Interpretation of stochastic events in single-molecule measurements of conductance and transition voltage spectroscopy

The first simultaneous measurements of transition voltage (V(t)) spectroscopy (TVS) and conductance (G) histograms (Guo et al., J. Am. Chem. Soc. 2011, 133, 19189) form a great case for studying stochastic effects, which are ubiquitous in molecular junctions. Here an interpretation of those data is proposed that emphasizes the different physical content of V(t) and G and reveals that fluctuations in the molecular orbital alignment have a significantly larger impact on G than initially claimed. The present study demonstrates the usefulness of corroborating statistical information on different transport properties and gives support to TVS as a valuable investigative tool.     

Propensity rules for inelastic electron tunneling spectroscopy of single-molecule transport junctions

Using a perturbative approach to simple model systems, we derive useful propensity rules for inelastic electron tunneling spectroscopy (IETS) of molecular wire junctions. We examine the circumstances under which this spectroscopy (that has no rigorous selection rules) obeys well defined propensity rules based on the molecular symmetry and on the topology of the molecule in the junction. Focusing on conjugated molecules of C(2h) symmetry, semiquantitative arguments suggest that the IETS is dominated by a(g) vibrations in the high energy region and by out of plane modes (a(u) and b(g)) in the low energy region. Realistic computations verify that the proposed propensity rules are strictly obeyed by medium to large-sized conjugated molecules but are subject to some exceptions when small molecules are considered. The propensity rules facilitate the use of IETS to help characterize the molecular geometry within the junction.     

Quantitative nanostructural and single-molecule force spectroscopy biomolecular analysis of human-saliva-derived exosomes

Exosomes are naturally occurring nanoparticles with unique structure, surface biochemistry, and mechanical characteristics. These distinct nanometer-sized bioparticles are secreted from the surfaces of oral epithelial cells into saliva and are of interest as oral-cancer biomarkers. We use high- resolution AFM to show single-vesicle quantitative differences between exosomes derived from normal and oral cancer patient's saliva. Compared to normal exosomes (circular, 67.4 ± 2.9 nm), our findings indicate that cancer exosome populations are significantly increased in saliva and display irregular morphologies, increased vesicle size (98.3 ± 4.6 nm), and higher intervesicular aggregation. At the single-vesicle level, cancer exosomes exhibit significantly (P < 0.05) increased CD63 surface densities. To our knowledge, it represents the first report detecting single-exosome surface protein variations. Additionally, high-resolution AFM imaging of cancer saliva samples revealed discrete multivesicular bodies with intraluminal exosomes enclosed. We discuss the use of quantitative, nanoscale ultrastructural and surface biomolecular analysis of saliva exosomes at single-vesicle- and single-protein-level sensitivities as a potentially new oral cancer diagnostic.     

An analysis of the current-voltage characteristics of thin film front wall illuminated and back wall illuminated liquid junction and schottky barrier solar cells

Analytical expressions for the current-voltage characteristics of thin film front illuminated and back illuminated liquid junc tion solar cells have been derived as a function of film thickness. The usual assumptions of a flat minority carrier quasi Fermi level across the semiconductor depletion region or a constant minority carrier concentration at the semiconductor surface have not been made. Depletion layer recombination is included in both expressions. The effects of various parameters, including thickness and back recombination velocity, are determined for both types of devices.     

Interplay between barrier width and height in electron tunneling: photoinduced electron transfer in porphyrin-based donor-bridge-acceptor systems

The rate of electron tunneling in molecular donor-bridge-acceptor (D-B-A) systems is determined both by the tunneling barrier width and height, that is, both by the distance between the donor and acceptor as well as by the energy gap between the donor and bridge moieties. These factors are therefore important to control when designing functional electron transfer systems, such as constructs for photovoltaics, artificial photosynthesis, and molecular scale electronics. In this paper we have investigated a set of D-B-A systems in which the distance and the energy difference between the donor and bridge states (DeltaEDB) are systematically varied. Zinc(II) and gold(III) porphyrins were chosen as electron donor and acceptor because of their suitable driving force for photoinduced electron transfer (-0.9 eV in butyronitrile) and well-characterized photophysics. We have previously shown, in accordance with the superexchange mechanism for electron transfer, that the electron transfer rate is proportional to the inverse of DeltaEDB in a series of zinc/gold porphyrin D-B-A systems with bridges of constant edge to edge distance (19.6 A) and varying DeltaEDB (3900-17 600 cm(-1)). Here, we use the same donor and acceptor but the bridge is shortened or extended giving a set of oligo-p-phenyleneethynylene bridges (OPE) with four different edge to edge distances ranging from 12.7 to 33.4 A. These two sets of D-B-A systems-ZnP-RB-AuP+ and ZnP-nB-AuP+-have one bridge in common, and hence, for the first time both the distance and DeltaEDB dependence of electron transfer can be studied simultaneously in a systematic way.     

Scanning tunneling microscopy, orbital-mediated tunneling spectroscopy, and ultraviolet photoelectron spectroscopy of metal(II) tetraphenylporphyrins deposited from vapor.

Thin films of vapor-deposited Ni(II) and Co(II) complexes of tetraphenylporphyrin (NiTPP and CoTPP) were studied supported on gold and embedded in Al-Al(2)O(3)-MTPP-Pb tunnel diodes, where M = Ni or Co. Thin films deposited onto polycrystalline gold were analyzed by ultraviolet photoelectron spectroscopy (UPS) using He I radiation. Scanning tunneling microscopy (STM) and orbital-mediated tunneling spectroscopy (STM-OMTS) were performed on submonolayer films of CoTPP and NiTPP supported on Au(111). Inelastic electron tunneling spectroscopy (IETS) and OMTS were measured in conventional tunnel diode structures. The highest occupied pi molecular orbital of the porphyrin ring was seen in both STM-OMTS and UPS at about 6.4 eV below the vacuum level. The lowest unoccupied pi molecular orbital of the porphyrin ring was observed by STM-OMTS and by IETS-OMTS to be located near 3.4 eV below the vacuum level. The OMTS spectra of CoTPP had a band near 5.2 eV (below the vacuum level) that was attributed to transient oxidation of the central Co(II) ion. That is, it is due to electron OMT via the half-filled d(z)(2) orbital present in Co(II) of CoTPP. The NiTPP OMTS spectra show no such band, consistent with the known difficulty of oxidation of the Ni(II) ion. The STM-based OMTS allowed these two porphyrin complexes to be easily distinguished. The present work is the first report of the observation of STM-OMTS, tunnel junction OMTS, and UPS of the same compounds. Scanning tunneling microscope-based orbital-mediated tunneling provides more information than UPS or tunnel junction-based OMTS and does so with molecular-scale resolution.     

Microfluidic separation and capture of analytes for single-molecule spectroscopy

A complex mixture of fluorescently labeled biological molecules is separated electrophoretically on a chip and the constituent molecules are confined in a sub-nanolitre microchamber, which allows analysis by various single-molecule techniques.     

Measurement of relative atomic transition probabilities using flame atomic absorption spectroscopy

A simple and straightforward technique has been developed to measure relative atomic transition probabilities (A_ji) using flame atomic absorption spectroscopy (FAAS). A_ji values for Cu, Ag, Mn and Mo are calculated from absorption measurements in an air—acetylene flame using both line emission (AAL) and continuum excitation (AAC) sources. For Mn and Mo, large discrepancies for A_ji are found between AAL and AAC, which may be attributed to hyperfine structure (hfs). For continuum source measurements the absorbance is independent of the absorption line profile thus eliminating the problems associated with hfs. Therefore, AAC provides a more accurate method for relative A_ji measurements, with determinations of better than 10% relative standard deviation for all elements.     

Initial observation of magnetization hysteresis and quantum tunneling in mixed manganese-lanthanide single-molecule magnets

The preparation of a new family of mixed transition metal/lanthanide clusters is reported. The reaction of [Mn3O(O2CPh)6(py)2(H2O)] with Ln(NO3)3 (Ln = Nd, Gd, Dy, Ho, and Eu) in a 1:2 molar ratio in MeOH/MeCN (1:20 v/v) leads to dark crystals in 55-60% isolated yield of complexes all containing the [Mn11Ln4]45+ core. The Dy compound has been found to give out-of-phase AC susceptibility signals, suggesting it might be a single-molecule magnet (SMM). This was confirmed by the observation of magnetization hysteresis loops. An Arrhenius plot constructed from magnetization decay data gave a barrier to relaxation of 9.3 K and showed the temperature-independent relaxation at very low temperatures indicative of quantum tunneling of magnetization. This is the initial demonstration of hysteresis and quantum behavior in a mixed 3d/4f SMM.     

Dispersion and current-voltage characteristics of helical polyacetylene single fibers

To study the transport properties of individual helical polyacetylene (PA) fibers, we developed a method to extract a single fiber from tightly entangled ropes of helical PA bulk film. After a few minutes of sonication of a piece of helical PA bulk film in an organic solution containing surfactant, a droplet of solution is deposited on the pre-pattened electrode under argon atmosphere. AFM images show that extracted helical PA fibers are typically 10 mum in length and 100-200 nm in diameter. We found that the helicity of bulk materials is conserved. We present the temperature dependencies of current-voltage characteristics of individual helical PA fibers doped with iodine.     

Ensemble and single-molecule fluorescence spectroscopy of a calcium-ion indicator dye

The spectroscopic properties of Calcium Green 2 (CG-2), a dual-fluorophore Ca(2+) indicator dye, were characterized by a combination of steady state and time-resolved ensemble spectroscopic measurements, molecular mechanics calculations and single-molecule fluorescence spectroscopy. It was found that in Ca(2+) free solutions, CG-2 exists primarily as a highly quenched intramolecular dimer, but when bound to Ca(2+), the molecule adopts an extended, fluorescent conformation. The difference in emission properties of these two CG-2 conformations is explained in terms of simple exciton theory. Through single-molecule fluorescence measurements, we have shown that the bulk increase in ensemble fluorescence intensity correlates with a simple statistical increase in the number of fluorescent molecules in solution. In addition, we have also observed that the majority of CG-2 molecules photobleach in a single step, despite the molecule possessing two distinct fluorophores. A small fraction of molecules photobleach in multiple steps or show a series of transitions between emissive and nonemissive fluorescent states ("blinking"). We rationalize these photophysical phenomena using a simple model based on dipole-dipole F?rster coupling between fluorophores in conjunction with irreversible photodamage to one of the constituent chromophores.     

Inelastic tunneling spectroscopy using scanning tunneling microscopy on trans-2-butene molecule: spectroscopy and mapping of vibrational feature

Inelastic tunneling spectroscopy (IETS) measurement using scanning tunneling microscopy (STM) with a commercially available STM set up is presented. The STM-IETS spectrum measured on an isolated trans-2-butene molecule on the Pd(110) shows a clear vibrational feature in d2I/dV2 at the bias voltage of 360 mV and -363 mV, which corresponds to the nu(C-H) mode (d2I/dV2 approximately 10 nA/V2). In addition, we have obtained an image by mapping the vibrational feature of nu(C-H) in d2I/dV2. The image is obtained by scanning the tip on the surface with the feedback loop activated while the modulation voltage is superimposed on the sample voltage. With the method that is readily performable with conventional software, we have clearly differentiated the molecules of trans-2-butene and butadiene through the mapping of the vibrational feature, demonstrating its capability of chemical identification in atomic scale.     

Thermally stimulated transition in tunneling characteristics of molecular junction of tin/octadecanol/tin

We report the observation of temperature dependent tunneling behavior of capillary tunnel junctions of octadecanol (C₁₈H₃₇OH) from room temperature to 130 °C. The molecular tunnel junction consists of octadecanol (C₁₈H₃₇OH) sandwiched between tin electrodes utilizing the naturally existing surface oxides. The resistance of the molecular junction was found to increase appreciably at temperatures around 80 °C . The transition of the junction resistance is suggested to be associated with the breaking of hydrogen bonding between molecular terminal groups with the electrode surface.     

Mechanically tightening,untying and retying a protein trefoil knot by single-molecule force spectroscopy

Knotted conformation is one of the most surprising topological features found in proteins, and understanding the folding mechanism of such knotted proteins remains a challenge. Here, we used optical tweezers (OT) to investigate the mechanical unfolding and folding behavior of a knotted protein Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD). We found that when stretched from its N- and C-termini, TrmD can be mechanically unfolded and stretched into a tightened trefoil knot, which is composed of ca. 17 residues. Stretching of the unfolded TrmD involved a compaction process of the trefoil knot at low forces. The unfolding pathways of the TrmD were bifurcated, involving two-state and three-state pathways. Upon relaxation, the tightened trefoil knot loosened up first, leading to the expansion of the knot, and the unfolded TrmD can then fold back to its native state efficiently. By using an engineered truncation TrmD variant, we stretched TrmD along a pulling direction to allow us to mechanically unfold TrmD and untie the trefoil knot. We found that the folding of TrmD from its unfolded polypeptide without the knot is significantly slower. The knotting is the rate-limiting step of the folding of TrmD. Our results highlighted the critical importance of the knot conformation for the folding and stability of TrmD, offering a new perspective to understand the role of the trefoil knot in the biological function of TrmD.

Optical tweezers are used to stretch a knotted protein along different directions to probe its unfolding–folding behaviors, and the conformational change of its knot structure.      

Measurement of sugar content by multidimensional analysis and mid-infrared spectroscopy

The advent of more and more powerful micro-computers has allowed the introduction of multidimensional analysis in research laboratories. Complex mathematical treatments are now possible within a few seconds. Prediction equations that linked sucrose, fructose, glucose, total sugars and reducing sugars concentrations to the spectral data, were established by regression on the principal components. Very high correlation coefficient values between the first ten axes and the chemical values were obtained. The bias and standard deviation (S.D.) values obtained between reference and predicted values were good. From such aqueous biological samples containing a ternary mixture of sucrose, fructose and glucose it was possible to (i) identify the characteristic IR bands of these different sugars (and their combination: reducing sugars, total sugars)-using spectral pattern; and (ii) to specifically measure their concentrations with good accuracy.     

Classification of the biological material with use of FTIR spectroscopy and statistical analysis

Rapid detection and discrimination of dangerous biological materials such as bacteria and their spores has become a security aim of considerable importance. Various analytical methods, including FTIR spectroscopy combined with statistical analysis have been used to identify vegetative bacteria, bacterial spores and background interferants. The present work discusses the application of FTIR technique performed in reflectance mode using Horizontal Attenuated Total Reflectance accessory (HATR) to the discrimination of biological materials. In comparison with transmission technique the HATR is more rapid and do not require the sample destruction, simultaneously giving similar absorbance bands. HATR-FTIR results combined with statistical analysis PCA and HCA demonstrate that this combination provides novel and accurate microbial identification technique.     

Influence of pulling handles and device stiffness in single-molecule force spectroscopy

In single-molecule force spectroscopy, individual molecules and complexes are often stretched by pulling devices via intervening molecular handles. Accurate interpretation of measurements from such experiments in terms of the underlying energy landscape, defined by activation barriers and intrinsic rates of transition, relies on our understanding, and proper theoretical treatment, of the effects of the pulling device and handle. Here, we present a framework based on Kramers' theory that elucidates the dependence of measured rupture forces and rates on the pulling device stiffness and attributes of the handle, contour length and persistence length. We also introduce a simple analytic model that improves prediction of activation barriers and intrinsic rates for all device stiffnesses and handle properties, thus allowing for a more reliable interpretation of experiments. Our analyses also suggests intuitive ways of displaying the measured force spectra for proper prognosis of device and handle effects and provides the range of device and handle attributes over which these effects can be neglected.     

Unfolding dynamics of cytochrome c revealed by single-molecule and ensemble-averaged spectroscopy

Denaturant-induced conformational change of yeast iso-1-cytochrome c (Cytc) has been comprehensively investigated in the single-molecule and bulk phases. By fluorescence-quenching experiments with dye-labelled heme-protein (Alexa 488-labelled Cytc, Cytc-A488), we clearly show that the fluorescence quenching observed from folded Cytc-A488 is due mainly to photoinduced electron transfer (PET) between electron-donating amino acids such as tryptophan and the dye attached to the protein. In addition, the unfolding process of Cytc-A488 observed in the single-molecule and bulk phases can be explained well in terms of a three-state model: Cytc unfolds through an intermediate with a native-like compactness. By quantitative analysis of fluorescence correlation spectroscopy (FCS) data, we were able to observe a relaxation time of ～1.5 μs corresponding to segmental motion and fast folding dynamics of 55 μs in the unfolded state of Cytc. The results presented here also suggest that a combination of single-molecule and ensemble-averaged spectroscopy is necessary to provide convincing and comprehensive assignments of protein kinetics.     

Conformational studies by Raman spectroscopy and statistical analysis of gauche interactions in n-butylamine

Raman spectra of n-butylamine recorded at different temperatures show pairs of bands whose temperature-dependent intensities clearly suggest their assignment to different conformers in simultaneous equilibria. Normal coordinate analysis and i.r. spectra of n-butylamine are also used to assign the spectra. These vibrational data are interpreted and correlated with structural information obtained from a statistical analysis of gauche skeletal arrangements in n-butylamine at different temperatures.