Epitaxially guided assembly of collagen layers on mica surfaces

Ordered assembly of collagen molecules on flat substrates has potential for various applications and serves as a model system for studying the assembly process. While previous studies demonstrated self-assembly of collagen on muscovite mica into highly ordered layers, the mechanism by which different conditions affect the resulting morphology remains to be elucidated. Using atomic force microscopy, we follow the assembly of collagen on muscovite mica at a concentration lower than the critical fibrillogenesis concentration in bulk. Initially, individual collagen molecules adsorb to mica and subsequently nucleate into fibrils possessing the 67 nm D-periodic bands. Emergence of fibrils aligned in parallel despite large interfibril distances agrees with an alignment mechanism guided by the underlying mica. The epitaxial growth was further confirmed by the formation of novel triangular networks of collagen fibrils on phlogopite mica, whose surface lattice is known to have a hexagonal symmetry, whereas the more widely used muscovite does not. Comparing collagen assembly on the two types of mica at different potassium concentrations revealed that potassium binds to the negatively charged mica surface and neutralizes it, thereby reducing the binding affinity of collagen and enhancing surface diffusion. These results suggest that collagen assembly on mica follows the surface adsorption, diffusion, nucleation, and growth pathway, where the growth direction is determined at the nucleation step. Comparison with other molecules that assemble similarly on mica supports generality of the proposed assembly mechanism, the knowledge of which will be useful for controlling the resulting surface morphologies.     

Temperature-dependent formation of octadecylsiloxane self-assembled monolayers on mica as studied by atomic force microscopy

We have investigated the growth of octadecylsiloxane (ODS) self-assembled monolayers on mica. Freshly cleaved muscovite mica and octadecyltrichlorosilane (OTS) dissolved in toluene (c = 1.0 mmol/L) have been used as substrate and precursor, respectively. The water content of the adsorption solution was between 14.6 and 16.6 mmol/L. Adsorption experiments were carried out in a temperature range between 5 and 45 degrees C, and the obtained submonolayer ODS films were characterized with atomic force microscopy (AFM). Besides the morphology of the films, also information on the surface coverage has been obtained by quantitative evaluation of the AFM images. Depending on the temperature, evidence for both ordered and disordered expanded ODS phases has been found. The pronounced maximum in surface coverage--in contrast to adsorption on silicon substrates--at a temperature of about 27 degrees C and the different morphology of the submonolayer films as compared to silicon substrates could be explained in terms of a deposition, diffusion, and aggregation (DDA) model.     

Robust self-assembled octadecylphosphonic acid monolayers on a mica substrate

As determined by scratch tests, self-assembled monolayers (SAMs) of octadecylphosphonic acid (OPA) on a muscovite mica substrate were found to be mechanically robust and to serve as a lubricant to protect the underlying mica substrate. For comparison purposes, three polymer films were subjected to scratch tests under the same conditions. The scratch tests were conducted using a diamond-tipped stylus, and the resultant scratches were examined using atomic force microscopy. The excellent mechanical strength of OPA SAMs is supported by analysis with time-of-flight secondary ion mass spectrometry, which suggests that the headgroup of the OPA is strongly bonded to the substrate atoms. The molecular lubrication provided by OPA SAMs suggests that the interaction between the headgroup and the substrate is sufficiently strong to endure significant shear force and that the hydrocarbon chains are able to dissipate shear energy.     

ToF-SIMS investigation of octadecylphosphonic acid monolayers on a mica substrate

In this paper, time-of-flight secondary ion mass spectrometry (ToF-SIMS) under static conditions was used to investigate self-assembled monolayers (SAMs) of octadecylphosphonic acid (OPA) formed on freshly cleaved muscovite mica substrates. The coverage of OPA on mica ranged from 20 to 100%, with a film thickness of 1.7+/-0.2 nm, which was determined by atomic force microscopy (AFM) imaging. The relative intensity of the specific secondary ion species associated with the OPA and with the exposed mica substrate exhibited good correlation with surface coverage. An excellent correlation was also observed (R2=0.98) between the relative SIMS [OPA-H]- intensity and the surface carbon concentration (OPA C 1s, in atomic %) from XPS at the prescribed surface coverage. The observation of positive and negative OPA molecular attachment of secondary ions involving the substrate species is discussed in terms of the chemical affinity of the OPA phosphonate headgroup for the cleaved mica surface as well as the sampling depth. In addition, the OPA molecular attachment species formed with the potassium ions on the cleaved mica substrate dominated the positive secondary ion mass spectrum in the high-mass range. A temperature-dependent, ToF-SIMS study employing in situ heating of a 100% coverage OPA monolayer revealed that the molecules begin to diffuse above approximately 80 degrees C, resulting in a decrease in the relative secondary ion yield of the OPA-specific secondary ions. This observation is hypothesized to be due to a decrease in the effective coverage of the substrate by the OPA molecules, which in turn could be due to the formation of multilayers upon heating in an effort to minimize the energy of the system. The interesting behavior of the novel OPA dimer species as a function of temperature is also reported. It was observed that the relative intensity of OPA and the mica-specific secondary ion peak intensities to that of Si (mica substrate) provides an effective means to estimate the change in coverage at elevated temperatures.     

Epitaxial Crystallization of Insulin on an Ordered 2D Polymer Template

Two-dimensional polymers (2DP) are a new class of materials that consist of a monolayer of ordered molecular building blocks, which have been covalently linked. One of these monomers was self-assembled on a flat muscovite mica scaffold and subsequently the organic layer was polymerized. The resulting flat and stable 2DP layer was used as a template for protein crystallization. Crystals of insulin were epitaxially grown on the template, whereas insulin crystals grown on clean muscovite mica had a random orientation. The template was selective, considering that no epitaxially ordered crystals formed of hen egg white lysozyme, bovine serum albumin, or talin.     

Investigation of surface changes on mica induced by atomic force microscopy imaging under liquids

Surface changes on muscovite mica induced by tip-surface interactions in atomic force microscopy (AFM) experiments under liquids are described. Investigations have been performed with AFM operated both in contact mode (CM-AFM) and in tapping mode (TM-AFM). Additionally, force-distance measurements have been carried out. In contrast to CM-AFM pronounced surface changes can be observed in TM-AFM experiments. However, TM-AFM images of areas previously scanned in contact mode show that imaging in contact mode changes the surface, too. An evaluation of force-distance measurements reveals that these changes depend on the adhesive interaction between tip and sample, which in turn strongly depends on the surrounding medium. The artefact can be avoided by changing the pH-value of the medium or by working with mixtures of ethanol and water. This greatly enhances the applicability of TM-AFM for in-situ investigation of surface processes on mica, which is a frequently used substrate for many technological and biological applications.     

From clusters to fibers: parameters for discontinuous para-hexaphenylene thin film growth

All relevant steps of discontinuous thin film growth of para-hexaphenylene on muscovite mica (0 0 1) from wetting layer over small and large clusters to nanofibers are observed and investigated in detail by a combined polarized fluorescence and atomic force microscopy study. From a variation of film thickness and surface temperature, we determine effective activation energies for cluster growth of 0.17 eV, for nanofiber length growth of 0.46 eV, for width growth of 0.19 eV, and for height growth of 0.07 eV. The corresponding exponential prefactors for the nanofiber growth are 1 x 10(9), 6 x 10(4), and 3 x 10(2) nm. Polarized fluorescence studies reveal that nanofibers grow along the grooves of the mica surface and that they do not change direction if they cross an even number of mica surface steps, while they change direction by 120 degrees for an odd number of steps. These results are taken as an input for a model of the unidirectional growth process on mica. Absolute parameters allowing one to grow nanofibers of predetermined morphology via organic molecular beam epitaxy are also given.     

Self-organization of guanosine 5'-monophosphate on mica

Self-assembled aggregates of guanosine 5′-monophosphate (GMP) on the surface of muscovite mica were investigated by atomic force microscopy (AFM). Aqueous solutions of sodium, potassium and ammonium GMP salts were studied. For solution concentrations c < 0.005 wt% only small islands of deposited material are present on the surface. For c  0.02 wt%, in addition to the islands and patches, also linear aggregates called G-wires are formed. The wire-like aggregates are on average 1.9 nm high and can be several micrometers long. They exhibit a profound directional growth along the six main crystallographic axes of the basal plane of mica. For c > 0.1 wt% flat terraces with the height of 2.5 nm appear. They are formed of G-wires lying with their long axis parallel to the substrate and stacking in a hexagonal arrangement. The morphology of the adsorbates is independent of the type of salt used to prepare the initial solution. This signifies that intrinsic potassium ions from the substrate play much more important role in the GMP adsorption than cations from the solution.     

Controlling the self-assembly of a deoxiguaninosine on mica

We report a tapping mode scanning force microscopy investigation on the growth from solutions of a lipophilic deoxiguanosine derivative on the basal plane of a muscovite mica surface. By varying the chemical composition of the substrate surface it is possible to drive the self-assembly towards either layered thin films, which are very likely to consist of guanosine quartets, or nanoribbons, comprising hydrogen bonded networks. Both these two architectures, which are peculiar to the guanine base itself, are of notable importance for the fabrication of molecular nanowires. Moreover, the first arrangement provides evidence that the guanosine is able to behave as ionophore, thus, suggesting its utilization in the development of solid-state ion selective sensors.     

Adsorption of polymers with crown ether substituents on muscovite mica

Ultrahigh specific surface area muscovite with different ions at the surface (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Ca²⁺, Sr²⁺, Ba²⁺, Cu²⁺) was treated with aqueous solutions of low molecular weight crown ethers and polymers with crown ether substituents. The adsorption was assessed by UV analysis of the supernatant solution, and with TGA and IR spectroscopy of the mica solids. In contrast to other layered silicates, the low molecular weight crown ethers show no affinity to any of the muscovite surfaces. The polymers can adsorb, however, depending on the type of surface cation. The results indicate that at least some of the crown ether moieties are complexed to surface cations and that the diameter of the ions at the surface plays an important role in the adsorption process.     

Organothiol Monolayer Formation Directly on Muscovite Mica

Organothiol monolayers on metal substrates (Au, Ag, Cu) and their use in a wide variety of applications have been extensively studied. Here, the growth of layers of organothiols directly onto muscovite mica is demonstrated using a simple procedure. Atomic force microscopy, surface X‐ray diffraction, and vibrational sum‐frequency generation IR spectroscopy studies revealed that organothiols with various functional endgroups could be self‐assembled into (water) stable and adaptable ultra‐flat organothiol monolayers over homogenous areas as large as 1 cm². The strength of the mica–organothiol interactions could be tuned by exchanging the potassium surface ions for copper ions. Several of these organothiol monolayers were subsequently used as a template for calcite growth.     

The Adsorption of Dodecyltrimethylammonium Bromide on Mica in Aqueous Solution Studied by X-Ray Diffraction and Atomic Force Microscopy

The adsorption of dodecyltrimethylammonium bromide (DTAB) onto natural muscovite mica and a synthetic expandable mica (EM) in aqueous solution has been investigated using both microscopic and macroscopic surface characterization techniques. The electrokinetic properties of the surfaces were monitored as a function of the concentration of DTAB using atomic force microscopy and microelectrophoresis. The adsorption isotherm of DTAB on EM was measured up to a solution concentration just below the critical micelle concentration of the surfactant. The thickness of the adsorbed layer on EM was determined using X-ray diffraction. Results indicate that the adsorbed layer consists of molecules lying quite flat on the mica surface at low concentrations and adsorbed in interleaved aggregate structures at concentrations approaching the critical micelle concentration of the surfactant in solution. Copyright 2001 Academic Press.     

Adsorption of alpha-chymotrypsin onto mica in laminar flow conditions. Adsorption kinetic constant as a function of tris buffer concentration at pH 8.6

We examined the adsorption kinetics of alpha-chymotrypsin (pH 8.6, 10(-2) to 0.5 M Tris buffer) on muscovite mica in conditions of laminar flow through a slit. The range of buffer concentrations is between two limits: (i) no adsorption in 1 M Tris and (ii) no desorption in 10(-3) M Tris. Studying the dependence of adsorption kinetics on the wall shear rate leads to the determination of the interfacial adsorption kinetic constant ka and the diffusion coefficient. The obtained value for the diffusion coefficient is close to the one expected from the molecular size of alpha-chymotrypsin. The interfacial adsorption kinetic constant of alpha-chymotrypsin decreases when ionic strength increases, while the initial desorption constant (over a part of all the adsorbed population) shows the contrary. Although alpha-chymotrypsin is almost at its isoelectric point, the effect of ionic strength on the adsorption kinetics suggests the importance of electrostatic interactions between the protein and mica. We observed an increase in the adsorption rate, at a surface coverage near 0.14 microg cm(-2), for adsorption in 10(-2) M Tris and the low wall shear rates (<300 s(-1)). This change in the adsorption rate suggests a structural transition, that we assume again to be due to electrostatic interactions, but between proteins. The large dipole moment of the protein may induce this transition, illustrated here by the ferroelectric/antiferroelectric pattern. The variation of the zeta potential with interfacial concentration seems to be in agreement with such a model.     

New insights on growth mechanisms of protein clusters at surfaces: an AFM and simulation study

Despite its relevance to a wide range of technological and fundamental areas, a quantitative understanding of protein surface clustering dynamics is often lacking. In inorganic crystal growth, surface clustering of adatoms is well described by diffusion-aggregation models. In such models, the statistical properties of the aggregate arrays often reveal the molecular scale aggregation processes. We investigate the potential of these theories to reveal hitherto hidden facets of protein clustering by carrying out concomitant observations of lysozyme adsorption onto mica surfaces, using atomic force microscopy, and Monte Carlo simulations of cluster nucleation and growth. We find that lysozyme clusters diffuse across the substrate at a rate that varies inversely with size. This result suggests which molecular scale mechanisms are responsible for the mobility of the proteins on the substrate. In addition the surface diffusion coefficient of the monomer can also be extracted from the comparison between experiments and simulations. While concentrating on a model system of lysozyme-on-mica, this 'proof of concept' study successfully demonstrates the potential of our approach to understand and influence more biomedically applicable protein-substrate couples.     

Imaging structured water and bound polysaccharide on mica surface at ambient temperature

The presence of a water layer on the surface of muscovite mica under ambient conditions is well established. The water molecules are well ordered and seem to be oriented, leading to an icelike monolayer (probably ferroelectric) in epitaxial relation with the mica surface. We have imaged and characterized the height and contact angle of ordered water layer(s) formed by wetting and de-wetting processes on mica surfaces at different states of hydration by tapping mode atomic force microscopy. Implications that the presence of such an ordered water layer may have for imaging of biological samples are also discussed, with consideration of data for the polysaccharide hyaluronan.     

Epitaxial morphologies of polyoxymethylene. I. Electron microscopy

Epitaxial crystallization of polyoxymethylene (POM) from 0.5% iodobenzene solution has been attempted between 150 and 165°C on 11 different substrates having surface energies ranging from 136 to 1240 ergs/cm². Included in this series are several substrates such as CaF₂, SrF₂, BaF₂, graphite, mica, and silicon which had not been previously tested as substrates for POM epitaxy. The fluorite series materials are not good substrates for the epitaxial crystallization of POM from solution. In contrast, both rodlike and fold-plane epitaxial morphologies have been found on the (001) surfaces of NaCl, NaF, and mica. A possible mechanism for the formation of fold-plane epitaxy is discussed which involves solution nucleation followed by adsorption and growth of the nuclei on the substrate. It is suggested that variations observed in nucleation density of rodlike epitaxial crystals from substrate to substrate are a result of differences in degree of preferential solvent adsorption. Substrates having higher surface energies should tend to adsorb solvent more strongly and thus inhibit profuse nucleation of polymer crystals on the surface.     

Surface-assisted assembly of an ionic-complementary peptide: controllable growth of nanofibers

Numerous studies have shown that a surface can direct and regulate molecular assembly. In this study, the nanofiber growth of an ionic-complementary peptide, EAK16-II, on a mica surface was investigated under various solution conditions via in situ atomic force microscopy. In comparison to the assembly in bulk solution, nanofiber growth of EAK16-II on mica is surface-assisted and involves two steps: (1) adsorption of nanofibers and fiber clusters (from the bulk solution) on the surface, serving as the "seeds"; (2) fiber elongation of the "seeds" from their active ends. The nanofiber growth can be controlled by adjusting the solution pH since it modulates the adsorption of the "seeds" on mica and their growth rates. The amount of the adsorbed "seeds" decreases with increasing solution pH, while the growth rate under different solution conditions is found to follow the order pure water > 1 mM HCl > 1 mM NaOH > 10 mM HCl approximately 10 mM NaOH approximately 0. The pH-dependent nanofiber growth is due to the surface charge of the peptides and peptide assemblies in various solutions as indicated by zeta-potential measurements. A simple model was proposed to describe surface-assisted nanofiber growth. This study provides insights into the assembly of peptide/protein on a surface, which is essential to understand such physiological protein aggregation systems as amyloid fibrillogenesis. In addition, the potential of this finding to construct biocompatible electrodes for biomolecular sensing is also discussed.     

Applying grazing incidence X-ray reflectometry (XRR) to characterising nanofilms on mica

Molecularly smooth mica has hitherto not been widely used as a substrate for the X-ray reflectometry (XRR) technique. That is largely due to the difficulty of achieving flatness over a sufficiently large area of mica. Here we show that this difficulty can be overcome by slightly bending the mica substrate over an underlying cylinder; the enhanced rigidity of the bent mica sheet along the axis of the cylinder provides sufficient flatness along this axis for XRR measurements. To test this method, we have employed it to characterise three types of nanofilms on mica in air: (A) Cr-Au thin films; (B) a surface-grown zwitterionic polymer brush; and (C) a Langmuir-Blodgett (LB) phospholipid monolayer, using a table-top X-ray reflectometer. Fitting the obtained reflectivity curves with the standard Parratt algorithm allows us to extract the structural information of the nanofilms (both thickness and apparent roughness). Our simple method points to how XRR may be exploited as a useful characterisation tool for nanofilms on mica.     

AFM Observation of Phospholipid Molecules Absorbed on Mica

An atomic force microscope was used to observe the adsorption of phospholipid molecules(phospha-tidylcholine, PC) on the mica substrate.The film-formation by PC molecules adsorption spontaneously on the specific substrate has been proved. It is revealed that the evident differences of morphology exist among different sampler with different concentrations of PC/decane solutions. different adsorption times, etc.The effect of tip-induced change in size of domains formed by adsorption of PC molecules on the substrate during the AFM scanning was observed.     

Fibrinogen adsorption on mica studied by AFM and in situ streaming potential measurements

Adsorption of fibrinogen from aqueous solutions on mica was studied using AFM and in situ streaming potential measurements. In the first stage, bulk physicochemical properties of fibrinogen and the mica substrate were characterized for various ionic strength and pH. The zeta potential and number of uncompensated (electrokinetic) charges on the protein surfaces were determined from microelectrophoretic measurements. Analogously, using streaming potential measurements, the electrokinetic charge density of mica was determined for pH range 3-10 and the NaCl background electrolyte concentration of 10(-3) and 10(-2) M. Next, the kinetics of fibrinogen adsorption at pH 3.5 and 7.4 in the diffusion cell was studied using a direct AFM determination of the number of molecules per unit area of the mica substrate. Then, streaming potential measurements were performed to determine the apparent zeta potential of fibrinogen-covered mica for different pH and ionic strength in terms of its surface concentration. A quantitative interpretation of these streaming potential measurements was achieved in terms of the theoretical model postulating a side-on adsorption of fibrinogen molecules as discrete particles. On the basis of these results, the maximum coverage of fibrinogen Θ close to 0.29 was predicted, in accordance with previous theoretical predictions. It was also suggested that anomalous adsorption for pH 7.4, where fibrinogen and the mica substrate were both negatively charged, can be explained in terms of a heterogeneous charge distribution on fibrinogen molecules. It was estimated that the positive charge was 12 e (for NaCl concentration of 10(-2) M and pH 7.4) compared with the net charge of fibrinogen at this pH, equal to -21 e. Results obtained in this work proved that the coverage of fibrinogen can be quantitatively determined using the streaming potential method, especially for Θ < 0.2, where other experimental methods become less accurate.