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Cell migration in fibrous extracellular matrix (ECM) is crucial to many physiological and pathological processes such as tissue regeneration, immune response, and cancer progression. During migration, individual cells can generate active pulling forces via actomyosin contraction, which are transmitted to the ECM fibers through focal adhesion complexes, remodel the ECM, and eventually propagate to and can be sensed by other cells in the system. The microstructure and physical properties of the ECM can also significantly influence cell migration, e.g., via durotaxis and contact guidance. Here, we develop a computational model for two-dimensional cell migration regulated by cell-ECM micromechanical coupling. Our model explicitly takes into account a variety of cellular-level processes, including focal adhesion formation and disassembly, active traction force generation and cell locomotion due to actin filament contraction, transmission and propagation of tensile forces in the ECM, as well as the resulting ECM remodeling. We validate our model by accurately reproducing single-cell dynamics of MCF-10A breast cancer cells migrating on collagen gels and show that the durotaxis and contact guidance effects naturally arise as a consequence of the cell-ECM micromechanical interactions considered in the model. Moreover, our model predicts strongly correlated multicellular migration dynamics, which are resulted from the ECM-mediated mechanical coupling among the migrating cell and are subsequently verified in in vitro experiments using MCF-10A cells. Our computational model provides a robust tool to investigate emergent collective dynamics of multicellular systems in complex in vivo microenvironment and can be utilized to design in vitro microenvironments to guide collective behaviors and self-organization of cells.

Single molecule techniques emerge as powerful and quantitative approaches for scientific investigations in last decades. Among them, single molecule fluorescence spectroscopy (SMFS) is able to non-invasively characterize and track samples at the molecular level. Here, applications of SMFS to fundamental biological questions have been briefly summarized in catalogues of single-molecule counting, distance measurements, force sensors, molecular tracking, and ultrafast dynamics. In these SMFS applications, statistics and physical laws are utilized to quantitatively analyze the behaviors of biomolecules in cellular signaling pathways and the mechanisms of biological functions. This not only deepens our understanding of bio-systems, but also provides a fresh angle to those fundamental questions, leading to a more quantitative thinking in life science.

The impact of the nanostructures of conjugated polymers on their electronic properties is significant. We notice that poly(3-hexylthiophene-2,5-diyl) (P3HT) can form nano-islands and nano-fibrils, and both of them show an similar single-layer thickness (∼1.5–1.6 nm) as evaluated by atomic force microscopy (AFM). The Raman spectra indicate the highest degree of chain planarity and crystallinity in the nano-islands. By using scanning Kelvin probe microscopy (SKPM) and electrostatic force microscopy (EFM), we image the surface potential at the nanometer scale and quantitatively estimate the charges on these nanostructures as modulated by light. Both results are consistent with each other: the surface potential increases on switching on the light, and the photo-induced charges are around 15–25% and positive. A similar amount of the photo-induced charges in both P3HT islands and fibril is consistent with the π–π stacking nature of these nanostructures. As P3HT is a hole-conducting molecule, positive charges are found easily injected into a single island by a positively biased EFM tip, and then the injected charges are able to propagate all over the island.

Based on mixed piezoelectric and ferroelectric nanostructures with polymers in various architectural forms, the flexible triboelectric nanogenerators (TENGs) have been extensively investigated. In this study, we design and fabricate lead-free ZnSnO 3 nanocubes @ polydimethylsiloxane (PDMS) based TENG by dispersing ZnSnO 3 nanocubes into PDMS elastomer at different ratios. Finally, the TENG can attain a maximum electrical output signal up to 400 V and 28 μA at a current density of 7 μA cm −2 , and an effective power to 3 mW at 6 wt% mass ZnSnO 3 mixed into PDMS. The harvested energy is utilized to drive 106 blue LEDs. In addition, this TENG generates stable electric power, even in the compressing-and-releasing process for over 500 cycles. Further investigation by electrostatic force microscopy shows clearly the maximum surface charge density appears at the optimal mixing ratio of ZnSnO 3 piezoelectric nanostructures into PDMS. This demonstrates that the optimization of the mixing ration between ZnSnO 3 piezoelectric nanostructures and PDMS is an effective method to increase the surface charges on the composite thin film and then improve the performance of the TENG. This TENG technology reveals a great candidate to be applied in large-scale device fabrications, flexible sensors and biological devices because of its easy fabrication process, high output power and biocompatibility.

Background Metastasis determines the lethality of cancer. In most clinical cases, patients are able to live with tumor proliferation before metastasis. Thus, the transition from tumor proliferation to metastasis/invasion is essential. However, the mechanism is still unclear and especially, the proliferation-to-metastasis/invasion transition point has not been well defined. Therefore, quantitative characterization of this transition is urgently needed. Methods We have successfully developed a home-built living-cell incubation system combined with an inverted optical microscope, and a systematic, quantitative approach to describing the major characteristic morphological parameters for the identification of the critical transition points for tumor-cell spheroids in a collagen fiber scaffold. Results The system focuses on in vitro tumor modeling, e.g. the development of tumor-cell spheroids in a collagen fiber scaffold and the monitoring of cell transition from proliferation to invasion. By applying this approach to multiple tumor spheroid models, such as U87 (glioma tumor), H1299 (lung cancer), and MDA-MB-231 (breast cancer) cells, we have obtained quantitative morphological references to evaluate the proliferation-to-invasion transition time, as well as differentiating the invasion potential of tumor cells upon environmental changes, i.e. drug application. Conclusions Our quantitative approach provides a feasible clarification for the proliferation-to-invasion transition of in vitro tumor models (spheroids). Moreover, the transition time is a useful reference for the invasive potential of tumor cells. General significance This quantitative approach is potentially applicable to primary tumor cells, and thus has potential applications in the fields of cancer metastasis investigations and clinical diagnostics.

In this report, a novel method towards synthesis of two-layer sandwiched graphene@(Li0.893Fe0.036)Co(PO4) nanoparticles (SG@LFCPO) has been presented. In the approach, the sheet-like precursor, as the sacrificial template, and glucose molecules, as the carbon source, are the key factors involved in forming the specific morphology in which both top and bottom graphene sheets tightly envelop the (Li0.893Fe0.036)Co(PO4) nanoparticles, just like a sandwich. Owing to the combination of various favorable conditions, such as Fe doping, graphene coating and morphology design, the as-prepared SG@LFCPO displays very promising performance in terms of rate performance (discharge capacity of 85 mA h g−1 at 20 C), cyclability (coulombic efficiency of around 92.6%), stability (capacity retention of 94.6% after 100 cycles) and fast kinetics.

A 3-D microfluidic system consisting of microchamber arrays embedded in a collagen hydrogel with tuneable biochemical gradients that mimics the tumour microenvironment of mammary glands was constructed for the investigation on the interactions between invasive breast cancer cells and stromal cells. The hollow microchambers in collagen provide a very similar 3-D environment to that in vivo that regulates collective cellular dynamics and behaviour, while the microfluidic channels surrounding the collagen microchamber arrays allow one to impose complex concentration gradients of specific biological molecules or drugs. We found that breast epithelial cells (MCF-10A) seeded in the microchambers formed lumen-like structures similar to those in epithelial layers. When MCF-10A cells were co-cultured with invasive breast cancer cells (MDA-MB-231), the formation of lumen-like structures in the microchambers was inhibited, indicating the capability of cancer cells to disrupt the structures formed by surrounding cells for further invasion and metastasis. Subsequent mechanism studies showed that down regulation of E-cad expression due to MMPs produced by the cancer cells plays a dominant role in determining the cellular behaviour. Our microfluidic system offers a robust platform for high throughput studies that aim to understand combinatorial effects of multiple biochemical and microenvironmental factors.

Cadherins, a large family of calcium-dependent adhesion molecules, are critical for intercellular adhesion. While crystallographic structures for several cadherins show clear structural similarities, their relevant adhesive strengths vary and their mechanisms of adhesion between types I and II cadherin subfamilies are still unclear. Here, stretching of cadherins was explored experimentally by atomic force microscopy and computationally by steered molecular dynamics (SMD) simulations, where partial unfolding of the E-cadherin ectodomains was observed. The SMD simulations on strand-swapping cadherin dimers displayed similarity in binding strength, suggesting contributions of other mechanisms to explain the strength differences of cell adhesion in vivo. Systematic simulations on the unfolding of the extracellular domains of type I and II cadherins revealed diverse pathways. However, at the earliest stage, a remarkable similarity in unfolding was observed for the various type I cadherins that was distinct from that for type II cadherins. This likely correlates positively with their distinct adhesive properties, suggesting that the initial forced deformation in type I cadherins may be involved in cadherin-mediated adhesion. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:25

Development of an in vitro three-dimensional (3D) model that closely mimics actual environment of tissue has become extraordinarily important for anti-cancer study. In recent years, various 3D cell culture systems have been developed, with multicellular tumor spheroids being the most popular and effective model. In this work, we present a microfluidic device used as a robust platform for generating core–shell hydrogel microspheres with precisely controlled sizes and varied components of hydrogel matrix. To gain a better understanding of the governing mechanism of microsphere formation, computational models based on multiphase flow were developed to numerically model the droplet generation and velocity field evolution process with COMSOL Multiphysics software. Our modeling results show good agreement with experiments in size dependence on flow rate as well as effect of vortex flow on microsphere formation. With real-time tuning of the flow rates of aqueous phase and oil phase, tumor cells were encapsulated into the microspheres with controllable core–shell structure and different volume ratios of core (comprised of alginate, Matrigel, and/or Collagen) and shell (comprised of alginate). Viability of cells in four different hydrogel matrices were evaluated by standard acridine orange (AO) and propidium iodide (PI) staining. The proposed microfluidic system can play an important role in engineering the in vitro micro-environment of tumor spheroids to better mimic the actual in vivo 3D spatial structure of a tumor and perfect the 3D tumor models for more effective clinical therapies.

A systematic understanding of the evolution and growth dynamics of invasive solid tumors in response to different chemotherapy strategies is crucial for the development of individually optimized oncotherapy. Here, we develop a hybrid three-dimensional (3D) computational model that integrates pharmacokinetic model, continuum diffusion-reaction model and discrete cell automaton model to investigate 3D invasive solid tumor growth in heterogeneous microenvironment under chemotherapy. Specifically, we consider the effects of heterogeneous environment on drug diffusion, tumor growth, invasion and the drug-tumor interaction on individual cell level. We employ the hybrid model to investigate the evolution and growth dynamics of avascular invasive solid tumors under different chemotherapy strategies. Our simulations reproduce the well-established observation that constant dosing is generally more effective in suppressing primary tumor growth than periodic dosing, due to the resulting continuous high drug concentration. In highly heterogeneous microenvironment, the malignancy of the tumor is significantly enhanced, leading to inefficiency of chemotherapies. The effects of geometrically-confined microenvironment and non-uniform drug dosing are also investigated. Our computational model, when supplemented with sufficient clinical data, could eventually lead to the development of efficient in silico tools for prognosis and treatment strategy optimization., Comment: 41 pages, 8 figures

We report Atomic Force Microscopy (AFM) and Scanning Kelvin Probe Microscopy (SKPM) studies on the surface morphology and surface potential properties of CH3NH3PbI3, CH3NH3PbI3-xClx, CH3NH3PbI3-xBrx and CH3NH3PbBr3-xClx, respectively. For CH3NH3PbI3 rod structure, its surface potential is independent of the precursor concentration, suggesting a robust electronic feature. Surface potential studies of CH3NH3PbI3 particle reveal that the Fermi level within CH3NH3PbI3 is strongly influenced by the substrate. In the case of CH3NH3PbI3-xClx, its surface potential depends on precursor concentrations and we suspect that chlorine concentrated solutions might lead to more chlorine incorporation in the final products, thus lowering its Fermi level. Also, we studied the surface potentials of CH3NH3PbI3-xBrx and CH3NH3PbBr3-xClxwith specified halide ratios. The surface potential differences between different samples are related to their work function variations. These results are helpful to the understanding of the structural and electronic properties of perovskite materials.

Increasing the triboelectric charge density on the friction layer of polydimethylsiloxane (PDMS) is a basic approach towards improving the output performance of a triboelectric nanogenerator (TENG). Most previous work focuses on the surface structure or dielectric properties, nonetheless, a few studies have focused on electronegative modification. NaNbO3-PDMS TENG (N-TENG) devices are fabricated by dispersing cubic NaNbO3, which is a lead-free piezoelectric material with molecular oxygen dangling bonds on the surface of the crystal, into the PDMS at different mass ratios. When the mass ratio is 7 wt%, the maximum output performance of the N-TENG is obtained. The open-circuit voltage is 550 V, the short-circuit current is 16 µA, and the effective power densities reach up to 5.5 W m−2 at a load resistance of ~100 MΩ. The N-TENG has been used to assemble self-powered electronic watches and illuminate commercial light-emitting diodes, respectively. Its fundamental mechanism has also been discussed in detail from the perspective of dielectric modulation and electronegative modification. This N-TENG technology is revealed to be a splendid candidate for application in large-scale device fabrication, flexible sensors and biological devices thanks to its easy fabrication process, low consumption, high output power density and biocompatibility.

The mechanism by which light is converted into chemical energy in a natural photosynthetic system has drawn considerable research interest. Using fluorescence spectroscopy and microscopic imaging, ...

Cadherin-mediated adhesion plays a crucial role in multicellular organisms. Dysfunction within this adhesion system has major consequences in many pathologies, including cancer invasion and metastasis. However, mechanisms controlling cadherin recognition and adhesive strengthening are only partially understood. Here, we investigated the homophilic interactions and mechanical stability of the extracellular (EC) domains of E-cadherin and cadherin 7 using atomic force microscopy and magnetic tweezers. Besides exhibiting stronger interactions, E-cadherin also showed more efficient force-induced self-strengthening of interactions than cadherin 7. In addition, the distributions of the unbinding forces for both cadherins partially overlap with those of the unfolding forces, indicating that partial unfolding/deformation of the cadherin EC domains may take place during their homophilic interactions. These conformational changes may be involved in cadherins physiology function and contribute to the significant differences in adhesive strength mediated by type I and type II cadherins.

Nature Communications 5: Article number: 4525 (2014); Published: 31 July 2014; Updated: 4 March 2015 The affiliation details for Jie Yan are incorrect in this Article. The correct affiliation details for this author are given below: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore.

Quantum chemical calculations and experiments on model compounds are used to study the effect of conformational changes on intramolecular electron transfer in perylenebisimide–biphenyl dyads. The molecular orbital energy level of the biphenyl moiety and thus the free energy change for electron transfer depends on the dihedral angle between the two phenyl rings. Dramatic changes in the orbital energy levels result in very different electron transfer behavior for three model compounds. These changes are accurately predicted by calculations using density functional theory, which also give accurate results for geometries, suggesting such calculations can be useful in designing molecular electronic species.

It is widely appreciated that single-molecule spectroscopy (SMS) can be used to measure properties of individual molecules which would normally be obscured in an ensemble-averaged measurement. In this report we show how SMS can be used to measure photoinduced interfacial electron transfer (IET) and back electron transfer rates in a prototypical chromophore-bridge-electrode nonadiabatic electron transfer system. N-(1-hexylheptyl)-N'-(12-carboxylicdodecyl)perylene-3,4,9,10-tetracarboxylbisimide was synthesized and incorporated into mixed self-assembled monolayers (SAMs) on an ITO (tin-doped indium oxide, a p-type semiconductor) electrode. Single-molecule fluorescence time trajectories from this system reveals "blinks", momentary losses in fluorescence (>20 ms to seconds in duration), which are attributed to discrete electron transfer events: electron injection from the perylene chromophore into the conduction band of the ITO leads to the loss of fluorescence, and charge recombination (back electron transfer) leads to the return of fluorescence. Such blinks are not observed when an electrode is not present. The fluorescence trajectories were analyzed to obtain the forward and back electron rates; the measured rates are found to lie in the millisecond to second regime. Different rates are observed for different molecules, but the lifetime distributions for the forward or back electron transfer for any given molecule are well fit by single exponential kinetics. The methodology used is applicable to a wide variety of systems and can be used to study the effects of distance, orientation, linker, environment, etc. on electron transfer rates. The results and methodology have implications for molecular electronics, where understanding and controlling the range of possible behaviors inherent to molecular systems will likely be as important as understanding the individual behavior of any given molecule.

It is widely appreciated that single-molecule spectroscopy (SMS) can be used to measure properties of individual molecules which would normally be obscured in an ensemble-averaged measurement. In this article, we show how SMS can be used to investigate intramolecular electron transfer (IET) processes in model dimer systems composed of two perylene chromophores connected via an adjustable bridge. The fluorescence behaviors of a large number of molecules are cataloged and the results statistically analyzed to gauge information about the range of behaviors of the ensemble. Single-molecule fluorescence time trajectories reveal “blinks”, momentary losses in fluorescence (>20 ms to seconds in duration), which are attributed to discrete IET excursions to the charge-separated (CS) state. We find that fluorescence blinking behavior is dependent on bridge length and chromophore geometry, which affect the electronic coupling and therefore the IET. The statistical trends observed in this analysis are used to corrobor...

Preferred-orientated 4H–SiC nanocrystalline films on silicon substrates were successfully prepared by the pyrolysis of the polyimide Langmuir–Blodgett films containing nanometer-sized amorphous silicon nitride powders. Fourier-transform infrared spectra revealed 4H–SiC nanoparticles with interfacial silicon oxide. Ultraviolet photoluminescence with energies above the band gap of bulk 4H–SiC was observed from the films in which the mean size of 4H–SiC particles was 10 nm, while no photoluminescence was observed from the films in which the mean size of particles was 17 nm. A quantum confinement/luminescence center model is suggested to explain the origin of the ultraviolet luminescence.

Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2–9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing., Author Summary Mechanical stretching of cells causes the substrate domain of p130Cas (CasSD) to be phosphorylated on 15 tyrosine residues embedded along its length. CasSD is rich in proline and surprisingly well conserved in placental mammals. Stretching of CasSD by atomic force microscopy has identified that it requires far less force than normal folded proteins. Classical biophysical analyses have determined that CasSD is a typical intrinsically disordered protein, a difficult-to-study group of molecules covering about 30% of human proteins. The average size of CasSD is larger and elongated than folded globular proteins but smaller than chemically denatured proteins. We have simulated a large number of all-atom protein structures using a fast all-atom sampling method. The result is in good agreement with the experimental observation. As it is already known that stretching somehow exposes the tyrosine residues to phosphorylation, a mechanism is proposed where straightening of the p130Cas substrate domain backbone conformation through mechanical stretching can lead to dissociation of p130Cas-binding LIM domain proteins and exposure of CasSD tyrosine residues for phosphorylation. This study has led to a new model of a protein-based mechanism of force sensing at the leading edge of cells that allows the cells to feel their way as they move.

Force sensing at cadherin-mediated adhesions is critical for their proper function. α-Catenin, which links cadherins to actomyosin, has a crucial role in this mechanosensing process. It has been hypothesized that force promotes vinculin binding, although this has never been demonstrated. X-ray structure further suggests that α-catenin adopts a stable auto-inhibitory conformation that makes the vinculin-binding site inaccessible. Here, by stretching single α-catenin molecules using magnetic tweezers, we show that the subdomains MI vinculin-binding domain (VBD) to MIII unfold in three characteristic steps: a reversible step at ~5 pN and two non-equilibrium steps at 10-15 pN. 5 pN unfolding forces trigger vinculin binding to the MI domain in a 1:1 ratio with nanomolar affinity, preventing MI domain refolding after force is released. Our findings demonstrate that physiologically relevant forces reversibly unfurl α-catenin, activating vinculin binding, which then stabilizes α-catenin in its open conformation, transforming force into a sustainable biochemical signal.

Intense ultraviolet (UV) photoluminescence (PL) was observed at room temperature from amorphous Si:O:C films synthesized at 200, 300, 400°C by the liquid–solution-phase (LSP) method. The intensity was as strong as that of visible emission band from porous silicon. In all the films, a band existed, centered at 340 nm, while a shoulder band centered at 380 nm was observed from the films deposited at 400°C. By analyzing the PL and photoluminescence excitation (PLE) spectra, Fourier-transform infrared (FTIR) absorption, X-ray photoelectronic spectroscopy (XPS) and X-ray diffraction (XRD), we suggest that the photoluminescence peak at 340 nm originates from the defects in silicon oxide network, while the peak at 380 nm may be related to Si–C and Si–Si bonds in the samples.

α -SiC thin layers were successfully grown on silicon (111) by pyrolysis of the organic liquid dip-coating films and polyimide Langmuir–Blodgett (LB) films containing nanometer sized amorphous silicon nitride powders (NASNP) at 900–1000°C in vacuum. The products were characterized by X-ray diffraction (XRD), infrared absorption (IR) and Raman spectra, the results of which strongly support the formation of preferred orientated 4H-SiC in the layers by pyrolysis of LB films containing NASNP. Based on the IR spectra of samples pyrolyzed at different temperatures and the computed values of the Gibbs free energy changes of the reactions, the mechanism of formation of α -SiC is discussed.

By combining nano-particles and the Langmuir–Blodgett method of depositing films, SiC thin films were successfully grown on Si substrates, the growth temperature for the synthesis of SiC/Si composites is lowered to 700°C. The growth process based on the fourier transform-infrared (FTIR) spectra is discussed. The films on Si(111) substrates exhibit preferred orientation and weak photo-luminescence is detected from samples grown on p-type Si substrates.

β-Silicon carbide layers have been prepared by high temperature pyrolysis of polyimide Langmuir-Blodgett films on porous silicon substrate in vacuum. The formation of silicon carbide is confirmed by the IR and XRD spectra. It is found that photoluminescence still exists and appears in the blue-green and ultraviolet regions after thermal treatment at 900°C. These results indicate that the silicon carbide layers, which are formed, are responsible for the blue-green luminescence.

P130Cas is a docking protein essentially coordinating tyrosine-kinase-based signaling pathways associated with cell adhesion and migration etc. Its central substrate domain (CasSD) can bind to Crk and includes 15 YxxP motifs, where most tyrosine phosphorylation happens. It has been shown that CasSD can be stretched to promote phosphorylation, the mechanism of which needs to be explored in detail. Thus, it is important to uncover the native structure(s) of CasSD and the structural changes associated with mechanical stretching, both of which are still unclear. Here, we used atomic force microscopy force mode and magnetic tweezers to stretch individual molecules of CasSD constructs. Our results showed that the CasSD domain was intrinsically disordered. Natively, CasSD domains took many conformations beside random coils, while most of these conformations possessed limited mechanical stability. In magnetic tweezers experiments, the intramolecular interactions stabilizing the varied native conformations of CasSD were found similar in strength. Such diversity in native conformations of CasSD domains, as discovered here, should play important role in their signaling functions and their limited strength should be relevant to the mechanical activation of those signaling pathways.

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

Mechanical manipulation at the single molecule level of proteins exhibiting mechanical stability poses a technical challenge that has been almost exclusively approached by atomic force microscopy (AFM) techniques. However, due to mechanical drift limitations, AFM techniques are restricted to experimental recordings that last less than a minute in the high-force regime. Here we demonstrate a novel combination of electromagnetic tweezers and evanescent nanometry that readily captures the forced unfolding trajectories of protein L at pulling forces as low as 10 ∼ 15 pN. Using this approach, we monitor unfolding and refolding cycles of the same polyprotein for a period of time longer than 30 min. From such long-lasting recordings, we obtain ensemble averages of unfolding step sizes and rates that are consistent with single-molecule AFM data obtained at higher stretching forces. The unfolding kinetics of protein L at low stretching forces confirms and extends the observations that the mechanical unfolding rate is exponentially dependent on the pulling force within a wide range of stretching forces spanning from 13 pN up to 120 pN. Our experiments demonstrate a novel approach for the mechanical manipulation of single proteins for extended periods of time in the low-force regime.

The molecular mechanism by which a mechanical stimulus is translated into a chemical response in biological systems is still unclear. We show that mechanical stretching of single cytoplasmic proteins can activate binding of other molecules. We used magnetic tweezers, total internal reflection fluorescence, and atomic force microscopy to investigate the effect of force on the interaction between talin, a protein that links liganded membrane integrins to the cytoskeleton, and vinculin, a focal adhesion protein that is activated by talin binding, leading to reorganization of the cytoskeleton. Application of physiologically relevant forces caused stretching of single talin rods that exposed cryptic binding sites for vinculin. Thus in the talin-vinculin system, molecular mechanotransduction can occur by protein binding after exposure of buried binding sites in the talin-vinculin system. Such protein stretching may be a more general mechanism for force transduction.

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

We report here on the systematic investigation of photoinduced intramolecular electron transfer (IET) in a series of donor-bridge-acceptor molecules as a means of understanding electron transport through the bridge. Perylenebisimide chromophores connected by various oligophenylene bridges are studied because their electron-transfer behavior can readily be monitored by following changes in the fluorescence intensity. We find dramatic switching of the IET behavior as the solvent polarity (dielectric constant) is increased. By combining steady-state and time-resolved fluorescence spectroscopy in a variety of solvents at multiple temperatures with standard theories of electron transfer, we determine parameters governing the IET behavior of these dimers, such as the electronic coupling through the bridges. We also deploy available ab initio quantum chemical methods to calculate the through-space component of the electronic coupling matrix element. Single-molecule investigations of the electron-transfer behavior also show that IET can be switched reversibly by a similar mechanism in an isolated individual molecule.

Detecting structure, dynamics, and chemical reactions at the single-molecule level represents the ultimate degree of sensitivity for sensing and imaging. There is a tremendous need to develop new molecular systems and methodology for single-molecule-based sensing. This work presents for the first time the single-molecule spectroscopy of a new molecular probe which uses an intramolecular electron transfer mechanism to detect binding, local structure, and interfacial processes. Moreover, we show how information about the interaction of these probes with their environment is obtained from an analysis of the intensity, duration and time-varying behavior of the single-molecule fluorescence.