Observation and analysis of single DNA nano-kinetics by pin-fiber video scope

The nano-kinetic movement of a single DNA molecule was observed and analyzed by a newly developed video-microscope system with an optical fiber, called a pin-fiber video scope. A single lambda-DNA molecule was put in focus using fiber-illumination, and the stretching and shrinking motion was measured. The molecule's kinetics were analyzed by numerical calculations and are discussed. A photocleavage phenomenon of DNA molecules was also visualized by the pin-fiber video scope. The new video-microscope system has the potential to observe and analyze the nano-kinetics of a single molecule.     

Nano-kinetics of probe-particles in solution visualized by a pin-fiber video scope.

The nano-kinetics of colloidal particles and living cells with the colloidal particles were visualized by a newly developed video scope. The system of the new video scope has a feature of fine controlling the illumination conditions by using a single optical fiber. This characteristic enables one to obtain clear images of living cells and the motions of colloidal particles by light-scattering effects. In the experiments, RBL-2H3 cells and gold colloidal particles were observed. Scattering images with high contrast and a dark background like in dark-field observations could be attained. In the experiments, a pulsed laser was also applied. The results obtained in this study could validate the effectiveness and possibility of a new video scope for applications to biological and biomedical fields.     

Live single-cell molecular analysis by video-mass spectrometry.

The direct molecular analysis of a live single cell viewed under a video-microscope has been developed. The cell contents are sucked into a nano-electrospray tip, and hundreds of peaks of ionic compounds of low molecular weight are detected by nano-ESI Q-TOF mass spectrometry (MS). Cell-specific MS peaks in a single mouse-embryonic fibroblasts cell are extracted by a t-test, and one of the peaks is proceeded to MS/MS analysis for molecular identification. This method is direct and quick to identify the molecules of a cell with simultaneous observation by a video-microscope.     

Direct drug metabolism monitoring in a live single hepatic cell by video mass spectrometry

The metabolism of anti-breast cancer drug, tamoxifen, in a single human hepatocellular carcinoma cell, HepG2, was directly monitored by a video-mass spectroscope. The cytoplasm, a vacuole or nucleus of the cell was directly sucked by a nano-spray tip under a video-microscope, and then was introduced into a mass spectrometer. Unchanged drug molecules were found in cytoplasm and a vacuole, but the metabolites were only found in the cytoplasm. This direct detection of drug metabolites in a live single cell is useful for speedy drug metabolism monitoring.     

Compression of Langmuir films composed of fine particles: collapse mechanism and wettability

The collapse mechanism of microparticulate Langmuir films was studied experimentally in the present work. Using a Wilhelmy film balance, surface pressure vs area isotherms were determined, and the particle removal during the compression was examined by video-microscope and by naked eye. Upon compressing partially wettable 75 microm diameter surface modified glass beads at liquid (water or aqueous surfactant solution)-air (or n-octane) interfaces, different collapse mechanisms were visualized depending on the wettability of the particles. At low contact angles (below 40 degrees ) irreversible particle removal was observed as a consequence of a particulate line-by-line collapse mechanism. At higher contact angles a buckling-type collapse mechanism was revealed without particle removal from the liquid interface. In the case of irreversible particle removal we assessed the contact angles from the nondissipative part of the isotherm. These values were found to be in reasonable agreement with those determined directly on the beads.     

Resin-packed nanoelectrospray in combination with video and mass spectrometry for the direct and real-time molecular analysis of mast cells

A nano-electrospray ionization (nanoESI) emitter for analysis of a biological solution was developed by packing a nanoESI needle with two types of resins for desalting and preconcentration of target molecules. Determination of secreted histamine and serotonin molecules in cell culture buffers was demonstrated using 5-methyltryptamine as internal standard. The results showed good linearity of target signals in the concentration range from 0.25 to 50.0 ng/mL of histamine or serotonin. These molecules were monitored to be secreted by A23187 (calcium ionophore) stimulant in rat peritoneal mast cells. Using a combination of a video-microscope and a mass spectrometer, we could visualize exocytotic moments and analyze secreted molecules by mass spectrometry simultaneously. Time-dependent release of histamine and serotonin from activated mast cells showed that significant production of these molecules occurred and reached a maximal level at 15 min for serotonin and at 30 min for histamine, respectively. These results showed that this method allows the direct and timely analysis of secreted molecules in biological responses.     

Molecular dynamics simulations of liquid crystal molecules on a polyimide monolayer

Preliminary results are presented on the molecular dynamics simulations of alignment of the liquid crystal molecule, 4-n-octyl-4'-cyanobiphenyl (8CB), on a polyimide (pyromelltic dianhydride-p-phenylene diamine) oligomer monolayer. We actually simulated a three-layer system, i.e., liquid crystal molecule/polyimide oligomer/a basal plane of graphite. First, simulations of the oligomers adsorbed on graphite were done in order to obtain reasonable adsorption structures, as the pre-stage simulation of the three-layer system. Then, by placing a liquid crystal layer on top, the three-layer system was simulated. The stable liquid crystal alignment direction on the polyimide monolayer was found roughly to be the polyimide chain direction with zero pretilt in this combination of liquid crystal and polymer materials. The calculated adsorption energy of an 8CB molecule to the polyimide monolayer was 128 kJ mol^-1 and the carbonyl group of the polyimide was the main adsorption site.     

一种双功能光折变模型分子的光电导性质

咔唑衍生物及含咔唑基团的聚合物由于在光信息传导、全息照相和光折变等高科技领域的潜在应用前景而越来越受到重视［１～３］,大量有机光折变材料已被合成．其中一些含有咔唑成分的光折变材料具有很优异的性能．１９９４年,一种具有低玻璃化温度的ＰＶＫ掺杂体系的净增益参数达到２２０ｃｍ－１,而衍射效率也近似达到１００％．这一结果超过了已知的绝大多数无机物［２］．大多数咔唑衍生物及含咔唑基团的聚合物都具有良好的光电导性质,并且主要以空穴传导为主．但它们的迁移率和机理由于引入不同的侧基和掺杂物出现很大差别［４～６］…     

Radiative and nonradiative decay rates of a molecule close to a metal particle of complex shape

We present a model to evaluate the radiative and nonradiative lifetimes of electronic excited states of a molecule close to a metal particle of complex shape and, possibly, in the presence of a solvent. The molecule is treated quantum mechanically at Hartree-Fock (HF) or density-functional theory (DFT) level. The metal/solvent is considered as a continuous body, characterized by its frequency dependent local dielectric constant. For simple metal shapes (planar infinite surface and spherical particle) a version of the polarizable continuum model based on the integral equation formalism has been used, while an alternative methodology has been implemented to treat metal particles of arbitrary shape. In both cases, equations have been numerically solved using a boundary element method. Excitation energies and nonradiative decay rates due to the energy transfer from the molecule to the metal are evaluated exploiting the linear response theory (TDHF or TDDFT where TD--time dependent). The radiative decay rate of the whole system (molecule + metal/solvent) is calculated, still using a continuum model, in terms of the response of the surrounding to the molecular transition. The model presented has been applied to the study of the radiative and nonradiative lifetimes of a lissamine molecule in solution (water) and close to gold spherical nanoparticles of different radius. In addition, the influence of the metal shape has been analyzed by performing calculations on a system composed by a coumarin-type molecule close to silver aggregates of complex shape.     

Synthesis of a pentalene centered polycyclic fused system

A novel pentalene-centered polycyclic 24π-electron system, IB1, was synthesized via a Pd-catalyzed homocoupling reaction. The geometry structure was studied by X-ray diffraction and theoretical method. The HOMO level of IB1 was studied by electrochemical experiment and DFT methods. The IB1 molecule shows a strong electro-donating property and can form a charge transfer complex with electro-acceptor TCNQ, indicating fascinating potential in the field of organic electronics.     

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

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

Multidimensional optical spectroscopy of a single molecule in a current-carrying state

The nonlinear optical signals from an open system consisting of a molecule connected to metallic leads, in response to a sequence of impulsive pulses, are calculated using a superoperator formalism. Two detection schemes are considered: coherent stimulated emission and incoherent fluorescence. The two provide similar but not identical information. The necessary superoperator correlation functions are evaluated either by converting them to ordinary (Hilbert space) operators which are then expanded in many-body states, or by using Wick's theorem for superoperators to factorize them into nonequilibrium two point Green's functions. As an example we discuss a stimulated Raman process that shows resonances involving two different charge states of the molecule in the same signal.     

Phase switching of a single isomeric molecule and associated characteristic rectification

Variations in molecular electronic structures related to conformational change are exceedingly attractive because of their key role in the understanding and development of functional processes in molecular electronics and biology. We observed, for the first time, the novel phase switching of a photoactive isomeric molecule, N-(2-mercaptoethyl)-4-phenylazobenzamide (Azo molecule) at a single-molecule level, which exhibits a distinctive change in the conductive characteristic under scanning tunneling microscope (STM) measurement. In comparison with the results obtained by the measurement of photoactive isomerization of the isolated Azo molecule, which was performed also for the first time, the observed characteristics are attributed to the results of the trans and cis phase transformation of the Azo molecule, under the condition of an external electric field and current flow. A specific point is that the potential landscape of the system is controllable by the electric field and provides a conformational stability with asymmetric bias dependence resulting in rectification.     

Orientation of a benzene molecule inside a carbon nanotube

Benzene molecules confined in carbon nanotubes of varying radii are employed as semiconductors in electronic nanodevices, and their orientation determines the electrical properties of the system. In this paper, we investigate the interaction energy of all the possible configurations of a benzene molecule inside various carbon nanotubes and then we determine the equilibrium configuration. We adopt the continuous approach together with the semi-empirical Lennard-Jones potential function to model van der Waals interaction between a benzene molecule and a carbon nanotube. This approach results in an analytical expression, which accurately approximates the interaction energy and can be readily used to generate numerical data. We find that horizontal, tilted and perpendicular configurations on the axis of the carbon nanotube are all possible equilibrium configurations of the benzene molecule when the radius of the carbon nanotube is less than 5.580 Å. However, when the radius of the carbon nanotube is larger than 5.580 Å an offset horizontal orientation is the only possible equilibrium configuration of the benzene molecule. In the limiting case, the orientation of a benzene molecule on a graphene sheet can be derived simply by letting the radius of the carbon nanotube tend to infinity.     

Desymmetrization of a centrosymmetric diepoxide: efficient synthesis of a key intermediate in a total synthesis of hemibrevetoxin B

The preparation of an established intermediate in a total synthesis of hemibrevetoxin B is described. The acid-catalyzed cyclization of trans-4,5-epoxyoctane-2,7-dione exhibited a valuable mixture of kinetic and thermodynamic control: stereospecific epoxide opening was followed by equilibration of the products to provide the required trans-fused octahydropyrano[3,2-b]pyran ring system. Two-directional elaboration, by acetal substitution, ozonolysis, and sulfur ylide-mediated epoxidation, provided a centrosymmetric diepoxide. The key step of the synthesis was the first desymmetrization of a centrosymmetric molecule in natural product synthesis: Jacobsen asymmetric epoxide hydrolysis and acetonization provided the known synthetic intermediate in 97% yield and >95% ee over two steps. The exploitation of the center of symmetry of the AB ring system of the natural product contributed greatly to the efficiency (eight steps, 34% overall yield) of the synthesis.     

Effect of Hydrogen on O2 Adsorption and Dissociation on a TiO2 Anatase (001) Surface

The effect of hydrogen on the adsorption and dissociation of the oxygen molecule on a TiO₂ anatase (001) surface is studied by first‐principles calculations coupled with the nudged elastic band (NEB) method. Hydrogen adatoms on the surface can increase the absolute value of the adsorption energy of the oxygen molecule. A single H adatom on an anatase (001) surface can lower dramatically the dissociation barrier of the oxygen molecule. The adsorption energy of an O₂ molecule is high enough to break the O?O bond. The system energy is lowered after dissociation. If two H adatoms are together on the surface, an oxygen molecule can be also strongly adsorbed, and the adsorption energy is high enough to break the O?O bond. However, the system energy increases after dissociation. Because dissociation of the oxygen molecule on a hydrogenated anatase (001) surface is more efficient, and the oxygen adatoms on the anatase surface can be used to oxidize other adsorbed toxic small gas molecules, hydrogenated anatase is a promising catalyst candidate.     

L-alanine in a droplet of water: a density-functional molecular dynamics study

We report the results of a Born-Oppenheimer molecular dynamics study on an L-alanine amino acid in neutral aqueous solution. The whole system, the L-alanine zwitterion and 50 water molecules, was treated quantum mechanically. We found that the hydrophobic side chain (R = CH3) defines the trajectory path of the molecule. Initially fully hydrated in an isolated droplet of water, the amino acid moves to the droplet's surface, exposing its hydrophobic methyl group and alpha-hydrogen out of the water. The structure of an L-alanine with the methyl group exposed to the water surface was found to be energetically favorable compared to a fully hydrated molecule. The dynamic behavior of the system suggests that the first hydration shell of the amino acid is localized around carboxylate (CO2-) and ammonium (NH3+) functional groups; it is highly ordered and quite rigid. In contrast, the hydration shell around the side chain is much less structured, suggesting a modest influence of the methyl group on the structure of water. The number of water molecules in the first hydration shell of an alanine molecule is constantly changing; the average number was found to equal 7. The molecular dynamics results show that L-alanine in water does not have a preferred conformation, as all three of the molecule's functional sites (i.e., CH3, NH3+, CO2-) perform rotational movements around the C(alpha)-site bond.     

Encapsulating a single G-quadruplex aptamer in a protein nanocavity

The alpha-hemolysin (alphaHL) protein pore has many applications in biotechnology. This article describes a single-molecule manipulation system that utilizes the nanocavity enclosed by this pore to noncovalently encapsulate a guest molecule. The guest is the thrombin-binding aptamer (TBA) that folds into the G-quadruplex in the presence of cations. Trapping the G-quadruplex in the nanocavity resulted in characteristic changes to the pore conductance that revealed important molecular processes, including spontaneous unfolding of the quartet structure and translocation of unfolded DNA in the pore. Through detection with Tag-TBA, we localized the G-quadruplex near the entry of the beta-barrel inside the nanocavity, where the molecule vibrates and rotates to different orientations. This guest-nanocavity supramolecular system has potential for helping to understand single-molecule folding and unfolding kinetics.     

Theoretical study of the acid-promoted hydrolysis of oxazolin-5-one: a microhydration model

The acid-promoted hydrolysis of 2,4,4-trimethyloxazolin-5-one (TMO) is studied employing the density functional theory (B3LYP) method in conjunction with the 6-31++G(d,p) basis set. Two types of reaction mechanism, N-protonated and O-protonated, are considered, involving protonation at the nitrogen and carbonyl oxygen of TMO to activate the C2 and C5 atoms, respectively, in favor of attack by water molecules. In the N-protonated pathway, the nucleophilic water molecule attacks the activated C2 atom, with a proton transfer from the water molecule to the oxygen atom attached to C2 and the fission of the C2-O bond, leading to a cis ring-opening product (N-acyl-alpha-amino isobutyric acid). While, in the O-protonated pathway, the nucleophilic water molecule attacks the activated carbonyl C5 atom, accompanied by a proton transfer from the water molecule toward the nitrogen atom of oxazole ring and the cleavage of C5-O bond; as a result, a corresponding trans product is generated. The water-assisted hydrolysis reactions are also examined together. A local microhydration model, in which an extra water molecule was added to obtain a continuous H-bond network around the reaction centers, was adopted to mimic the system for the two types of reaction processes. In addition, bulk solvent effect is introduced by use of the conductor-like polarizable continuum model (CPCM). Our computational results in kinetics and thermodynamics clearly manifest that the O-protonated pathway with the nucleophilic attack at the carbonyl C5 atom is more favorable than the N-protonated one, in nice agreement with the available experimental conclusion.     

Characteristics of a liposome immunoassay on a poly(methyl methacrylate) surface

Liposome immunoassay (LIA) is based on enzyme immunoassay (EIA) but the detection sensitivity could be significantly enhanced by using antibody-coupled immunoliposomes encapsulating HRP (horse radish peroxidase). Here, we applied LIA to non-porous poly(methyl methacrylate) (PMMA) and polystyrene (PS) surfaces to compare its detection sensitivity with that of EIA, using rabbit IgG (a ligand molecule) and anti-rabbit IgG antibody (a capture molecule) as the model system. LIA developed much stronger color signals than EIA, especially at a lower concentration range (< ca. 1 μg mL⁻¹). PMMA showed higher affinity toward rabbit IgG than the PS surface, and the anti-rabbit IgG antibody adsorbed on PMMA was more stable than that on PS. Furthermore, the effects of spot volume and antibody concentration on the signal density were analyzed. The signal density increased as the antibody concentration increased, but it was not significantly affected by the spot volume (2.5–20 μL). In conclusion, LIA on PMMA as a solid support is a very useful, highly sensitive microarray detection system. Sang Youn Hwang and Yoichi Kumada have same rights on this paper.