A tetrairon(III) single-molecule magnet is deposited using a thermal evaporation technique in high vacuum. The chemical integrity is demonstrated by time-of-flight secondary ion mass spectrometry on a film deposited on Al foil, while superconducting quantum interference device magnetometry and alternating current susceptometry of a film deposited on a kapton substrate show magnetic properties identical to the pristine powder. High-frequency electron paramagnetic resonance spectra confirm the characteristic behavior for a system with S = 5 and a large Ising-type magnetic anisotropy. All these results indicate that the molecules are not damaged during the deposition procedure keeping intact the single-molecule magnet behavior.

Unraveling secrets of telomeres: one molecule at a time

PubMed Central

Lin, Jiangguo; Kaur, Parminder; Countryman, Preston; Opresko, Patricia L.; Wang, Hong

2016-01-01

Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins. PMID:24569170

Fluorescence Microscopy of Single Molecules

ERIC Educational Resources Information Center

Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

2004-01-01

The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

Basic concepts of quantum interference and electron transport in single-molecule electronics.

PubMed

Lambert, C J

2015-02-21

This tutorial outlines the basic theoretical concepts and tools which underpin the fundamentals of phase-coherent electron transport through single molecules. The key quantity of interest is the transmission coefficient T(E), which yields the electrical conductance, current-voltage relations, the thermopower S and the thermoelectric figure of merit ZT of single-molecule devices. Since T(E) is strongly affected by quantum interference (QI), three manifestations of QI in single-molecules are discussed, namely Mach-Zehnder interferometry, Breit-Wigner resonances and Fano resonances. A simple MATLAB code is provided, which allows the novice reader to explore QI in multi-branched structures described by a tight-binding (Hückel) Hamiltonian. More generally, the strengths and limitations of materials-specific transport modelling based on density functional theory are discussed.

Single-molecule spectroscopic methods.

PubMed

Haustein, Elke; Schwille, Petra

2004-10-01

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms. Copyright 2004 Elsevier Ltd.

Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time

PubMed Central

Hohng, Sungchul

2010-01-01

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. PMID:20808851

Single Molecule Study of Metalloregulatory Protein-DNA Interactions

NASA Astrophysics Data System (ADS)

Sarkar, Susanta; Benitez, Jaime; Huang, Zhengxi; Wang, Qi; Chen, Peng

2007-03-01

Control of metal concentrations is essential for living body. Metalloregulatory proteins respond to metal concentrations by regulating transcriptions of metal resistance genes via protein-DNA interactions. It is thus necessary to understand interactions of metalloregulatory proteins with DNA. Ensemble measurements provide average behavior of a vast number of biomolecules. In contrast, single molecule spectroscopy can track single molecules individually and elucidate dynamics of processes of short time scales and intermediate structures not revealed by ensemble measurements. Here we present single molecule study of interactions between PbrR691, a MerR-family metalloregulatory protein and DNA. We presume that the dynamics of protein/DNA conformational changes and interactions are important for the transcription regulation and kinetics of these dynamic processes can provide useful information about the mechanisms of these metalloregulatory proteins.

The sensing mechanism of N-doped SWCNTs toward SF6 decomposition products: A first-principle study

NASA Astrophysics Data System (ADS)

Gui, Yingang; Tang, Chao; Zhou, Qu; Xu, Lingna; Zhao, Zhongyong; Zhang, Xiaoxing

2018-05-01

In order to monitor the insulation status of SF6-insulated equipment on-line, SOF2 and SO2F2, two typical decomposition products of SF6 under electric discharge condition, are chosen as the target gases to evaluate the type and severity of discharge. In this work, single N atom doping method is adopted to improve the gas sensitivity of single wall carbon nanotubes to SOF2 and SO2F2. Single and double gas molecules adsorptions are considered to completely analyze the adsorption properties of N-doped single wall carbon nanotubes. Calculation results show that N atom doping enhances the surface activity of carbon nanotubes. When gas molecules physically adsorbed on N-doped single wall carbon nanotubes, the weak interaction between gas molecules and N-doped single wall carbon nanotubes nearly not changes the electrical property according to analysis of the density of states and molecular orbitals. While the chemisorption between gas molecules and N-doped single wall carbon nanotubes distinctly decreases the conductivity of adsorption system.

Enhanced post wash retention of combed DNA molecules by varying multiple combing parameters.

PubMed

Yadav, Hemendra; Sharma, Pulkit

2017-11-01

Recent advances in genomics have created a need for efficient techniques for deciphering information hidden in various genomes. Single molecule analysis is one such technique to understand molecular processes at single molecule level. Fiber- FISH performed with the help of DNA combing can help us in understanding genetic rearrangements and changes in genome at single DNA molecule level. For performing Fiber-FISH we need high retention of combed DNA molecules post wash as Fiber-FISH requires profuse washing. We optimized combing process involving combing solution, method of DNA mounting on glass slides and coating of glass slides to enhance post-wash retention of DNA molecules. It was found that average number of DNA molecules observed post-wash per field of view was maximum with our optimized combing solution. APTES coated glass slides showed lesser retention than PEI surface but fluorescent intensity was higher in case of APTES coated surface. Capillary method used to mount DNA on glass slides also showed lesser retention but straight DNA molecules were observed as compared to force flow method. Copyright © 2017 Elsevier Inc. All rights reserved.

Spin squeezing a cold molecule

NASA Astrophysics Data System (ADS)

Bhattacharya, M.

2015-12-01

In this article we present a concrete proposal for spin squeezing the cold ground-state polar paramagnetic molecule OH, a system currently under fine control in the laboratory. In contrast to existing work, we consider a single, noninteracting molecule with angular momentum greater than 1 /2 . Starting from an experimentally relevant effective Hamiltonian, we identify an adiabatic regime where different combinations of static electric and magnetic fields can be used to realize the single-axis twisting Hamiltonian of Kitagawa and Ueda [M. Kitagawa and M. Ueda, Phys. Rev. A 47, 5138 (1993), 10.1103/PhysRevA.47.5138], the uniform field Hamiltonian proposed by Law et al. [C. K. Law, H. T. Ng, and P. T. Leung, Phys. Rev. A 63, 055601 (2001), 10.1103/PhysRevA.63.055601], and a model of field propagation in a Kerr medium considered by Agarwal and Puri [G. S. Agarwal and R. R. Puri, Phys. Rev. A 39, 2969 (1989), 10.1103/PhysRevA.39.2969]. We then consider the situation in which nonadiabatic effects are quite large and show that the effective Hamiltonian supports spin squeezing even in this case. We provide analytical expressions as well as numerical calculations, including optimization of field strengths and accounting for the effects of field misalignment. Our results have consequences for applications such as precision spectroscopy, techniques such as magnetometry, and stereochemical effects such as the orientation-to-alignment transition.

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

PubMed Central

Yang, Weidong; Musser, Siegfried M.

2008-01-01

The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells. PMID:16879979

Using a nanopore for single molecule detection and single cell transfection.

PubMed

Nelson, Edward M; Kurz, Volker; Shim, Jiwook; Timp, Winston; Timp, Gregory

2012-07-07

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

Modeling the effect of glutamate diffusion and uptake on NMDA and non-NMDA receptor saturation.

PubMed Central

Holmes, W R

1995-01-01

One- and two-dimensional models of glutamate diffusion, uptake, and binding in the synaptic cleft were developed to determine if the release of single vesicles of glutamate would saturate NMDA and non-NMDA receptors. Ranges of parameter values were used in the simulations to determine the conditions when saturation could occur. Single vesicles of glutamate did not saturate NMDA receptors unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. However, the release of eight vesicles at 400 Hz caused NMDA receptor saturation for all parameter values tested. Glutamate uptake was found to reduce NMDA receptor saturation, but the effect was smaller than that of changes in the diffusion coefficient or in the number of glutamate molecules in a vesicle. Non-NMDA receptors were not saturated unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. The release of eight vesicles at 400 Hz caused significant non-NMDA receptor desensitization. The results suggest that NMDA and non-NMDA receptors are not saturated by single vesicles of glutamate under usual conditions, and that tetanic input, of the type typically used to induce long-term potentiation, will increase calcium influx by increasing receptor binding as well as by reducing voltage-dependent block of NMDA receptors. Images FIGURE 1 PMID:8580317

Preface: Charge transport in nanoscale junctions

NASA Astrophysics Data System (ADS)

Albrecht, Tim; Kornyshev, Alexei; Bjørnholm, Thomas

2008-09-01

Understanding the fundamentals of nanoscale charge transfer is pivotal for designing future nano-electronic devices. Such devices could be based on individual or groups of molecular bridges, nanotubes, nanoparticles, biomolecules and other 'active' components, mimicking wire, diode and transistor functions. These have operated in various environments including vacuum, air and condensed matter, in two- or three-electrode configurations, at ultra-low and room temperatures. Interest in charge transport in ultra-small device components has a long history and can be dated back to Aviram and Ratner's letter in 1974 (Chem. Phys. Lett. 29 277-83). So why is there a necessity for a special issue on this subject? The area has reached some degree of maturity, and even subtle geometric effects in the nanojunction and noise features can now be resolved and rationalized based on existing theoretical concepts. One purpose of this special issue is thus to showcase various aspects of nanoscale and single-molecule charge transport from experimental and theoretical perspectives. The main principles have 'crystallized' in our minds, but there is still a long way to go before true single-molecule electronics can be implemented. Major obstacles include the stability of electronic nanojunctions, reliable operation at room temperature, speed of operation and, last but not least, integration into large networks. A gradual transition from traditional silicon-based electronics to devices involving a single (or a few) molecule(s) therefore appears to be more viable from technologic and economic perspectives than a 'quantum leap'. As research in this area progresses, new applications emerge, e.g. with a view to characterizing interfacial charge transfer at the single-molecule level in general. For example, electrochemical experiments with individual enzyme molecules demonstrate that catalytic processes can be studied with nanometre resolution, offering a route towards optimizing biosensors at the molecular level. Nanoscale charge transport experiments in ionic liquids extend the field to high temperatures and to systems with intriguing interfacial potential distributions. Other directions may include dye-sensitized solar cells, new sensor applications and diagnostic tools for the study of surface-bound single molecules. Another motivation for this special issue is thus to highlight activities across different research communities with nanoscale charge transport as a common denominator. This special issue gathers 27 articles by scientists from the United States, Germany, the UK, Denmark, Russia, France, Israel, Canada, Australia, Sweden, Switzerland, the Netherlands, Belgium and Singapore; it gives us a flavour of the current state-of-the-art of this diverse research area. While based on contributions from many renowned groups and institutions, it obviously cannot claim to represent all groups active in this very broad area. Moreover, a number of world-leading groups were unable to take part in this project within the allocated time limit. Nevertheless, we regard the current selection of papers to be representative enough for the reader to draw their own conclusions about the current status of the field. Each paper is original and has its own merit, as all papers in Journal of Physics: Condensed Matter special issues are subjected to the same scrutiny as regular contributions. The Guest Editors have deliberately not defined the specific subjects covered in this issue. These came out logically from the development of this area, for example: 'Traditional' solid state nanojunctions based on adsorbed layers, oxide films or nanowires sandwiched between two electrodes: effects of molecular structure (aromaticity, anchoring groups), symmetry, orientation, dynamics (noise patterns) and current-induced heating. Various 'physical effects': inelastic tunnelling and Coulomb blockade, polaron effects, switching modes, and negative differential resistance; the role of many particle excitations, new surface states in semiconductor electrodes, various mechanisms for single molecule rectification of the current, inelastic electron spectra and SERS spectroscopy. Three terminal architectures allowing (electrochemical) gating and transistor effects. Electrochemical nanojunctions and gating: intermolecular electron transfer in multi-redox metalloproteins, contact force modulation, characteristic current-noise patterns due to conformational fluctuations, resonance effects and electrocatalysis. Novel architectures: linear coupled quantum-dot-bridged junctions, electrochemical redox mediated transfer in two center systems leading to double maxima current-voltage plots and negative differential resistance, molecular-nanoparticle hybrid junctions and unexpected mesoscopic effects in polymeric wires. Device integration: techniques for creating stable metal/molecule/metal junctions using 'nano-alligator clips' and integration with 'traditional' silicon-based technology. The Guest Editors would like to thank all of the authors and referees of this special issue for their meticulous work in making each paper a valuable contribution to this research area, the early-bird authors for their patience, and Journal of Physics: Condensed Matter editorial staff in Bristol for their continuous support.

Charge transport in nanoscale junctions.

PubMed

Albrecht, Tim; Kornyshev, Alexei; Bjørnholm, Thomas

2008-09-03

Understanding the fundamentals of nanoscale charge transfer is pivotal for designing future nano-electronic devices. Such devices could be based on individual or groups of molecular bridges, nanotubes, nanoparticles, biomolecules and other 'active' components, mimicking wire, diode and transistor functions. These have operated in various environments including vacuum, air and condensed matter, in two- or three-electrode configurations, at ultra-low and room temperatures. Interest in charge transport in ultra-small device components has a long history and can be dated back to Aviram and Ratner's letter in 1974 (Chem. Phys. Lett. 29 277-83). So why is there a necessity for a special issue on this subject? The area has reached some degree of maturity, and even subtle geometric effects in the nanojunction and noise features can now be resolved and rationalized based on existing theoretical concepts. One purpose of this special issue is thus to showcase various aspects of nanoscale and single-molecule charge transport from experimental and theoretical perspectives. The main principles have 'crystallized' in our minds, but there is still a long way to go before true single-molecule electronics can be implemented. Major obstacles include the stability of electronic nanojunctions, reliable operation at room temperature, speed of operation and, last but not least, integration into large networks. A gradual transition from traditional silicon-based electronics to devices involving a single (or a few) molecule(s) therefore appears to be more viable from technologic and economic perspectives than a 'quantum leap'. As research in this area progresses, new applications emerge, e.g. with a view to characterizing interfacial charge transfer at the single-molecule level in general. For example, electrochemical experiments with individual enzyme molecules demonstrate that catalytic processes can be studied with nanometre resolution, offering a route towards optimizing biosensors at the molecular level. Nanoscale charge transport experiments in ionic liquids extend the field to high temperatures and to systems with intriguing interfacial potential distributions. Other directions may include dye-sensitized solar cells, new sensor applications and diagnostic tools for the study of surface-bound single molecules. Another motivation for this special issue is thus to highlight activities across different research communities with nanoscale charge transport as a common denominator. This special issue gathers 27 articles by scientists from the United States, Germany, the UK, Denmark, Russia, France, Israel, Canada, Australia, Sweden, Switzerland, the Netherlands, Belgium and Singapore; it gives us a flavour of the current state-of-the-art of this diverse research area. While based on contributions from many renowned groups and institutions, it obviously cannot claim to represent all groups active in this very broad area. Moreover, a number of world-leading groups were unable to take part in this project within the allocated time limit. Nevertheless, we regard the current selection of papers to be representative enough for the reader to draw their own conclusions about the current status of the field. Each paper is original and has its own merit, as all papers in Journal of Physics: Condensed Matter special issues are subjected to the same scrutiny as regular contributions. The Guest Editors have deliberately not defined the specific subjects covered in this issue. These came out logically from the development of this area, for example: 'Traditional' solid state nanojunctions based on adsorbed layers, oxide films or nanowires sandwiched between two electrodes: effects of molecular structure (aromaticity, anchoring groups), symmetry, orientation, dynamics (noise patterns) and current-induced heating. Various 'physical effects': inelastic tunnelling and Coulomb blockade, polaron effects, switching modes, and negative differential resistance; the role of many particle excitations, new surface states in semiconductor electrodes, various mechanisms for single molecule rectification of the current, inelastic electron spectra and SERS spectroscopy. Three terminal architectures allowing (electrochemical) gating and transistor effects. Electrochemical nanojunctions and gating: intermolecular electron transfer in multi-redox metalloproteins, contact force modulation, characteristic current-noise patterns due to conformational fluctuations, resonance effects and electrocatalysis. Novel architectures: linear coupled quantum-dot-bridged junctions, electrochemical redox mediated transfer in two center systems leading to double maxima current-voltage plots and negative differential resistance, molecular-nanoparticle hybrid junctions and unexpected mesoscopic effects in polymeric wires. Device integration: techniques for creating stable metal/molecule/metal junctions using 'nano-alligator clips' and integration with 'traditional' silicon-based technology. The Guest Editors would like to thank all of the authors and referees of this special issue for their meticulous work in making each paper a valuable contribution to this research area, the early-bird authors for their patience, and Journal of Physics: Condensed Matter editorial staff in Bristol for their continuous support.

Raman spectroelectrochemistry of molecules within individual electromagnetic hot spots.

PubMed

Shegai, Timur; Vaskevich, Alexander; Rubinstein, Israel; Haran, Gilad

2009-10-14

The role of chemical enhancement in surface-enhanced Raman scattering (SERS) remains a contested subject. We study SERS spectra of 4-mercaptopyridine molecules excited far from the molecular resonance, which are collected from individual electromagnetic hot spots at concentrations close to the single-molecule limit. The hot spots are created by depositing Tollen's silver island films on a transparent electrode incorporated within an electrochemical cell. Analysis of the intensity of the spectra relative to those obtained from individual rhodamine 6G molecules on the same surface provides a lower limit of approximately 3 orders of magnitude for the chemical enhancement. This large enhancement is likely to be due to a charge transfer resonance involving the transfer of an electron from the metal to an adsorbed molecule. Excitation at three different wavelengths, as well as variation of electrode potential from 0 to -1.2 V, lead to significant changes in the relative intensities of bands in the spectrum. It is suggested that while the bulk of the enhancement is due to an Albrecht A-term resonance Raman effect (involving the charge transfer transition), vibronic coupling provides additional enhancement which is sensitive to electrode potential. The measurement of potential-dependent SERS spectra from individual hot spots opens the way to a thorough characterization of chemical enhancement, as well to studies of redox phenomena at the single-molecule level.

Probing Electronic and Thermoelectric Properties of Single Molecule Junctions

NASA Astrophysics Data System (ADS)

Widawsky, Jonathan R.

In an effort to further understand electronic and thermoelectric phenomenon at the nanometer scale, we have studied the transport properties of single molecule junctions. To carry out these transport measurements, we use the scanning tunneling microscope-break junction (STM-BJ) technique, which involves the repeated formation and breakage of a metal point contact in an environment of the target molecule. Using this technique, we are able to create gaps that can trap the molecules, allowing us to sequentially and reproducibly create a large number of junctions. By applying a small bias across the junction, we can measure its conductance and learn about the transport mechanisms at the nanoscale. The experimental work presented here directly probes the transmission properties of single molecules through the systematic measurement of junction conductance (at low and high bias) and thermopower. We present measurements on a variety of molecular families and study how conductance depends on the character of the linkage (metal-molecule bond) and the nature of the molecular backbone. We start by describing a novel way to construct single molecule junctions by covalently connecting the molecular backbone to the electrodes. This eliminates the use of linking substituents, and as a result, the junction conductance increases substantially. Then, we compare transport across silicon chains (silanes) and saturated carbon chains (alkanes) while keeping the linkers the same and find a stark difference in their electronic transport properties. We extend our studies of molecular junctions by looking at two additional aspects of quantum transport -- molecular thermopower and molecular current-voltage characteristics. Each of these additional parameters gives us further insight into transport properties at the nanoscale. Evaluating the junction thermopower allows us to determine the nature of charge carriers in the system and we demonstrate this by contrasting the measurement of amine-terminated and pyridine-terminated molecules (which exhibit hole transport and electron transport, respectively). We also report the thermopower of the highly conducting, covalently bound molecular junctions that we have recently been able to form, and learn that, because of their unique transport properties, the junction power factors, GS2, are extremely high. Finally, we discuss the measurement of molecular current-voltage curves and consider the electronic and physical effects of applying a large bias to the system. We conclude with a summary of the work discussed and an outlook on related scientific studies.

Rapid ultrasensitive single particle surface-enhanced Raman spectroscopy using metallic nanopores.

PubMed

Cecchini, Michael P; Wiener, Aeneas; Turek, Vladimir A; Chon, Hyangh; Lee, Sangyeop; Ivanov, Aleksandar P; McComb, David W; Choo, Jaebum; Albrecht, Tim; Maier, Stefan A; Edel, Joshua B

2013-10-09

Nanopore sensors embedded within thin dielectric membranes have been gaining significant interest due to their single molecule sensitivity and compatibility of detecting a large range of analytes, from DNA and proteins, to small molecules and particles. Building on this concept we utilize a metallic Au solid-state membrane to translocate and rapidly detect single Au nanoparticles (NPs) functionalized with 589 dye molecules using surface-enhanced resonance Raman spectroscopy (SERRS). We show that, due to the plasmonic coupling between the Au metallic nanopore surface and the NP, signal intensities are enhanced when probing analyte molecules bound to the NP surface. Although not single molecule, this nanopore sensing scheme benefits from the ability of SERRS to provide rich vibrational information on the analyte, improving on current nanopore-based electrical and optical detection techniques. We show that the full vibrational spectrum of the analyte can be detected with ultrahigh spectral sensitivity and a rapid temporal resolution of 880 μs.

Wafer-scale metasurface for total power absorption, local field enhancement and single molecule Raman spectroscopy

PubMed Central

Wang, Dongxing; Zhu, Wenqi; Best, Michael D.; Camden, Jon P.; Crozier, Kenneth B.

2013-01-01

The ability to detect molecules at low concentrations is highly desired for applications that range from basic science to healthcare. Considerable interest also exists for ultrathin materials with high optical absorption, e.g. for microbolometers and thermal emitters. Metal nanostructures present opportunities to achieve both purposes. Metal nanoparticles can generate gigantic field enhancements, sufficient for the Raman spectroscopy of single molecules. Thin layers containing metal nanostructures (“metasurfaces”) can achieve near-total power absorption at visible and near-infrared wavelengths. Thus far, however, both aims (i.e. single molecule Raman and total power absorption) have only been achieved using metal nanostructures produced by techniques (high resolution lithography or colloidal synthesis) that are complex and/or difficult to implement over large areas. Here, we demonstrate a metasurface that achieves the near-perfect absorption of visible-wavelength light and enables the Raman spectroscopy of single molecules. Our metasurface is fabricated using thin film depositions, and is of unprecedented (wafer-scale) extent. PMID:24091825

Single Molecule Visualization of Protein-DNA Complexes: Watching Machines at Work

NASA Astrophysics Data System (ADS)

Kowalczykowski, Stephen

2013-03-01

We can now watch individual proteins acting on single molecules of DNA. Such imaging provides unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the application of two complementary procedures. In one, single DNA molecules are attached to a polystyrene bead and are then captured by an optical trap. The DNA, a worm-like coil, is extended either by the force of solution flow in a micro-fabricated channel, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Protein action is visualized by fluorescent reporters: fluorescent dyes that bind double-stranded DNA (dsDNA), fluorescent biosensors for single-stranded DNA (ssDNA), or fluorescently-tagged proteins. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Using these approaches, we imaged the search for DNA sequence homology conducted by the RecA-ssDNA filament. The manner by which RecA protein finds a single homologous sequence in the genome had remained undefined for almost 30 years. Single-molecule imaging revealed that the search occurs through a mechanism termed ``intersegmental contact sampling,'' in which the randomly coiled structure of DNA is essential for reiterative sampling of DNA sequence identity: an example of parallel processing. In addition, the assembly of RecA filaments on single molecules of single-stranded DNA was visualized. Filament assembly requires nucleation of a protein dimer on DNA, and subsequent growth occurs via monomer addition. Furthermore, we discovered a class of proteins that catalyzed both nucleation and growth of filaments, revealing how the cell controls assembly of this protein-DNA complex.

Random walk on a leash: a simple single-molecule diffusion model for surface-tethered redox molecules with flexible linkers.

PubMed

Huang, Kuan-Chun; White, Ryan J

2013-08-28

We develop a random walk model to simulate the Brownian motion and the electrochemical response of a single molecule confined to an electrode surface via a flexible molecular tether. We use our simple model, which requires no prior knowledge of the physics of the molecular tether, to predict and better understand the voltammetric response of surface-confined redox molecules when motion of the redox molecule becomes important. The single molecule is confined to a hemispherical volume with a maximum radius determined by the flexible molecular tether (5-20 nm) and is allowed to undergo true three-dimensional diffusion. Distance- and potential-dependent electron transfer probabilities are evaluated throughout the simulations to generate cyclic voltammograms of the model system. We find that at sufficiently slow cyclic voltammetric scan rates the electrochemical reaction behaves like an adsorbed redox molecule with no mass transfer limitation; thus, the peak current is proportional to the scan rate. Conversely, at faster scan rates the diffusional motion of the molecule limits the simulated peak current, which exhibits a linear dependence on the square root of the scan rate. The switch between these two limiting regimes occurs when the diffusion layer thickness, (2Dt)(1/2), is ~10 times the tether length. Finally, we find that our model predicts the voltammetric behavior of a redox-active methylene blue tethered to an electrode surface via short flexible single-stranded, polythymine DNAs, allowing the estimation of diffusion coefficients for the end-tethered molecule.

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

PubMed Central

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

Exploring the folding pattern of a polymer chain in a single crystal by combining single-molecule force spectroscopy and steered molecular dynamics simulations.

PubMed

Song, Yu; Feng, Wei; Liu, Kai; Yang, Peng; Zhang, Wenke; Zhang, Xi

2013-03-26

Understanding the folding pattern of a single polymer chain within its single crystal will shed light on the mechanism of crystallization. Here, we use the combined techniques of atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations to study the folding pattern of a polyethylene oxide (PEO) chain in its single crystal. Our results show that the folding pattern of a PEO chain in the crystal formed in dilute solution follows the adjacent re-entry folding model. While in the crystal obtained from the melt, the nonadjacent folding with large and irregular loops contributes to big force fluctuations in the force-extension curves. The method established here can offer a novel strategy to directly unravel the chain-folding pattern of polymer single crystals at single-molecule level.

Imaging mRNA In Vivo, from Birth to Death.

PubMed

Tutucci, Evelina; Livingston, Nathan M; Singer, Robert H; Wu, Bin

2018-05-20

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

Combining nanofluidics and plasmonics for single molecule detection

NASA Astrophysics Data System (ADS)

West, Melanie M.

Single molecule detection is limited by the small scattering cross-section of molecules which leads to weak optical signals that can be obscured by background noise. The combination of plasmonics and nanofluidics in an integrated nano-device has the potential to provide the signal enhancement necessary for the detection of single molecules. The purpose of this investigation was to optimize the fabrication of an optofluidic device that integrates a nanochannel with a plasmonic bowtie antenna. The fluidic structure of the device was fabricated using UV-nanoimprint lithography, and the gold plasmonic antennas were fabricated using a shadow evaporation and lift-off process. The effect of electron beam lithography doses on the resolution of antenna-nanochannel configurations was studied to minimize antenna gap size while maintaining the integrity of the imprinted features. The smallest antenna gap size that was achieved was 46 nm. The antennas were characterized using dark field spectroscopy to find the resonance shift, which indicated the appropriate range for optical signal enhancement. The dark field scattering results showed antennas with a broad and well-defined resonance shift that ranged from 650--800 nm. The Raman scattering results showed the highest enhancement factor (EF = 2) for antennas with an "inverted configuration," which involved having the triangles of the antenna facing back-to-back rather than the more conventional tip-to-tip bowtie arrangement.

Identification of vibrational signatures from short chains of interlinked molecule-nanoparticle junctions obtained by inelastic electron tunnelling spectroscopy

NASA Astrophysics Data System (ADS)

Jafri, S. H. M.; Löfås, H.; Fransson, J.; Blom, T.; Grigoriev, A.; Wallner, A.; Ahuja, R.; Ottosson, H.; Leifer, K.

2013-05-01

Short chains containing a series of metal-molecule-nanoparticle nanojunctions are a nano-material system with the potential to give electrical signatures close to those from single molecule experiments while enabling us to build portable devices on a chip. Inelastic electron tunnelling spectroscopy (IETS) measurements provide one of the most characteristic electrical signals of single and few molecules. In interlinked molecule-nanoparticle (NP) chains containing typically 5-7 molecules in a chain, the spectrum is expected to be a superposition of the vibrational signatures of individual molecules. We have established a stable and reproducible molecule-AuNP multi-junction by placing a few 1,8-octanedithiol (ODT) molecules onto a versatile and portable nanoparticle-nanoelectrode platform and measured for the first time vibrational molecular signatures at complex and coupled few-molecule-NP junctions. From quantum transport calculations, we model the IETS spectra and identify vibrational modes as well as the number of molecules contributing to the electron transport in the measured spectra.Short chains containing a series of metal-molecule-nanoparticle nanojunctions are a nano-material system with the potential to give electrical signatures close to those from single molecule experiments while enabling us to build portable devices on a chip. Inelastic electron tunnelling spectroscopy (IETS) measurements provide one of the most characteristic electrical signals of single and few molecules. In interlinked molecule-nanoparticle (NP) chains containing typically 5-7 molecules in a chain, the spectrum is expected to be a superposition of the vibrational signatures of individual molecules. We have established a stable and reproducible molecule-AuNP multi-junction by placing a few 1,8-octanedithiol (ODT) molecules onto a versatile and portable nanoparticle-nanoelectrode platform and measured for the first time vibrational molecular signatures at complex and coupled few-molecule-NP junctions. From quantum transport calculations, we model the IETS spectra and identify vibrational modes as well as the number of molecules contributing to the electron transport in the measured spectra. Electronic supplementary information (ESI) available: Methods and materials. Details of the ab initio calculation of molecular vibrations and inelastic spectra of ODT between two Au electrodes. A model of carrier transport through the molecular junctions. See DOI: 10.1039/c3nr00505d

Proposed alteration of images of molecular orbitals obtained using a scanning tunneling microscope as a probe of electron correlation.

PubMed

Toroz, Dimitrios; Rontani, Massimo; Corni, Stefano

2013-01-04

Scanning tunneling spectroscopy (STS) allows us to image single molecules decoupled from the supporting substrate. The obtained images are routinely interpreted as the square moduli of molecular orbitals, dressed by the mean-field electron-electron interaction. Here we demonstrate that the effect of electron correlation beyond the mean field qualitatively alters the uncorrelated STS images. Our evidence is based on the ab initio many-body calculation of STS images of planar molecules with metal centers. We find that many-body correlations alter significantly the image spectral weight close to the metal center of the molecules. This change is large enough to be accessed experimentally, surviving to molecule-substrate interactions.

Distinguishing between protein dynamics and dye photophysics in single-molecule FRET experiments.

PubMed

Chung, Hoi Sung; Louis, John M; Eaton, William A

2010-02-17

Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small alpha/beta protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers-donor(10)/acceptor(57) and donor(57)/acceptor(10)-which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol-coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects-a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single molecule experimentation in biological physics: exploring the living component of soft condensed matter one molecule at a time.

PubMed

Harriman, O L J; Leake, M C

2011-12-21

The soft matter of biological systems consists of mesoscopic length scale building blocks, composed of a variety of different types of biological molecules. Most single biological molecules are so small that 1 billion would fit on the full-stop at the end of this sentence, but collectively they carry out the vital activities in living cells whose length scale is at least three orders of magnitude greater. Typically, the number of molecules involved in any given cellular process at any one time is relatively small, and so real physiological events may often be dominated by stochastics and fluctuation behaviour at levels comparable to thermal noise, and are generally heterogeneous in nature. This challenging combination of heterogeneity and stochasticity is best investigated experimentally at the level of single molecules, as opposed to more conventional bulk ensemble-average techniques. In recent years, the use of such molecular experimental approaches has become significantly more widespread in research laboratories around the world. In this review we discuss recent experimental approaches in biological physics which can be applied to investigate the living component of soft condensed matter to a precision of a single molecule. © 2011 IOP Publishing Ltd Printed in the UK & the USA

Enhancement of Raman scattering signal of a few molecules using photonic nanojet mediated SERS technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, G. M.; Parit, M. K.; Laha, R.

2016-05-06

Now a days, single molecule surface enhanced Raman spectroscopy (SMSERS) has become a fascinating tool for studying the structural properties, static and dynamic events of single molecules (instead of ensemble average), with the help of efficient plasmonic nanostructures. This is extremely useful in the field of proteomics because the structural properties of protein molecules are heterogeneous. Even though, SMSERS provides wealthy information about single molecules, it demands high quality surface enhanced Raman scattering (SERS) substrates. So far, a very few researchers succeeded in demonstrating the single molecule Raman scattering using conventional SERS technique. However, the experimental S/N of the Ramanmore » signal has been found to be very poor. Recently, with the help of photonic nanojet of an optical microsphere, we were able to enhance the SERS signal of a few molecules adsorbed on the SERS substrates (gold symmetric and asymmetric nanodimers and trimers dispersed on a glass slide). Herein, we report a few details about photonic nanojet mediated SERS technique, a few experimental results and a detailed theoretical study on symmetric and asymmetric nanosphere dimers to understand the dependence of localised surface plasmon resonance (LSPR) wavelength of a nanodimer on the nanogap size and polarization of the excitation light.« less

Ab initio studies of electronic transport through amine-Au-linked junctions of photoactive molecules

NASA Astrophysics Data System (ADS)

Strubbe, David A.; Quek, Su Ying; Venkataraman, Latha; Choi, Hyoung Joon; Neaton, J. B.; Louie, Steven G.

2008-03-01

Molecules linked to Au electrodes via amine groups have been shown to result in reproducible molecular conductance values for a wide range of single-molecule junctions [1,2]. Recent calculations have shown that these linkages result in a junction conductance relatively insensitive to atomic structure [3]. Here we exploit these well-defined linkages to study the effect of isomerization on conductance for the photoactive molecule 4,4'-diaminoazobenzene. We use a first-principles scattering-state method based on density-functional theory to explore structure and transport properties of the cis and trans isomers of the molecule, and we discuss implications for experiment. [1] L Venkataraman et al., Nature 442, 904-907 (2006); [2] L Venkataraman et al., Nano Lett. 6, 458-462 (2006); [3] SY Quek et al., Nano Lett. 7, 3477-3482 (2007).

Optical Manipulation of Single Magnetic Beads in a Microwell Array on a Digital Microfluidic Chip.

PubMed

Decrop, Deborah; Brans, Toon; Gijsenbergh, Pieter; Lu, Jiadi; Spasic, Dragana; Kokalj, Tadej; Beunis, Filip; Goos, Peter; Puers, Robert; Lammertyn, Jeroen

2016-09-06

The detection of single molecules in magnetic microbead microwell array formats revolutionized the development of digital bioassays. However, retrieval of individual magnetic beads from these arrays has not been realized until now despite having great potential for studying captured targets at the individual level. In this paper, optical tweezers were implemented on a digital microfluidic platform for accurate manipulation of single magnetic beads seeded in a microwell array. Successful optical trapping of magnetic beads was found to be dependent on Brownian motion of the beads, suggesting a 99% chance of trapping a vibrating bead. A tailor-made experimental design was used to screen the effect of bead type, ionic buffer strength, surfactant type, and concentration on the Brownian activity of beads in microwells. With the optimal conditions, the manipulation of magnetic beads was demonstrated by their trapping, retrieving, transporting, and repositioning to a desired microwell on the array. The presented platform combines the strengths of digital microfluidics, digital bioassays, and optical tweezers, resulting in a powerful dynamic microwell array system for single molecule and single cell studies.

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

PubMed

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

2018-01-10

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-5 nm 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 ≥4 × 10 3 with high quantum yield (≥50%). 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 ±1.5 nm. Our approach introduces a straightforward noninvasive way to measure and quantify confined optical modes on the nanoscale.

Orientation and Rotational Motions of Single Molecules by Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM)

PubMed Central

Beausang, John F.; Sun, Yujie; Quinlan, Margot E.; Forkey, Joseph N.; Goldman, Yale E.

2013-01-01

In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin. PMID:22550303

Adding remnant magnetization and anisotropic exchange to propeller-like single-molecule magnets through chemical design.

PubMed

Westrup, Kátia Cristina M; Boulon, Marie-Emmanuelle; Totaro, Pasquale; Nunes, Giovana G; Back, Davi F; Barison, Andersson; Jackson, Martin; Paulsen, Carley; Gatteschi, Dante; Sorace, Lorenzo; Cornia, Andrea; Soares, Jaísa F; Sessoli, Roberta

2014-10-13

The selective replacement of the central iron(III) ion with vanadium(III) in a tetrairon(III) propeller-shaped single-molecule magnet has allowed us to increase the ground spin state from S=5 to S=13/2. As a consequence of the pronounced anisotropy of vanadium(III), the blocking temperature for the magnetization has doubled. Moreover, a significant remnant magnetization, practically absent in the parent homometallic molecule, has been achieved owing to the suppression of zero-field tunneling of the magnetization for the half-integer molecular spin. Interestingly, the contribution of vanadium(III) to the magnetic anisotropy barrier occurs through the anisotropic exchange interaction with iron(III) spins and not through single ion anisotropy as in most single-molecule magnets. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microscopie de fluorescence de protéines autofluorescentes uniques pour la biologie cellulaire

NASA Astrophysics Data System (ADS)

Cognet, Laurent; Coussen, Françoise; Choquet, Daniel; Lounis, Brahim

In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells. To cite this article: L. Cognet et al., C. R. Physique 3 (2002) 645-656.

Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability

NASA Astrophysics Data System (ADS)

Schwartz, Jerrod J.; Stavrakis, Stavros; Quake, Stephen R.

2010-02-01

Although single-molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures (1.8 K), the field has since advanced to include room-temperature observations, largely owing to the use of objective lenses with high numerical aperture, brighter fluorophores and more sensitive detectors. This has opened the door for many chemical and biological systems to be studied at native temperatures at the single-molecule level both in vitro and in vivo. However, it is difficult to study systems and phenomena at temperatures above 37 °C, because the index-matching fluids used with high-numerical-aperture objective lenses can conduct heat from the sample to the lens, and sustained exposure to high temperatures can cause the lens to fail. Here, we report that TiO2 colloids with diameters of 2 µm and a high refractive index can act as lenses that are capable of single-molecule imaging at 70 °C when placed in immediate proximity to an emitting molecule. The optical system is completed by a low-numerical-aperture optic that can have a long working distance and an air interface, which allows the sample to be independently heated. Colloidal lenses were used for parallel imaging of surface-immobilized single fluorophores and for real-time single-molecule measurements of mesophilic and thermophilic enzymes at 70 °C. Fluorophores in close proximity to TiO2 also showed a 40% increase in photostability due to a reduction of the excited-state lifetime.

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

PubMed

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.

Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

PubMed

Koh, Hye Ran; Wang, Xinlei; Myong, Sua

2016-08-01

TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. Copyright © 2016 Elsevier Inc. All rights reserved.

Influences of Electromagnetic Energy on Bio-Energy Transport through Protein Molecules in Living Systems and Its Experimental Evidence

PubMed Central

Pang, Xiaofeng; Chen, Shude; Wang, Xianghui; Zhong, Lisheng

2016-01-01

The influences of electromagnetic fields (EMFs) on bio-energy transport and its mechanism of changes are investigated through analytic and numerical simulation and experimentation. Bio-energy transport along protein molecules is performed by soliton movement caused by the dipole–dipole electric interactions between neighboring amino acid residues. As such, EMFs can affect the structure of protein molecules and change the properties of the bio-energy transported in living systems. This mechanism of biological effect from EMFs involves the amino acid residues in protein molecules. To study and reveal this mechanism, we simulated numerically the features of the movement of solitons along protein molecules with both a single chain and with three channels by using the Runge–Kutta method and Pang’s soliton model under the action of EMFs with the strengths of 25,500, 51,000, 76,500, and 102,000 V/m in the single-chain protein, as well as 17,000, 25,500, and 34,000 V/m in the three-chain protein, respectively. Results indicate that electric fields (EFs) depress the binding energy of the soliton, decrease its amplitude, and change its wave form. Also, the soliton disperses at 102,000 V/m in a single-chain protein and at 25,500 and 34,000 V/m in three-chain proteins. These findings signify that the influence of EMFs on the bio-energy transport cannot be neglected; however, these variations depend on both the strength and the direction of the EF in the EMF. This direction influences the biological effects of EMF, which decrease with increases in the angle between the direction of the EF and that of the dipole moment of amino acid residues; however, randomness at the macroscopic level remains. Lastly, we experimentally confirm the existence of a soliton and the validity of our conclusion by using the infrared spectra of absorption of the collagens, which is activated by another type of EF. Thus, we can affirm that both the described mechanism and the corresponding theory are correct and that EMFs or EFs can influence the features of energy transport in living systems and thus have certain biological effects. PMID:27463708

Influences of Electromagnetic Energy on Bio-Energy Transport through Protein Molecules in Living Systems and Its Experimental Evidence.

PubMed

Pang, Xiaofeng; Chen, Shude; Wang, Xianghui; Zhong, Lisheng

2016-07-25

The influences of electromagnetic fields (EMFs) on bio-energy transport and its mechanism of changes are investigated through analytic and numerical simulation and experimentation. Bio-energy transport along protein molecules is performed by soliton movement caused by the dipole-dipole electric interactions between neighboring amino acid residues. As such, EMFs can affect the structure of protein molecules and change the properties of the bio-energy transported in living systems. This mechanism of biological effect from EMFs involves the amino acid residues in protein molecules. To study and reveal this mechanism, we simulated numerically the features of the movement of solitons along protein molecules with both a single chain and with three channels by using the Runge-Kutta method and Pang's soliton model under the action of EMFs with the strengths of 25,500, 51,000, 76,500, and 102,000 V/m in the single-chain protein, as well as 17,000, 25,500, and 34,000 V/m in the three-chain protein, respectively. Results indicate that electric fields (EFs) depress the binding energy of the soliton, decrease its amplitude, and change its wave form. Also, the soliton disperses at 102,000 V/m in a single-chain protein and at 25,500 and 34,000 V/m in three-chain proteins. These findings signify that the influence of EMFs on the bio-energy transport cannot be neglected; however, these variations depend on both the strength and the direction of the EF in the EMF. This direction influences the biological effects of EMF, which decrease with increases in the angle between the direction of the EF and that of the dipole moment of amino acid residues; however, randomness at the macroscopic level remains. Lastly, we experimentally confirm the existence of a soliton and the validity of our conclusion by using the infrared spectra of absorption of the collagens, which is activated by another type of EF. Thus, we can affirm that both the described mechanism and the corresponding theory are correct and that EMFs or EFs can influence the features of energy transport in living systems and thus have certain biological effects.