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Herein, we compared the ability of linear and cyclic peptides generated in silico to target different protein sites: internal pockets and solvent-exposed sites. We selected human lysozyme (HuL) as a model target protein combined with the computational evolution of linear and cyclic peptides. The sequence evolution of these peptides was based on the PARCE algorithm. The generated peptides were screened based on their aqueous solubility and HuL binding affinity. The latter was evaluated by means of scoring functions and atomistic molecular dynamics (MD) trajectories in water, which allowed prediction of the structural features of the protein-peptide complexes. The computational results demonstrated that cyclic peptides constitute the optimal choice for solvent exposed sites, while both linear and cyclic peptides are capable of targeting the HuL pocket effectively. The most promising binders found in silico were investigated experimentally by surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS) techniques. All tested peptides displayed dissociation constants in the micromolar range, as assessed by SPR; however, both NMR and ESI-MS suggested multiple binding modes, at least for the pocket binding peptides. A detailed NMR analysis confirmed that both linear and cyclic pocket peptides correctly target the binding site they were designed for.

Background: DNA hybridization is at the basis of most current technologies for genotyping and sequencing, due to the unique properties of DNA base-pairing that guarantee a high grade of selectivity. Nonetheless the presence of single base mismatches or not perfectly matched sequences can affect the response of the devices and the major challenge is, nowadays, to distinguish a mismatch of a single base and, at the same time, unequivocally differentiate devices read-out of fully and partially matching sequences.Results: We present here two platforms based on different sensing strategies, to detect mismatched and/or perfectly matched complementary DNA strands hybridization into ssDNA oligonucleotide monolayers. The first platform exploits atomic force microscopy-based nanolithography to create ssDNA nano-arrays on gold surfaces. AFM topography measurements then monitor the variation of height of the nanostructures upon biorecognition and then follow annealing at different temperatures. This strategy allowed us to clearly detect the presence of mismatches. The second strategy exploits the change in capacitance at the interface between an ssDNA-functionalized gold electrode and the solution due to the hybridization process in a miniaturized electrochemical cell. Through electrochemical impedance spectroscopy measurements on extended ssDNA self-assembled monolayers we followed in real-time the variation of capacitance, being able to distinguish, through the difference in hybridization kinetics, not only the presence of single, double or triple mismatches in the complementary sequence, but also the position of the mismatched base pair with respect to the electrode surface.Conclusion: We demonstrate here two platforms based on different sensing strategies as sensitive and selective tools to discriminate mismatches. Our assays are ready for parallelization and can be used in the detection and quantification of single nucleotide mismatches in microRNAs or in genomic DNA.

We studied the lateral forces arising during the vertical indentation of the cell membrane by an optically trapped microbead, using back focal plane interferometry to determine force components in all directions. We analyzed the cell-microbead interaction and showed that indeed the force had also lateral components. Using the Hertz model, we calculated and compared the elastic moduli resulting from the total and vertical forces, showing that the differences are important and the total force should be considered. To confirm our results we analyzed cells from two breast cancer cell lines: MDA-MB-231 and HBL-100, known to have different cancer aggressiveness and hence stiffness. 2016 Copyright © Taylor & Francis Group, LLC

The number of circulating tumor cells (CTCs) in blood is strongly correlated with the progress of metastatic cancer. Current methods to detect CTCs are based on immunostaining or discrimination of physical properties. Herein, a label-free method is presented exploiting the abnormal metabolic behavior of cancer cells. A single-cell analysis technique is used to measure the secretion of acid from individual living tumor cells compartmentalized in microfluidically prepared, monodisperse, picoliter (pL) droplets. As few as 10 tumor cells can be detected in a background of 200 000 white blood cells and proof-of-concept data is shown on the detection of CTCs in the blood of metastatic patients.

Micro and nanomechanical resonators represent a promising platform for proteins label-free detection because of their extreme sensitivity, fast response and low cost. Micro-pillars are columnar resonators that can be easily arranged in dense arrays of several thousand sensors in a squared mm. To exploit such a large density, however, a method for tracking independently micropillars resonance frequency is required. Here we present a detection method based on CCD imaging and software image analysis, which can measure the resonance frequency of tens of pillars in parallel. Acquiring simultaneously the frequency shift of up to 40 sensors and applying a proper statistical analysis, we were able to overcome the variability of the single measures improving the device sensitivity at low analyte concentration range. As a proof of concept, this method has been tested for the detection of a tumor marker, the Prostate Specific Membrane Antigen (PSMA). Pillars have been functionalized with an antibody against PSMA. The tumor marker (PSMA) has been detected in a range of concentrations between 300 pM and 100 nM, in buffer and in diluted bovine serum. The sensitivity of our method was limited only by the affinity constant of the antigen–antibody recognition. Moreover, this detection technique demonstrated to be effective in the 1–6 nM range, which is the window of PSMA concentration of clinical interest.

Micro- and nano-mechanical resonators such as cantilevers and pillars are more and more used as molecular sensors, due to their high sensitivity, ease of fabrication and high throughput. In order to modify their wetting properties and to prepare them for the selective adsorption of the molecules of interest, the sensor active surface has to be properly functionalized. This can significantly affect their mechanical performances. In this paper we report on the effect on a micropillar array of different hydrophobic treatments, based on plasma deposition of a fluorocarbon film (FCF) and on the formation of an alkanosilane coating, to obtain a superhydrophobic state. Each treatment was characterized by measuring the change of the quality factor in vacuum, the static contact angle and evaluating the compatibility with gold deposition, to create a bio-functional layer. As case study, DNA self assembled monolayer (SAM) formation and hybridization efficiency were measured. Finally, the superhydrophobicity was tested with diluted human plasma, as preliminary step toward the use of micropillars for biomolecular detection in human samples. (C) 2014 Elsevier B.V. All rights reserved.

The mechanical properties of cells are influenced by their microenvironment. Here we report cell stiffness alteration by changing the cell substrate stiffness for isolated cells and cells in contact with other cells. Polydimethylsiloxane (PDMS) is used to prepare soft substrates with three different stiffness values (173, 88 and 17 kPa respectively). Breast cancer cells lines, namely HBL-100, MCF-7 and MDA-MB-231 with different level of aggressiveness are cultured on these substrates and their local elasticity is investigated by vertical indentation of the cell membrane. Our preliminary results show an unforeseen behavior of the MDA-MB-231 cells. When cultured on glass substrate as isolated cells, they are less stiff than the other two types of cells, in agreement with the general statement that more aggressive and metastatic cells are softer. However, when connected to other cells the stiffness of MDA-MB-231 cells becomes similar to the other two cell lines. Moreover, the stiffness of MDA-MB-231 cells cultured on soft PDMS substrates is significantly higher than the stiffness of the other cell types, demonstrating thus the strong influence of the environmental conditions on the mechanical properties of the cells.

The oriented immobilization of proteins, key for the development of novel responsive biomaterials, relies on the availability of effective probes. These are generally provided by standard approaches based on in vivo maturation and in vitro selection of antibodies and/or aptamers. These techniques can suffer technical problems when a non-immunogenic epitope needs to be targeted. Here we propose a strategy to circumvent this issue by in silico design. In our method molecular binders, in the form of cyclic peptides, are computationally evolved by stochastically exploring their sequence and structure space to identify high-affinity peptides for a chosen epitope of a target globular protein: here a solvent-exposed site of β2-microglobulin (β2m). Designed sequences were screened by explicit solvent molecular dynamics simulations (MD) followed by experimental validation. Five candidates gave dose-response surface plasmon resonance signals with dissociation constants in the micromolar range. One of them was further analyzed by means of isothermal titration calorimetry, nuclear magnetic resonance, and 250 ns of MD. Atomic-force microscopy imaging showed that this peptide is able to immobilize β2m on a gold surface. In short, we have shown by a variety of experimental techniques that it is possible to capture a protein through an epitope of choice by computational design.

When I was facing the daunting task of recounting my approximately 70 years of scientific career and some aspects of my personal life, I decided to write this autobiographical piece in a “different” way. After a section on the almost bare facts of my life (Section 1), I include several other parts, loosely connected in time, in each of which I make an almost self-contained point, decreasing in this way the need for consistency and coherence in the whole article. I discuss, for example, the importance of original ideas, the independence of junior colleagues, and my work with molecular beams and in nanomedicine. I end by acknowledging those I was lucky enough to learn from in my intellectual development as a research scientist and with a couple of recommendations that may be useful to my junior and not-so-junior colleagues.

Background Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data. Methods This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45pos leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45neg cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively. Results By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45neg subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45neg cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment. Conclusion This study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0687-3) contains supplementary material, which is available to authorized users.

We have developed a micromechanical sensor based on vertically oriented oscillating beams, in which contrary to what is normally done (for example with oscillating cantilevers) the sensitive area is located at the free end of the oscillator. In the micropillar geometry used here, analyte adsorption is confined only to the tip of the micropillar, thus reducing the volume from which the analyte molecules must diffuse to saturate the surface to a sphere of radius more than 2 orders of magnitude smaller than the corresponding linear distance valid for adsorption on a macroscopic surface. Hence the absorption rate is 3 orders of magnitude faster than on a typical 200 × 20 square micrometer cantilever. Pillar oscillations are detected by means of an optical lever method, but the geometry is suitable for multiplexing with compact integrated detection. We demonstrate our technology by investigating the formation of a single-strand DNA self-assembled monolayer (SAM) consisting of less than 10(6) DNA molecules and by measuring their hybridization efficiency. We show that the binding rate is 1000 times faster than on a "macroscopic" surface. We also show that the hybridization of a SAM of maximum density DNA is 40% or 4 times the value reported in the literature. These results suggest that the lower values previously reported in the literature can be attributed to incomplete saturation of the surface due to the slower adsorption rate on the "macroscopic" surfaces used.

We use conductive-probe atomic force microscopy (CP-AFM) and an AFM based nanolithographic technique, called nanografting, to investigate the electron transport mediating properties of self-assembled monolayers (SAMs) of thiols on gold surfaces. We investigated many factors that can influence the measurements, including the contact nature, the roughness of the substrate, the magnitude of the applied normal load, the diversity of different probes, and the tip wearing during the measurements. All these factors influence the measured current value but to a very variable extent. Molecules and impurities adsorbed at the tip/substrate interface are found to be the major cause of the spread of the experimental results. Our work results in the clarification of a series of phenomena to which more attention should be paid in future experimentation.

We demonstrate that scattering scanning near-field infrared microscopy (s-SNIM) is a label free analytical method allowing hybridization detection of nanografted DNA patches on a sub-μM scale. On the basis of their distinct dielectric properties in the IR, we can distinguish between single stranded and double stranded DNA. The sensitivity of s-SNIM is found to be increased by 7 orders of magnitude compared to conventional FTIR spectroscopy due to the tip enhanced interaction with the substrate.

A new series of dendron-functionalized multiwalled carbon nanotube (MWNT) derivatives, characterized by the presence of numerous positively charged tetraalkyl ammonium salts at the periphery of the dendron, has been synthesized. The positive charges on the MWNT surface, coupled with the unique ability of carbon nanotubes (CNTs) to penetrate cell membranes, make the new derivatives potentially ideal vectors for siRNA delivery. Using a fluorescently labeled, noncoding siRNA sequence, we demonstrate that cytoplasmic delivery of the nucleic acid is remarkably increased throughout the different dendron generations. The work reported here highlights the fact that dendron-functionalized CNTs can be rationally designed as efficient carriers of siRNA that can eventually lead to gene silencing.

To understand better enzyme/DNA interactions and to design innovative detectors based on DNA nanoarrays, we need to study the effect of nanometric confinement on the biochemical activity of the DNA molecules. We focus on the study of the restriction enzyme reactions (DpnII) within DNA nanostructures on flat gold films by atomic force microscopy (AFM). Typically we work with a few patches of DNA self assembled monolayers (SAMs) that are hundred nm in size and are lithographically fabricated within alkylthiol SAMs by AFM nanografting. We start by nanografting a few patches of a single-stranded DNA (ssDNA) molecule of 44 base pairs (bps) with a 4 bps recognition sequence (specific for DpnII) in the middle. Afterwards, reaction-ready DNA nanopatches are obtained by hybridization with a complementary 44bps ssDNA sequence. The enzymatic reactions were carried out over nanopatches with different density. By carrying out AFM height measurements, we are able to show that the capability of the DpnII enzyme to reach and react at the recognition site is easily varied by controlling the DNA packing in the nanostructures. We have found strong evidence that inside our ordered DNA nanostructures the enzyme (that works as a dimer) can operate down to the limit in which the space between adjacent DNA molecules is equal to the size of the DNA/enzyme complex. Similar experiments were carried out with a DNA sequence without the recognition site, clearly finding that in that case the enzymatic reaction did not lead to digestion of the molecules. These findings suggest that it is possible to tune the efficiency of an enzymatic reaction on a surface by controlling the steric hindrance inside the DNA nanopatches without vary any further physical or chemical variable. These findings are opening the door to novel applications in both the fields of biosensing and fundamental biophysics.

We use atomic force microscopy (AFM) based force-compression measurements to probe the ligand-induced functional conformational changes in surface-immobilized dicysteine-terminated maltose binding proteins (dicys-MBPs). The proteins are immobilized at well-defined locations directly on Au substrates using the previously reported technique of nanografting. By measuring the difference between the ligand-free and ligand-bound mechanical work performed by the AFM-tip during the protein compression, we determine the open-closed transition energy for dicys-MBPs to be DeltaE0 = (8 +/- 4) Kcal/mol. We also compare the binding kinetics of two different ligands (maltose and maltotriose) to dicys-MBPs by performing AFM-friction measurements. We show that our results are consistent with a simple model for the surface-immobilized dicys-MBPs: the protein consists of two rigid lateral lobes connected by a hinge-loaded spring.

Roger Miller, perhaps the most outstanding experimental physical chemist of his generation, was sadly taken from us at the age of53 years, at the peak of his career. Roger Ervin Miller was born in Kitchener, Ontario, Canada, on 23 July 1952, the son of Ruth and Donald R. Miller. Roger had one older brother, Gerry, and one younger sister, Patricia. Roger's father worked in a tyre factory and was very innovative and creative in mending items around the house, a trait that he passed on to his children. From a very early age Roger was curious about the world around him. He was also a hard worker, raising rabbits or picking dew worms to earn money so that he could purchase model rockets, the bigger the better. This was the time of Sputnik and the US space programme, Roger being just 5 years old when the first Sputnik satellite was launched in 1957. As Roger explained in a memoir that he began to write in 2000 (but never finished; extracts are reproduced by courtesy of Mrs. Debbie Miller)

The in vivo reparative potential of Cardiac Stem Cells (CSC), cultured from explanted failing hearts (E-), is impaired by cellular senescence. Moreover, E-CSC are characterized, with respect to CSC obtained from healthy donors (D-), by an arrest in the autophagic degradation. Although the lysosome plays a pivotal role in cellular homeostasis and defects of this organelle may be associated with aging and heart failure, the lysosomal function of CSC has never been investigated. The aim of this work was to focus on the Lysosomal Compartment (LC) of E-CSC, evaluating elements that could jeopardize lysosome functionality. Methods and results Bioinformatics analysis conducted on genes differentially expressed between D- and E-CSC identified lysosomal-related gene sets as significantly enriched. Moreover, 29 differentially expressed genes were part of CLEAR (Coordinated Lysosomal Expression and Regulation) gene network, by which Transcription Factor EB (TFEB) regulates cellular clearance. Consistently, live cell imaging and flow cytometry analyses showed that the lysosomes of E-CSC are less acidic than the D-CSC ones. Furthermore, confocal microscopy showed in E-CSC: an accumulation of intralysosomal lipofuscins, a reduction of cathepsin B activity, evidence of lysosome membrane permeabilization, and the reduction of the nuclear active TFEB. The use of Rapamycin (TORC1 inhibitor) was able on one hand to increase TFEB activation and, on the other hand, to reduce lipofuscin mass, potentiating the lysosomal functionality. Conclusions This study demonstrated for the first time that E-CSC are characterized by a blunted activation of TFEB and an altered proteostasis. TORC1 hyperactivation plays a central role in this phenomenon.

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy.

In the last decade, cell elasticity has been widely investigated as a potential label free indicator for cellular alteration in different diseases, cancer included. Cell elasticity can be locally measured by pulling membrane tethers, stretching or indenting the cell using optical tweezers. In this paper, we propose a simple approach to perform cell indentation at pN forces by axially moving the cell against a trapped microbead. The elastic modulus is calculated using the Hertz-model. Besides the axial component, the setup also allows us to examine the lateral cell bead interaction. This technique has been applied to measure the local elasticity of HBL-100 cells, an immortalized human cell line, originally derived from the milk of a woman with no evidence of breast cancer lesions. In addition, we have studied the influence of substrate stiffness on cell elasticity by performing experiments on cells cultured on two substrates, bare and collagen-coated, having different stiffness. The mean value of the cell elastic modulus measured during indentation was 26 +/- 9 Pa for the bare substrate, while for the collagen-coated substrate it diminished to 19 +/- 7 Pa. The same trend was obtained for the elastic modulus measured during the retraction of the cell: 23 +/- 10 Pa and 13 +/- 7 Pa, respectively. These results show the cells adapt their stiffness to that of the substrate and demonstrate the potential of this setup for low-force probing of modifications to cell mechanics induced by the surrounding environment (e.g. extracellular matrix or other cells). (C) 2015 Elsevier Ltd. All rights reserved.

Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young’s modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines’ elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM.

We demonstrate that the Atomic Force Microscope (AFM) can be used to immobilize a dicysteine-terminated protein (Maltose Binding Protein, MBP-cys-cys for short) at well-defined locations directly on gold substrates via nanografting and characterize the in situ bioactivity of these proteins within the fabricated nanopatterns. This method exploits the high spatial and orientational control of the protein monolayer assembly allowed by nanografting, combined with the high sensitivity of the AFM for detecting ligand-binding events. The maltose-mediated conformational changes within the MBP have been found to change the AFM-tip-protein interaction, therefore causing the frictional signal to change. Our measurements show that the protein ligand-binding function is maintained upon the immobilization process and is not affected by (a) the addition of the cysteine dipeptide, (b) the spatial confinement associated with nanografting, and (c) the interaction between the protein and the Au substrate. These surface-confined proteins can also be regenerated, and their frictional response is reproducible through several maltose exposure/washing cycles. By measuring the change in the frictional force above the protein nanopatterns as a function of maltose concentration, we determined the dissociation constant for the MBP-cys-cys/maltose system to be kd = (1 +/- 0.04) microM. Our results show that the MBP-cys-cys system provides a very sensitive surface-based, protein nanobiosensor for maltose detection at the attogram level (approximately 100 nM concentration). The implications of our study for the fabrication of molecular-scale biological sensors are discussed at the end of the paper.

In this paper, we use atomic force microscopy (AFM) to nanostructure and image 1,10-decanedithiol (DDT) and biphenyl 4,4‘-dithiol (BPDT) layers on Au(111) surfaces comparing them to those prepared by self-assembly. First, layers of dithiols are self-assembled from solution onto gold surfaces and are imaged in situ with an AFM to examine the roughness of the layers. Second, 100 nm × 100 nm monolayer patches made of dithiol molecules are nanografted into a self-assembled monolayer (SAM) inert matrix made of 1-decanethiol (DT). Although nanografting of thiols routinely generates very compact layers with good height uniformity, nanostructuring of dithiols using this method always yields multilayers that form through intermolecular S−S bonds. We demonstrate in this paper two possible ways of tailoring, layer by layer, the structure of dithiols. First, we form multilayers by nanografting, using then the AFM tip to gradually shave away the top layers. In the second way, we add antioxidant to the solution while d...

The design of new strong and selective binders is a key step towards the development of new sensing devices and effective drugs. Both affinity and selectivity can be increased through chelation and here we theoretically explore the possibility of coupling two binders through a flexible linker. We prove the enhanced ability of double binders of keeping their target with a simple model where a polymer composed by hard spheres interacts with a spherical macromolecule, such as a protein, through two sticky spots. By Monte Carlo simulations and thermodynamic integration we show the chelating effect to hold for coupling polymers whose radius of gyration is comparable to size of the chelated particle. We show the binding free energy of flexible double binders to be higher than that of two single binders and to be maximized when the binding sites are at distances comparable to the mean free polymer end-to-end distance. The affinity of two coupled binders is therefore predicted to increase non linearly and in turn, by targeting two non-equivalent binding sites, this will lead to higher selectivity.

Certain organic molecules, such as alkanethiols, can adsorb on metals to form monolayers. Sometimes domains appear in the monolayers. For example, an incomplete monolayer may form islands, and a mixed-composition monolayer may separate into distinct phases. During annealing, the molecules diffuse on the metal surface. The domain boundary energy drives the domains to coarsen. The contact potential between the dissimilar domains drives the domains to refine. On the basis of existing experimental information, we suggest that the competition between coarsening and refining should stabilize certain domain patterns. We formulate a free energy functional to include the effects of mixed species, domain boundary, and contact potential. An approximate energy minimization estimates the equilibrium domain size. We derive a diffusion equation consistent with the free energy functional. The numerical solution of the diffusion equation follows the evolution of the monolayers from a random initial concentration field to patterns of dots and stripes. We also discuss the practical implications of the theory and, in particular, the possibility of guided self-assembly.

We have been able to grow well-ordered crystalline overlayers of either alkylthiols or dodecane on the surface of self-assembled monolayers (SAMs) of alkylthiols on Au(111) by exposing the surface ...

The threshold region of vibrational energy redistribution (IVR) presents a great experimental and computational challenge for organic molecules with more than 10 degrees of freedom. The density of states ρtot is high and requires high resolution measurements over a wide range to cover all relevant timescales experimentally. Yet ρtot is sufficiently low that IVR quantities like the initial relaxation time τIVR or the number of participating states Neff are very sensitive to the coupling structure. To highlight the competing effects of molecular symmetry and mode localization on the accessible density of states, this work complements a study of benzene (Callegari, A., Merker, U., Engels, P., Srivastava, H. K., Lehmann, K. K., and Scoles, G., 2000, J. chem. Phys., 113, 10583) by measuring the CH overtone spectra of pyrrole (C4H4NH) and 1,2,3-triazine (C3N3H3) using eigenstate-resolved double-resonance spectroscopy. Large scale computations of IVR dynamics were undertaken, applying filter diagonalization to a...

We study the binding of molecular oxygen to a graphene sheet and to a (8,0) single walled carbon nanotube, by means of spin-unrestricted density-functional calculations. We find that triplet oxygen retains its spin-polarized state when interacting with graphene or the nanotube. This leads to the formation of a weak bond with essentially no charge transfer between the molecule and the sheet or tube, as one would expect for a physisorptive bond. This result is independent on the approximation used for the exchange-correlation functional. The binding strength, however, depends strongly on the functional, reflecting the inability of current approximation functionals to deal correctly with dispersion forces. Gradient-corrected functionals yield very weak binding at distances around 4 A, whereas local density functional results yield substantially stronger binding for both graphene and the nanotube at distances of less than 3 A. The picture of oxygen physisorption is not substantially altered by the presence of topological defects such as 5–7 Stone–Wales pairs.

Short peptides can be designed in silico and synthesized through automated techniques, making them advantageous and versatile protein binders. A number of docking-based algorithms allow for a computational screening of peptides as binders. Here we developed ex-novo peptides targeting the maltose site of the Maltose Binding Protein, the prototypical system for the study of protein ligand recognition.We used a Monte Carlo based protocol, to computationally evolve a set of octapeptides starting from a polialanine sequence. We screened in silico the candidate peptides and characterized their binding abilities by surface plasmon resonance, fluorescence and electrospray ionization mass spectrometry assays. These experiments showed the designed binders to recognize their target with micromolar affinity. We finally discuss the obtained results in the light of further improvement in the exnovo optimization of peptide based binders. Copyright

Parallel three-helix bundle metalloproteins incorporating C-terminal thiol groups have been designed to orient vertically on a gold(111) surface. A small area of pre-assembled octadecanethiol was exchanged with the proteins under the action of an AFM tip. The resulting “nanografted” metalloprotein was imaged at low applied force. The measured height of the octadecanethiol monolayer was 2.2(1) nm and the mean height above this of the grafted metalloprotein layer was 3.1(4) nm. Thus the total height of the grafted metalloprotein is 5.3(4) nm. The predicted height of the vertically oriented metalloprotein is 5.2 nm. We take this as good evidence for spatial control of monolayer assembly of three-helix bundles with predictable orientation.

The structure and the electronic properties of a series of CH3- and CF3-terminated alkanethiol monolayers on Au(1 1 1) have been studied by scanning tunneling microscopy (STM) and surface X-ray and helium scattering. At full coverage, the CH3-terminated monolayers form long-range ordered domains of a ð p 3 � p 3ÞR30 and a ð2 p 3 � 3ÞR30 standing-up phase. By thermal desorption, distinct lying-down phases of intermediate density as well as the ðpp 3Þ lying-down phase were generated. In contrast, the CF3-terminated monolayers at full coverage form a standing-up phase of hexagonal symmetry that exhibits no long-range order at room temperature. Even after annealing, the domain sizes are smaller by more than one order of magnitude compared to the CH3-terminated thiol monolayers. A com- parison of the low-density lying-down phases suggests no measurable influence of the CF3-group on the ordering. The current-voltage dependence (I-V -curves) measured by scanning tunneling spectroscopy (STS) shows no voltage gap for CH3-terminated decanethiols. In contrast, in the I-V -curves for CF3-terminated decanethiol monolayers, an asym- metric voltage gap of about 2 V can be clearly observed. The latter results are discussed in terms of a microscopic model that includes the formation of an interfacial Coulomb barrier at the CF3/vacuum boundary. In addition, the effects of the tunneling conditions on the STM image contrast were examined. These studies demonstrate that the nature of the STM images and thus, the respective apparent lateral order of the films, strongly depends on the choice of the tunneling parameters. 2001 Elsevier Science B.V. All rights reserved.

Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular α-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of α-Synuclein on gold to study the early stages of α-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine-α-Synuclein adducts., Scientific Reports, 4, ISSN:2045-2322

While the attempts currently in progress in several groups for the rigorous inclusion of dispersion interactions in density functional theory (DFT) calculations mature and evolve into practical methodology, we contribute to the debate on the applicability of current functionals to the calculation of weak interaction with a systematic investigation of a few, typical, weakly bound systems. We have used both pure DFT and a hybrid approach in which the total interaction energy is partitioned into two parts: (a) the dispersion energy which, in a first approximation is the contribution due to intermonomer correlations and (b) all other interactions. The first component is accurately obtained at all distances of interest by means of a well-known damped multipolar expansion of the dispersion energy while for the second component different approximations will be evaluated. The need to avoid double counting a fraction of the correlation energy when using the hybrid approach and the choice of the appropriate functio...

The origin of the superlattice present in the commensurate hexagonal structure of self-assembled monolayers of n-alkanethiols on gold and the question of whether the thiols are adsorbed onto the surface as dimers (disulfides) or monomers (thiolates) have been under debate for many years. Looking for a better understanding of the structural properties of these systems, we have performed a theoretical study of the molecular and dissociative adsorption of dimethyl disulfide on Au(111) as a function of coverage (0.25 e £ e 1), using gradient-corrected density functional (DFT) calculations with a slab geometry. For the dissociated state, our results indicate that the hcp hollow site is much less favorable than the fcc site. For the latter site, we find that, because of surface gold atom relaxation, the adsorption energy depends strongly on £, changing from 18 kcal/mol at £ ) 0.25 to 3 kcal/mol at £ ) 1. For the bridge site, instead, the adsorption energy is a weak function of £, and for all investigated coverages, this site is by far the most stable. According to our DFT approach, the adsorption of dimethyl disulfide is dissociative with a thermodynamic gain, at £ ) 1, of 13 kcal/mol with respect to the adsorbed molecular state. We also find, however, that the energy of c(4 2) structures containing at least two inequivalent CH 3S groups per unit cell (with a minimum S-S distance of 3.7 A) is, within the accuracy of our approach, indistinguishable from the pure (x3 x3) hexagonal structure. Our results suggest that the full solution of this thorny problem will require, also for the shortest chains, an estimate of the energetic contribution of dispersion forces that are not included in the DFT calculations.

The state-selective dissociative sticking coefficient on Pt(111) surfaces for CH4 in the 2ν3 J=1,2 ro-vibrational levels has been measured using thermal energy atomic scattering. Continuous wave laser excitation of a molecular beam of CH4 seeded in He with a 1.5 μm color center laser, tunable around 6000 cm−1 and coupled to the beam by means of a resonant build-up cavity, allows pumping of up to 11% of the molecules to the excited ro-vibrational state. The laser/molecular beam combination allows precise control over the translational energy as well as the vibrational state of the methane that impinges on the clean Pt(111) surface. The intensity of the specular reflection of the incident helium beam is used to quantitatively monitor the coverage of chemisorbed methane on the platinum surface as a function of time (exposure). The sticking coefficient (S0) of CH4 with 5.4 kJ/mol normal translational energy is found to increase from 6.2×10−6 to 1.8×10−4 upon 2ν3 excitation (the overtone of the asymmetric stre...

The development of novel molecular and biomolecular devices relies on the understanding of charge transport across molecule-substrate interfaces. However, different strategies adopted so far for fabricating and studying transport through metal-molecule-metal junctions yield values for the transport coefficients that differ by up to orders of magnitude even for the same junction. Conductive tip atomic force microscopy (CT-AFM) allows for the simultaneous measurement of transport and morphological properties of molecular assemblies, but absolute transport measurements depend on the nature of the AFM tip-molecule contact. In this work we present a differential approach to the study of metal-molecule-metal junctions based on the combination of AFM-driven nanolithography and CT-AFM. We nanograft patches of alkanethiol molecules in a self-assembled monolayer of alkanethiol molecules of different chain length and measure by CT-AFM the morphology and the transport properties of the nanopatches and of the reference layer. The method allows for the determination of the differential resistance between the two molecular layers and is thus independent of environmental factors. The validity of this approach is demonstrated by measuring the tunneling decay constant of alkanethiols as a function of their length.

We have used infrared–infrared double resonance spectroscopy to record a rovibrational eigenstate resolved spectrum of benzene in the region of the CH stretch first overtone. This experiment is the first of a series aimed at investigating intramolecular vibrational energy redistribution (IVR) in aromatic molecules. The experiment has been carried out in a supersonic molecular beam apparatus using bolometric detection. A tunable resonant cavity was used to enhance the on-beam intensity of the 1.5 μm color center laser used to pump the overtone, and a fixed frequency [R(30)] 13CO2 laser was used to saturate the coinciding ν18 rQ(2) transition of benzene. After assigning the measured lines of the highly IVR fractionated spectrum to their respective rotational quantum number J, analysis of the data reveals that the dynamics occurs on several distinct time scales and is dominated by anharmonic coupling with little contribution from Coriolis coupling. After the fast (∼100 fs) redistribution of the energy among ...

High-resolution helium nanodroplet isolation spectra of the first overtone (2ν1) of the acetylenic stretch of several substituted acetylenes (RC≡C–H) at T=0.38 K, have been observed for the first time. A tunable 1.5 μm laser is coupled, using a power buildup cavity, to a beam of He droplets seeded with the molecule to be studied. Absorption spectra are recorded by monitoring the beam depletion as a function of laser frequency with a thermal detector. The spectra of hydrogen cyanide (HCN), monodeuteroacetylene (DCCH), cyanoacetylene (NCCCH), propyne (CH3CCH), trifluoropropyne (CF3CCH), 3,3-dimethylbutyne ((CH3)3CCCH), and trimethylsilylacetylene ((CH3)3SiCCH) have been recorded. Due to the superfluid nature of the droplet, rotational resolution is achieved despite the presence of some solvent-induced broadening. The spectroscopic constants have been extracted by means of spectral simulations. The resulting rotational constants are smaller than for the bare molecule by a factor which depends on the molecule...

Dispersed emission spectra collected upon the 4 2P3/2,1/2←4 2S1/2 optical excitation of K atoms attached to helium nanodroplets include broad, structured, red-shifted features which are shown to be due to K*He exciplex formation, paralleling our former observation of Na*He [J. Reho, C. Callegari, J. Higgins, W. E. Ernst, K. K. Lehmann, and G. Scoles, Discuss. Faraday Soc. 108, 161 (1997)]. The exciplex formation is demonstrated by the agreement obtained in comparing the K*He A(1) 2Π→X(1) 2Σ emission spectra with the predictions derived from available ab initio potential energy surfaces. Recent analysis of both exciplex emissions also points to the possibility of triatomic (Na*He2 and K*He2) exciplex formation for a small fraction of the alkali atoms. The lack of fluorescence quenching, which is present when the spectra are taken in bulk liquid helium, is due to the surface location of the alkali atoms on the helium droplets that allows the nascent Na*He and K*He exciplexes to desorb from the droplet and e...

We have monitored the time evolution of the fluorescence of K*He exciplexes formed on the surface of helium nanodroplets using reversed time-correlated single photon counting. In modeling the present data and that from our previous work on Na*He, we find that partial spin–orbit coupling as well as the extraction energy of helium atoms from the droplet contribute to the observed dynamics of both K*He and Na*He formation, which differ considerably after either D1(n 2P1/2←n 2S1/2) or D2(n 2P3/2←n 2S1/2) excitation for both K(n=4) and Na(n=3). Our quantitative prediction of the Na*He formation dynamics coupled with preliminary data on and modeling of the formation dynamics of K*He allow for extrapolation to the case of Rb*He. Spin–orbit considerations combined with a simple model of helium atom extraction from the matrix reveal the following predicted trend: as the choice of the alkali guest atom is moved down the periodic table, alkali atom–He exciplex formation along the 1 2Π3/2 surface occurs faster while ...

We recorded rovibrational spectra of the 006+ level of 12C2H2 and the 2131 11–1 level of 13C2H2 in the ground electronic state using a two-photon sequential double resonance technique with a resolution of 15 MHz. Owing to the g/u symmetry of acetylene, the levels that we observe are inaccessible from the ground state by single photon techniques, and observation of these levels is reported here for the first time. Upper state rotational constants were derived from whole band fits of the observed lines, and compare favorably with expected values. Both spectra exhibit signs of local perturbations, and a density of states analysis leads us to believe that we are observing couplings to the full density of vibrational states one would expect from acetylene in this energy region. Despite the high resolution of our spectrometer, and the high excitation energy, no evidence for acetylene hydrogen permutation exchange isomerization (which is predicted to proceed through the vinylidene minimum on the potential) has been observed, implying that the rate of exchange isomerization is more than four orders-of-magnitude below the rate predicted by RRKM (Rice, Ramsperger, Kassel, and Marcus) theory.

Microwave-infrared double-resonance spectroscopy has been used to probe the solvation environment and its influence on the rotational relaxation of a cyanoacetylene molecule embedded in a superfluid 4He nanodroplet. The results support a model in which (within any given rotational state) the guest molecules are distributed over a set of spectroscopically inequivalent states which are most likely “particle-in-a-box” states originating from the confinement of the guest molecule within the droplet. Revisitation of previously collected microwave–microwave double-resonance data suggests that transitions between these states occur at a rate which is comparable to the rotational relaxation rate, but not fast enough as to produce motionally narrowed, homogeneous absorption lines. The relative intensities of the rotational lines in the microwave-infrared double-resonance spectra are observed to depend strongly on the average droplet size. In the large droplet limit we can explain the observed pattern by invoking a...

An X-ray study of a crystalline organic–organic heterostructure, consisting of a film of PTCDA (3,4,9,10-perylenetetracarboxylic dianhydride) grown on a self-assembled monolayer (SAM) of decanethiol on Au(1 1 1), is presented. The sandwich structure acts as a “molecular interferometer”, where the interference between the Laue function of the PTCDA film and the crystal truncation rod (CTR) of the substrate is governed by the SAM spacer thickness, d 0 . A pronounced destructive interference feature is observed, which allows the determination of d 0 with great sensitivity.

In an attempt to quantify the role played by the rigidity of the molecular backbone on the self-assembly process, monolayers of 4-methyl-4′-mercaptobiphenyl assembled on Au(111) were characterized by grazing incidence X-ray diffraction and low-energy atomic beam diffraction. Two phases of different density were observed. In the low-density ‘striped’ phase, the diffraction pattern is consistent with a commensurate rectangular (8×2 3 ) surface lattice. Systematic absences and the intensity modulation in the diffraction pattern suggest that the structure can be composed by staggered molecular rows arranged in a head-to-head orientation with their molecular axes close to the surface. The diffraction pattern of the high-density phase can be described by a commensurate ( 3 × 3 )R30° surface lattice. The measured intensity variation along the (1, 1) Bragg rod is consistent with a tilt angle of at most 19° from the surface normal. Therefore, both similarities and differences with the diffraction patterns of the low-density phase and the high-density phase of the monolayers of n -alkanethiol on Au(111) have been detected. The thermal behavior of the monolayers of 4-methyl-4′-mercaptobiphenyl was also examined. Both phases are found to be thermally more stable than the corresponding phases of monolayers of n -alkanethiols. Finally, the growth behavior of the monolayers of 4-methyl-4′-mercaptobiphenyl was investigated and various growth protocols were tried. Compared with the case of n -alkanethiol monolayers, the high-density phase of the monolayers of 4-methyl-4′-mercaptobiphenyl is more difficult to prepare.