Electrochemical detection of single molecules

The electrochemical behavior of a single molecule can be observed by trapping a small volume of a dilute solution of the electroactive species between an ultramicroelectrode tip with a diameter of approximately 15 nanometers and a conductive substrate. A scanning electrochemical microscope was used to adjust the tip-substrate distance ( approximately 10 nanometers), and the oxidation of [(trimethylammonio)methyl] ferrocene (Cp(2)FeTMA(+)) to Cp(2)FeTMA(2+) was carried out. The response was stochastic, and anodic current peaks were observed as the molecule moved into and out of the electrode-substrate gap. Similar experiments were performed with a solution containing two redox species, ferrocene carboxylate (Cp(2)FeCOO(-)) and Os(bpy)(3)(2+) (bpy is 2,2'-bipyridyl).     

Illuminating single molecules in condensed matter

Efficient collection and detection of fluorescence coupled with careful minimization of background from impurities and Raman scattering now enable routine optical microscopy and study of single molecules in complex condensed matter environments. This ultimate method for unraveling ensemble averages leads to the observation of new effects and to direct measurements of stochastic fluctuations. Experiments at cryogenic temperatures open new directions in molecular spectroscopy, quantum optics, and solid-state dynamics. Room-temperature investigations apply several techniques (polarization microscopy, single-molecule imaging, emission time dependence, energy transfer, lifetime studies, and the like) to a growing array of biophysical problems where new insight may be gained from direct observations of hidden static and dynamic inhomogeneity.     

Intramolecular long-distance electron transfer in organic molecules

Intramolecular long-distance electron transfer (EI) has been actively studied in recent years in order to test existing theories in a quantitative way and to provide the necessary constants for predicting ET rates from simple structural parameters. Theoretical predictions of an "inverted region," where increasing the driving force of the reaction will decrease its rate, have begun to be experimentally confirmed. A predicted nonlinear dependence of ET rates on the polarity of the solvent has also been confirmed. This work has implications for the design of efficient photochemical charge-separation devices. Other studies have been directed toward determining the distance dependence of ET reactions. Model studies on different series of compounds give similar distance dependences. When different stereochemical structures are compared, it becomes apparent that geometrical factors must be taken into account. Finally, the mechanism of coupling between donor and acceptor in weakly interacting systems has become of major importance. The theoretical and experimental evidence favors a model in which coupling is provided by the interaction with the orbitals of the intervening molecular fragments, although more experimental evidence is needed.     

Imaging precessional motion of the magnetization vector

We report on imaging of three-dimensional precessional orbits of the magnetization vector in a magnetic field by means of a time-resolved vectorial Kerr experiment that measures all three components of the magnetization vector with picosecond resolution. Images of the precessional mode taken with submicrometer spatial resolution reveal that the dynamical excitation in this time regime roughly mirrors the symmetry of the underlying equilibrium spin configuration and that its propagation has a non-wavelike character. These results should form the basis for realistic models of the magnetization dynamics in a largely unexplored but technologically increasingly relevant time scale.     

Electron tunneling through organic molecules in frozen glasses

Reaction rates extracted from measurements of donor luminescence quenching by randomly dispersed electron acceptors reveal an exponential decay constant of 1.23 per angstrom for electron tunneling through a frozen toluene glass (with a barrier to tunneling of 1.4 electron volts). The decay constant is 1.62 per angstrom (the barrier, 2.6 electron volts) in a frozen 2-methyl-tetrahydrofuran glass. Comparison to decay constants for tunneling across covalently linked xylyl (0.76 per angstrom) and alkyl (1.0 per angstrom) bridges leads to the conclusion that tunneling between solvent molecules separated by approximately 2 angstroms (van der Waals contact) is 20 to 50 times slower than tunneling through a comparable length of a covalently bonded bridge. Our results provide experimental confirmation that covalently bonded pathways can facilitate electron flow through folded polypeptide structures.     

Sequence-dependent pausing of single lambda exonuclease molecules

Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.     

Inclusion systems of organic molecules in restacked single-layer molybdenum disulfide

Novel materials have been obtained by restacking single-layer molybdenum disulfide (MoS(2)) with organic molecules included between the layers. A large variety of organic molecules can be included between layers of MoS(2) and other transition-metal dichalcogenides. The films with the included organics are formed at the interface between an aqueous suspension of the MoS(2) and a water-immiscible organic liquid. The organic molecules are not necessarily electron donors. A highly oriented, conducting film of restacked MoS(2) containing ferrocene is presented as an example.     

Correlating structural dynamics and function in single ribozyme molecules

We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.     

The complex folding network of single calmodulin molecules

Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.     

Nanoscale science of single molecules using local probes

Experiments on individual molecules using scanning probe microscopies have demonstrated an exciting diversity of physical, chemical, mechanical, and electronic phenomena. They have permitted deeper insight into the quantum electronics of molecular systems and have provided unique information on their conformational and mechanical properties. Concomitant developments in experimentation and theory have allowed a diverse range of molecules to be studied, varying in complexity from simple diatomics to biomolecular systems. At the level of an individual molecule, the interplays of mechanical and electronical behavior and chemical properties manifest themselves in an unusually clear manner. In revealing the crucial role of thermal, stochastic, and quantum-tunneling processes, they suggest that dynamics is inescapable and may play a decisive role in the evolution of nanotechnology.     

Sequence-specific molecular lithography on single DNA molecules

Recent advances in the realization of individual molecular-scale electronic devices emphasize the need for novel tools and concepts capable of assembling such devices into large-scale functional circuits. We demonstrated sequence-specific molecular lithography on substrate DNA molecules by harnessing homologous recombination by RecA protein. In a sequence-specific manner, we patterned the coating of DNA with metal, localized labeled molecular objects and grew metal islands on specific sites along the DNA substrate, and generated molecularly accurate stable DNA junctions for patterning the DNA substrate connectivity. In our molecular lithography, the information encoded in the DNA molecules replaces the masks used in conventional microelectronics, and the RecA protein serves as the resist. The molecular lithography works with high resolution over a broad range of length scales from nanometers to many micrometers.     

Phosphorescence of adsorbed ionic organic molecules at room temperature

Many salts of polynuclear carboxylic acids, phenols, amines, and sulfonic acids adsorbed on paper, silica, alumina, and other substrates exhibit strong triplet phosphorescence at room temperature, with no evidence of quenching by oxygen. No phosphorescence has been observed with nonionic materials. The spectra are similar to those of frozen solutions at -196 degrees C, and the technique provides a simple means of demonstrating phosphorescence phenomena, identifying unknown materials, and investigating the spectra of triplet states.     

Pre-unfolding resonant oscillations of single green fluorescent protein molecules

Fluorescence spectroscopy of a green fluorescent protein mutant at single-molecule resolution has revealed a remarkable oscillatory behavior that can also be driven by applied fields. We show that immediately before unfolding, several periodic oscillations among the chemical substates of the protein chromophore occur. We also show that applied alternating electric or acoustic fields, when tuned to the protein characteristic frequencies, give rise to strong resonance effects.     

Reversible unfolding of single RNA molecules by mechanical force

Here we use mechanical force to induce the unfolding and refolding of single RNA molecules: a simple RNA hairpin, a molecule containing a three-helix junction, and the P5abc domain of the Tetrahymena thermophila ribozyme. All three molecules (P5abc only in the absence of Mg2+) can be mechanically unfolded at equilibrium, and when kept at constant force within a critical force range, are bi-stable and hop between folded and unfolded states. We determine the force-dependent equilibrium constants for folding/unfolding these single RNA molecules and the positions of their transition states along the reaction coordinate.     

Echinoderms: an autoradiographic study of assimilation of dissolved organic molecules

In a holothurian and an ophiuroid, tritiated glucose and glycine in great dilution are removed from seawater by uptake through the skin. Cells differ in their competence to metabolize specific nutrients, an indication that there are specialized cellular responses to exogenous organic molecules. Embryonic ophiuroid tissues have an exceptional capacity for assimilation.     

Inducing and viewing the rotational motion of a single molecule

Tunneling electrons from the tip of a scanning tunneling microscope were used to induce and monitor the reversible rotation of single molecules of molecular oxygen among three equivalent orientations on the platinum(111) surface. Detailed studies of the rotation rates indicate a crossover from a single-electron process to a multielectron process below a threshold tunneling voltage. Values for the energy barrier to rotation and the vibrational relaxation rate of the molecule were obtained by comparing the experimental data with a theoretical model. The ability to induce the controlled motion of single molecules enhances our understanding of basic chemical processes on surfaces and may lead to useful single-molecule devices.     

Algae: amounts of DNA and organic carbon in single cells

An analysis of ten different unicellular algae, varying in size and containing from 10 to 6000 picograms of carbon per cell, indicates that the amount of DNA per cell is in direct proportion to cell size. The content of DNA is equal to approximately 1 to 3 percent af the cellular organic carbon.