Silica colloidal crystals as three-dimensional scaffolds for supported lipid films

This report describes the assembly of laterally diffusive lipid layers within the pores of colloidal crystals for potential application in membrane-based sensing. The amount of lipid encapsulated within colloidal crystals depends upon the method used to introduce the lipid to the crystalline substrate. Relative to a planar supported lipid bilayer, lipid loading in a 6.6 microm thick crystal was 15-73 times greater, as observed by fluorescence microscopy. Protein adsorption studies indicate that the crystal pores are open and that the silica surface of the crystal is passivated with respect to adsorption of a model protein when coated with POPC. Furthermore, the mesoporous environment of the colloidal crystal is found to protect lipid films from drying and rehydration processes that destroy planar supported lipid bilayers. The potential of colloidal crystal encapsulated lipid films for chemical sensing is demonstrated by a model protein binding assay.     

Tuning Chain Relaxation from an Amorphous Biopolymer Film to Crystals by Removing Air/Water Interface Limitations

A promising route to the synthesis of protein-mimetic materials that are capable of strong mechanics and complex functions is provided by intermolecular β-sheet stacking. An understanding of the assembly mechanism on β-sheet stacking at molecular-level and the related influencing factors determine the potential to design polymorphs of such biomaterials towards broad applications. Herein, we quantitatively reveal the air/water interface (AWI) parameters regulating the transformation from crowding amorphous aggregates to ordered phase and show that the polymorph diversity of β-sheet stacking is regulated by the chain relaxation-crystallization mechanism. An amorphous macroscale amyloid-like nanofilm is formed at the AWI, in which unfolded protein chains are aligned in a short-range manner to form randomly packed β-sheets. The subsequent biopolymer chain relaxation-crystallization to form nanocrystals is further triggered by removing the limitations of energy and space at the AWI.     

Microstructure of beta-lactoglobulin-stabilized emulsions containing non-ionic surfactant and excess free protein: influence of heating

The influence of the non-ionic surfactant Tween 20 on the microstructure of beta-lactoglobulin-stabilized emulsions with substantial excess free protein present was investigated via confocal microscopy. The separate distributions of oil droplets and protein were determined using two different fluorescent dyes. In the emulsion at ambient temperature the excess protein and protein-coated oil droplets were associated together in a reversibly flocculated state. The pore-size distribution of the initial flocculated emulsion was found to depend on the surfactant/protein ratio R, and at higher values of R the system became more inhomogeneous due to areas of local phase separation. Evidence for competitive displacement of protein from the oil-water interface by surfactant was obtained only on heating (from 25 to 85 degrees C) during the process of formation of a heat-set emulsion gel. By measuring fluorescence intensities of the protein dye inside and outside of the oil-droplet-rich areas, we have been able to quantify the evolving protein distribution during the thermal processing. The results are discussed in relation to previous work on the competitive adsorption of proteins and surfactants in emulsions and the effect of emulsion droplets on the rheology of heat-set protein gels.     

Sorption of Anionic Surfactants on Layered Double Hydroxides

In the present study we investigated the adsorption of sodium dodecyl- and octylsulfate, and sodium dodecyl- and octylbenzenesulfonate, on a layered double hydroxide (LDH) under controlled conditions. The results were compared to those obtained for the adsorption of surfactants on mineral oxides and on LDHs, showing that the behavior of surfactant adsorption on LDHs can be approximately explained by the models proposed for surfactant adsorption on mineral oxides. The electrokinetic potential of the surfactant-adsorbed LDH particles in suspension was measured in order to monitor the variation of this potential as a function of the adsorption. The results showed a characteristic behavior of the surfactant-adsorbed LDH that can be correlated to a rearrangement of the adsorbed layer at concentrations above the CMC. Surfactant sorption was also studied, using SDS and the calcined LDH, showing the regeneration of the calcined material by intercalating the DS anions. Two processes can occur during the removal of surfactants from aqueous solutions by LDHs, depending on the starting material: (i) adsorption itself, when a noncalcined LDH intercalated with a nonexchangeable anion, such as carbonate, is used; and (ii) adsorption and intercalation, which occurs when a calcined LDH, like Mg-Al-CO(3), is used. The potential application of this material for surfactant removal is one of the reasons for the need for a better understanding of the mechanisms involved. As we demonstrate here, LDHs are very efficient in removing surfactants from aqueous solutions. Copyright 2000 Academic Press.     

Adaptive Chemoenzymatic Microreactors Composed of Inorganic Nanoparticles and Bioinspired Intrinsically Disordered Proteins

The assembly of protein and inorganic nanoparticles represents an attractive approach to generate composite materials with multiple functions. Herein, we functionalize inorganic nanoparticles with intrinsically disordered protein domains associated with the formation of membraneless compartments in cells. These protein sequences, defined as low complexity domains (LCDs), encode intermolecular interactions that drive highly controlled, dynamic self‐assembly in response to environmental changes. We show that the properties of the LCDs can be transferred to inorganic nanoparticles, inducing controlled phase separation that is dynamic and responsive to ionic strength and pH. Specifically, we hybridize magnetic nanoparticles with multi‐domain proteins consisting of LCD domains and a globular enzyme, generating dynamic protein‐composite compartments that locally confine hybrid chemoenzymatic reactions and respond to external magnetic fields and changes in solution conditions.     

DNA hybridization-induced reorientation of liquid crystal anchoring at the nematic liquid crystal/aqueous interface

Interactions between DNA and an adsorbed cationic surfactant at the nematic liquid crystal (LC)/aqueous interface were investigated using polarized and fluorescence microscopy. The adsorption of octadecyltrimethylammonium bromide (OTAB) surfactant to the LC/aqueous interface resulted in homeotropic (untilted) LC alignment. Subsequent adsorption of single-stranded DNA (ssDNA) to the surfactant-laden interface modified the interfacial structure, resulting in a reorientation of the LC from homeotropic alignment to an intermediate tilt angle. Exposure of the ssDNA/OTAB interfacial complex to its ssDNA complement induced a second change in the interfacial structure characterized by the nucleation, growth, and coalescence of lateral regions that induced homeotropic LC alignment. Fluorescence microscopy showed explicitly that the complement was colocalized in the same regions as the homeotropic domains. Exposure to noncomplementary ssDNA caused no such response, suggesting that the homeotropic regions were due to DNA hybridization. This hybridization occurred in the vicinity of the interface despite the fact that the conditions in bulk solution were such that hybridization did not occur (high stringency), suggesting that the presence of the cationic surfactant neutralized electrostatic repulsion and allowed for hydrogen bonding between DNA complements. This system has potential for label-less and portable DNA detection. Indeed, LC response to ssDNA target was detected with a lower limit of approximately 50 fmol of complement and was sufficiently selective to differentiate a one-base-pair mismatch in a 16-mer target.     

Ellipsometric approach for the real-time detection of label-free protein adsorption by second harmonic generation

Second harmonic generation (SHG) was performed using a novel ellipsometric detection approach to selectively probe the real-time surface binding kinetics of an unlabeled protein. The coherence of nonlinear optical processes introduces new possibilities for exploiting polarization that are unavailable with incoherent methods, such as absorbance and fluorescence. Adsorption of bovine serum albumin (BSA) at silica/aqueous solution interfaces resulted in changes in the polarization state of the frequency-doubled light through weak, dynamic interactions with a coadsorbed nonlinear optical probe molecule (rhodamine 6G). Using a remarkably simple instrumental approach, signals arising exclusively from surface interactions with BSA were spatially isolated and selectively detected with high signal-to-noise. The relative intensities acquired during the kinetics experiments using both circularly and linearly polarized incident beams were in excellent agreement with the responses predicted from SHG ellipsometry polarization measurements. Analysis of the polarization-dependent SHG generated during BSA adsorption at glass/aqueous solution interfaces provided direct evidence for slow conformational changes within the protein layer after adsorption, consistent with protein denaturation. This polarization selection approach is sufficiently general to be easily extended to virtually all coherent nonlinear optical processes and a variety of different surface interactions and architectures.     

Influence of pH on colloidal properties and surface activity of polyglycerol fatty acid ester vesicles

Certain polyglycerol esters of fatty acids (PGE) form dispersions of uni- or multilamellar vesicles in dilute aqueous solution. These self-assembled aggregates reduce the surface-activity of PGE monomers such that interfacial films may take several hours to form. This is undesirable for processes, which rely on rapid surfactant adsorption, for example foaming. In the present work, we study the effect of pH on the colloidal (size distribution, morphology, surface charge) and interfacial (adsorption kinetics) properties of a commercial, non-purified PGE. Using dynamic light scattering, zeta-potential measurements and cryo-SEM, we show that changing the pH of the dispersion media can cause agglomeration and eventually osmotic rupture of PGE vesicles. The change in dispersion state also impacts the adsorption behavior at the water surface. Direct evidence that destabilized vesicle dispersion are more surface-active is provided by comparing the dynamic surface tension of solutions of different pH. The faster adsorption kinetics at low pH correlate with a remarkably increased foaming power. We suggest that an osmotic shock induced by changes in pH causes vesicles to deform and partially open, so that their hydrocarbon core is exposed to the dispersion media. This energetically unfavorable condition promotes the hydrophobically driven adsorption of surfactant monomers at surfaces and hence stimulates the foaming ability.     

Electrochemical surface plasmon resonance investigation of dodecyl sulfate adsorption to electroactive self-assembled monolayers via ion-pairing interactions

The redox-induced assembly of amphiphilic molecules and macromolecules at electrode surfaces is a potentially attractive means of electrochemically modulating the organization of materials and nanostructures on solid substrates via ion-pairing interactions or charge-transfer complexation. In this regard, we have investigated the potential-induced adsorption and aggregation of dodecyl sulfate, a common anionic surfactant, at a ferrocenylundecanethiolate (FcC11SAu) self-assembled monolayer (SAM)/aqueous solution interface by electrochemical surface plasmon resonance (ESPR) spectroscopy. The surfactant anions adsorb onto the electroactive SAM by specific ion-pairing interactions with the oxidized ferricinium species. The ferricinium charge density (QFc+) obtained by cyclic voltammetry and surface coverage measured by SPR indicate that the dodecyl sulfate forms an interdigitated monolayer, where half of the surfactant molecules have their sulfate headgroups paired to the surface and half have their headgroups exposed to the aqueous solution. The surface coverage of dodecyl sulfate was found to depend on both the ferricinium surface concentration and the surfactant aggregation state in solution. A maximum coverage of dodecyl sulfate on the ferricinium surface is obtained below the critical micelle concentration (cmc), in contrast to dodecyl sulfate adsorption to SAM surfaces of static positive charge. This marked difference in adsorption behavior is attributed to the dynamic generation of ferricinium by potential cycling and the specific nature of the ion-pairing interactions versus pure electrostatic ones. The results presented point to a new way of organizing molecules via electrical stimulus.     

Adsorption of myoglobin onto porous zirconium phosphate and zirconium benzenephosphonate obtained with template synthesis

Porous zirconium phosphate (P-ZrP) and zirconium benzenephosphonate (P-ZrBP) were prepared in the presence of an anionic surfactant acting as a template. Poorly crystalline materials with a P/Zr molar ratio equal to 2 and having a relatively high surface area and micro/mesoporosity have been obtained. The interaction of myoglobin with the two types of surfaces, the hydrophobic P-ZrBP and the hydrophilic P-ZrP, was investigated, and the adsorption isotherms were determined at different pH and temperature values. A model was proposed for the mechanism of the interaction of the protein with the surface based on the shape of the adsorption isotherm and the physical-chemical properties of myoglobin. The pH has been found to be an important parameter for determining the maximum adsorption capacity of P-ZrBP and P-ZrP for myoglobin molecules because of the changes that occur in the type and net charge of the protein surface as the pH of the medium changes. Protein binding affinity and capacity increase when the temperature is increased. This phenomenon occurs because myoglobin varies its conformation at high temperature with an increase in the exposed hydrophobic region. This process causes a stronger hydrophobic interaction between the protein and the adsorbent and reduces the repulsion between the adsorbed molecules. Studies on the activities of the obtained biocomposites are in progress.     

Chemical oscillation with periodic adsorption and desorption of surfactant ions at a water/nitrobenzene interface.

Chemical oscillations with periodic adsorption and desorption of surfactant ions, alkyl sulfate ions, at a water/nitrobenzene interface have been investigated. The interfacial tension was measured with a quasi elastic laser scattering (QELS) method and the interfacial electrical potential was obtained. We found that this oscillation consists of a series of abrupt adsorptions of ions, followed by a gradual desorption. In addition, we observed that each abrupt adsorption was always accompanied by a small waving motion of the liquid interface. From the analysis of the video images of the liquid interface or bulk phase, we could conclude that each abrupt adsorption is caused by nonlinear amplification of mass transfer of ions from the bulk phase to the liquid interface by a Marangoni convection, which was generated due to local adsorption of the surfactant ions at the liquid interface that resulted in the heterogeneity of the interfacial tension. In the present paper, we describe the mechanism of the chemical oscillation in terms of the hydrodynamic effect on the ion adsorption processes, and we also show the interfacial chemical reaction with ion exchange during the ion desorption process.     

In situ fluorescence probing of the chemical and structural changes during formation of hexagonal phase cetyltrimethylammonium bromide and lamellar phase CTAB/Poly(dodecylmethacrylate) sol–gel silica thin films

Surfactant-templated mesostructured sol–gel films formed by evaporation induced self assembly (EISA) exhibit highly-ordered hexagonal, lamellar, and cubic structures. The steady-state dip-coating configuration allows both the chemistry and the dynamics of the EISA process to be traced in real time because the steps involved in the formation of the mesostructured material are separated both spatially and temporally in the dip-coating direction. The dynamic processes occurring during film formation can be conveniently monitored by the combination of interferometry and fluorescence spectroscopy of incorporated molecular probes. The selected probes respond to changes in their rotational mobility and the surrounding solvent composition and report these changes through their fluorescence characteristics. By taking in situ fluorescence spectra at various positions within the progressively thinning film, changes in the solvent composition, onset of micelle formation and further organization to the final mesophase structure can be followed. The luminescence of the probe molecule is measured with a spatial resolution of 100 μm. Two categories of surfactant-templated mesostructured sol–gel films were examined. Cetyltrimethylammonium bromide (CTAB) systems assemble into a 2-D hexagonal surfactant/silica mesophase with the surfactant concentration used in this study. CTAB dodecylmethacrylate systems assemble into a lamellar mesophase, which can be further polymerized to form a poly(dodecylmethacrylate)/silica hybrid nanocomposite that mimics nacre. X-ray diffraction patterns, transmission electron microscopy images, and other techniques are used to characterize the final films.     

Properties of mixed protein/surfactant adsorption layers

The adsorption isotherms, adsorption kinetics and surface rheological properties of β-lactoglobulin, β-casein, in the absence and presence of Tween 20 were measured. To study the adsorption process (isotherms and kinetics) at the water–air interface the pendant drop technique (axial drop shape analysis, ADSA), and ring tensiometry were used. The surface shear rheological parameters were measured with a torsion pendulum set-up. Also, data of the equilibrium film thickness and surface diffusion coefficients obtained from fluorescence recovery after photobleaching (FRAP) measurements are used to understand the competitive adsorption mechanism. The adsorption process and shear rheological behaviour of the studied systems show a rather complex behaviour which depends most of all on the system's composition. At high protein or surfactant content the behaviour is controlled by the main component while for the more mixed systems the adsorption process is complex and consists of partial adsorption, surfactant–protein interaction and protein rearrangement as a function of surface coverage. The results obtained illustrate that all these processes must be taken into account in future new theoretical models to be derived for such systems.     

Study on the Surface Property of Surfactant Ionic Liquids Solutions

The surfactant TX-100 can be dissolved in ionic liquid bmimPF6 and decrease the surface tension of 1-buty1-3-methylimidazolium hexafluorophosphate (bmimPF6) solutions. Here, we confirmed that in this new system, the pure solvents need rearrangement at the air-wate rinterface at the initial stage. The dynamic surface tension (DST) study shows that at the initial adsorption stage, the adsorption model of surfactant accords with the diffusion-controlled adsorption mechanism, and the dilute ionic liquids solutions is further close to the diffusion-controlled adsorption.     

Interfacial behavior of recombinant forms of human pulmonary surfactant protein SP-C

The behavior at air-liquid interfaces of two recombinant versions of human surfactant protein SP-C has been characterized in comparison with that of native palmitoylated SP-C purified from porcine lungs. Both native and recombinant proteins promoted interfacial adsorption of dipalmitoylphosphatidylcholine bilayers to a limited extent, but catalyzed very rapid formation of films from different lipid mixtures containing both zwitterionic and anionic phospholipids. Once at the interface, the recombinant variants exhibited compression-driven structural transitions, consistent with changes in the orientation of the deacylated N-terminal segment, which were not observed in the native protein. Compression isotherms of lipid/protein films suggest that the recombinant SP-C forms promote expulsion at high pressures of a higher number of lipid molecules per mole of protein than does native SP-C. A more dynamic conformation of the N-terminal segment in recombinant SP-C forms is likely also responsible for facilitating compression-driven condensation of domains in anionic phospholipid films as observed by epifluorescence microscopy. Finally, both native palmitoylated SP-C and the phenylalanine-containing recombinant versions facilitate similarly the repetitive compression-expansion dynamics of lipid/protein films, which were able to reach maximal surface pressures with practically no hysteresis along multiple quasi-static or dynamic cycles.     

Ligand-induced reorganization and assembly in synthetic lipid membranes

Membrane targetting of soluble ligands accompanied by assembly of membrane components into functional superstructures underlies biological signal transduction and a variety of other processes ranging from blood coagulation to biomineralization. Protein or lipid components provide the interactions required for targetting and specific orientation of bound molecules; the membrane's fluidity allows reorganization and sampling of intermolecular contacts required for assembly into superstructures. We are developing synthetic membrane-based recognition systems capable of reproducing important features of biological targetting and assembly. Systems such as these may open up new routes to controlling molecular architecture in materials and devices. Specially designed metal-chelating receptor/reporter lipids have been used to study lipid reorganization induced by binding of metal-complexing ligands. Proteins and peptides are targetted to the Cu²⁺- and Ni²⁺-complexing lipids via coordination interactions with surface histidines. Binding and assembly of multivalent ligands are accompanied by reorganization of the lipid receptors, as measured by fluorescence spectroscopy and fluorescence microscopy. Coordination interactions between protein and chelating lipid components can be used for direct assembly into superstructures such as patterned lipid monolayers and two-dimensional protein crystals.     

Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy

Phototropins are light-activated kinases from plants that utilize light-oxygen-voltage (LOV) domains as blue light photosensors. Illumination of these domains leads to the formation of a covalent linkage between the protein and an internally bound flavin chromophore, destabilizing the surrounding protein and displacing an alpha-helix from its surface. Here we use a combination of spectroscopic tools to monitor the kinetic processes that spontaneously occur in the dark as the protein returns to the noncovalent ground state. Using time-resolved two-dimensional (2D) NMR methods, we measured the rate of this process at over 100 independent sites throughout the protein, establishing that regeneration of the dark state occurs cooperatively within a 1.6-fold range of observed rates. These data agree with other spectroscopic measurements of the kinetics of protein/FMN bond cleavage and global conformational changes, consistent with these processes experiencing a common rate-limiting step. Arrhenius analyses of the temperature dependence of these rates suggest that the transition state visited during this regeneration has higher energy than the denatured form of this protein domain despite the fact that there is no global unfolding of the domain during this process.     

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.     

Temperature and pH dual-responsive coatings of oligoperoxide-graft-poly(N-isopropylacrylamide): Wettability, morphology, and protein adsorption

Poly(N-isopropylacrylamide) (PNIPAM) coatings attached to glass with novel approach involving polymerization from oligoperoxide grafted to surface with (3-aminopropyl)triethoxysilane exhibit not only temperature- but also pH-dependence of wettability and protein adsorption. Wettability and composition of coatings, fabricated with different polymerization times, were determined using contact angle measurements and Time Of Flight-Secondary Ion Mass Spectrometry (TOF-SIMS), respectively. Thermal response of wettability, measured between 20 and 40 °C, was prominent at pH 9 and 7 and diminished or absent at pH 5 and 3. This indicates a transition between hydrated loose coils and hydrophobic collapsed chains that is blocked at low pH. Higher surface roughness and dramatically increased adsorption of model protein (lentil lectin labeled with fluorescein isothiocyanate) were observed with AFM and fluorescence microscopy to occur in hydrophobic phases (at pH 3, for pH varied at constant temperature of 22 °C and at ∼33 °C, for temperature varied at constant pH 9). Protein adsorption response to pH was confirmed by TOF-SIMS and Principal Component Analysis.     

Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays

One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.