Enzymic thin film coatings for bioactive materials

Bioactive materials have been explored for a broad range of applications including biocatalysts, biosensors, antifouling membranes and other functional and smart materials. We report herein a unique method for preparation of bioactive materials through a spin coating process. Specifically, we investigated the preparation of protease Subtilisin Carlsberg-coated plastic films and examined their activities for hydrolysis of chicken egg albumin (CEA). The process generated enzymic coatings with a typical loading of 13 microg/cm2, retaining 46% of the enzyme activity for hydrolysis of CEA in aqueous solutions. Interestingly, the surface-coated protease thin film not only catalyzed the hydrolysis of CEA in aqueous solutions, but also showed good activity for solid-state CEA that was coated on top of the enzyme thin film.     

Fabrication and electromechanical characterization of freestanding asymmetric membranes

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner in which specific properties and processes can be isolated and examined. Here, we describe an approach to fabricate and characterize planar, freestanding, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (DOPG) content as compared with previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC-DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison with symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.     

Protection of sensors for biological applications by photoinitiated chemical vapor deposition of hydrogel thin films

We report photoinitiated chemical vapor deposition (piCVD), a gentle synthetic method for the preparation of ultrathin films (approximately 100 nm) of the hydrogel poly(hydroxyethyl methacrylate) (pHEMA). piCVD occurs near room temperature and requires only mild vacuum conditions. The deposited films swell rapidly and reversibly in buffer solution, and the swelling properties can be controlled via the deposition conditions. Analysis of the swelling data indicates that the mesh size of the hydrogel creates a selectively permeable coating. The mesh is large enough to allow small molecule analytes to permeate the film but small enough to prevent the transport of large biomolecules such as proteins. X-ray photoelectron spectroscopy (XPS) shows that the films decrease nonspecific adhesion of the protein albumin by nearly 8-fold over bare silicon. A dry process, piCVD is suitable for coating particles with diameters as small as 5 microm. The absence of solvents and plasmas in piCVD allows films to be directly synthesized on optode sensors without degradation of sensitivity or response time.     

Hybrid materials from amphiphilic block copolymers and membrane proteins

Self-assembly of reactive amphiphilic block copolymers is used to prepare nanostructured hydrogels with exceptional permeability properties, vesicular structures and planar, freestanding membranes in aqueous solution. Although the underlying block copolymer membranes are two-three-fold thicker than conventional lipid bilayers, they can be regarded as mimetic of biological membranes and can be used as a matrix for membrane-spanning proteins. Surprisingly, the proteins remain functional, despite the extreme thickness of the membranes and even after polymerization of the reactive block copolymers. The unique combination of block copolymers with membrane proteins allows the preparation of mechanically stable, defect-free membranes and nanocapsules that have highly selective permeability and/or specific recognition sites. This is documented by some representative examples.     

Development of MoS2–CNT Composite Thin Film from Layered MoS2 for Lithium Batteries

Layered MoS₂ prepared by liquid‐phase exfoliation has been blended with single‐walled carbon nanotubes (SWNTs) to form novel composite thin films for lithium battery applications. The films were formed by vacuum filtration of blended dispersions onto nitrocellulose membranes. The resulting composite films were transferred onto Cu foil electrodes via a facile filtration/wet transfer technique from nitrocellulose membranes. The morphology of the film was characterised by field emission scanning electron microscopy, which suggests that the MoS₂‐SWNT composite film shows good adherence to the Cu foil substrate. The MoS₂‐SWNT composite thin films show strong electrochemical performance at different charge‐discharge rates. The capacity of a MoS₂‐SWNT composite film with thickness of 1 μm is approximately 992 mAh g^?1 after 100 cycles. The morphology study showed that the MoS₂‐SWNT thin film retains structural integrity after 100 cycles, while the MoS₂ thin film without SWNTs displays significant cracking. In addition, the novel composite thin film preparation and transfer protocols developed in this study could be extended to the preparation of various layered‐material‐based composite films, with the potential for new device designs for energy applications.     

Composition Engineering in Doctor‐Blading of Perovskite Solar Cells

Organic–inorganic halide perovskite (OIHP) solar cells with efficiency over 18% power conversion efficiency (PCE) have been widely achieved with lab scale spin‐coating method which is however not scalable for the fabrication of large area solar panels. The PCEs of OIHP solar cells made by scalable deposition methods, such as doctor‐blading or slot‐die coating, have been lagging far behind than spin‐coated devices. In this study the authors report composition engineering in doctor‐bladed OIHP solar cells with p–i–n planar heterojunction structure to enhance their efficiency. Phase purer OIHP thin films are obtained by incorporating a small amount of cesium (Cs⁺) and bromine (Br^?) ions into perovskite precursor solution, which also reduces the required film formation temperature. Pinhole free OIHP thin films with micrometer‐sized grains have been obtained assisted by a secondary grain growth with added methylammonium chloride into the precursor solution. The OIHP solar cells using these bladed thin films achieved PCEs over 19.0%, with the best stabilized PCE reaching 19.3%. This represents a significant step toward scalable manufacture of OIHP solar cells.     

Optical Absorption Enhancement in Freestanding GaAs Thin Film Nanopyramid Arrays

Although III–V compound semiconductor multi‐junction cells show the highest efficiency among all types of solar cells, their cost is quite high due to expensive substrates, long epitaxial growth and complex balance of system components. To reduce the cost, ultra‐thin films with advanced light management are desired. Here effective light trapping in freestanding thin film nanopyramid arrays is demonstrated and multiple‐times light path enhancement is realized, where only 160 nm thick GaAs with nanopyramid structures is equivalent to a 1 μm thick planar film. The GaAs nanopyramids are fabricated using a combination of nanosphere lithography, nanopyramid metal organic chemical vapor deposition (MOCVD) growth, and gas‐phase substrate removal processes. Excellent optical absorption is demonstrated over a broad range of wavelengths, at various incident angles and at large‐curvature bending. Compared to an equally thick planar control film, the overall number of photons absorbed is increased by about 100% at various incident angles due to significant antireflection and light trapping effects. By implementing these nanopyramid structures, III–V material usage and deposition time can be significantly reduced to produce high‐efficiency, low‐cost thin film III–V solar cells.     

Nanoscale reduction in surface friction of polymer surfaces modified with Sc3 hydrophobin from Schizophyllum commune

Hydrophobins are amphipathic self-assembling proteins secreted by filamentous fungi that exhibit remarkable ability to modify synthetic surfaces. Thin coatings of Sc3 hydrophobin isolated from the wood-rotting fungus Schizophyllum commune were prepared via spin coating and adsorption techniques onto polymeric surfaces. Surface morphology and nanotribological characteristics of the films were evaluated using lateral force microscopy (LFM) and nanoindentation techniques. This paper reports the first observation of reduction in nanoscale relative surface friction of Sc3 hydrophobin protein modified polymeric surfaces. Relative friction coefficients were dramatically reduced and hydrophilicity increased for polymer surfaces modified with Sc3 hydrophobin thin films. Morphology of the protein films as well as degree of surface modification was observed to be a function of film formation technique and composition of the substrate.     

Development and application of a nonradioactive phosphorescent autoradiograph marker

A simple and rapid method for permanently marking autoradiographs is described. This procedure is based on the phosphorescence of light-activated zinc sulfide and the subsequent exposure of x-ray films by this light emission. A lacquer-based carrier allows the zinc sulfide to remain in suspension and permits permanent marking onto diverse laboratory substrates such as x-ray films, paper, plastic wraps and nitrocellulose- and nylon-based membranes. An analogous wax-based carrier allows marking onto paper and dried acrylamide gels, which is useful for processing large numbers of radioactive DNA sequencing gels on a high-throughput scale. These inexpensive and nonhazardous markers will be useful in protocols that use x-ray films, regardless of whether radioactive or nonradioactive detection systems are used.     

S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Phase coexistence in films composed of DLPC and DPPC: A comparison between different model membrane systems

For the biophysical study of membranes, a variety of model systems have been used to measure the different parameters and to extract general principles concerning processes that may occur in cellular membranes. However, there are very few reports in which the results obtained with the different models have been compared. In this investigation, we quantitatively compared the phase coexistence in Langmuir monolayers, freestanding bilayers and supported films composed of a lipid mixture of DLPC and DPPC. Two-phase segregation was observed in most of the systems for a wide range of lipid proportions using fluorescence microscopy. The lipid composition of the coexisting phases was determined and the distribution coefficient of the fluorescent probe in each phase was quantified, in order to explore their thermodynamic properties. The comparison between systems was carried out at 30 mN/m, since it is accepted that at this or higher lateral pressures, the mean molecular area in bilayers is equivalent to that observed in monolayers. Our study showed that while Langmuir monolayers and giant unilamellar vesicles had a similar phase behavior, supported films showed a different composition of the phases with the distribution coefficient of the fluorescent probe being close to unity. Our results suggest that, in supported membranes, the presence of the rigid substrate may have led to a stiffening of the liquid-expanded phase due to a loss in the degrees of freedom of the lipids as a consequence of the proximity of the solid material.     

Optimized Electroless Silver Coating for Optical and Plasmonic Applications

Electroless metal deposition is a simple and convenient technique to fabricate metallic films and to provide isotropic metal functionalization of 3D structures with complex geometries. In this work, we describe the synthesis of silver coatings by means of a modified Tollens reaction and their use as optical coating. The chemical composition of the metallization bath is here addressed to optimize the metal coating deposition. The synthesis parameters have been tailored in order to deposit very smooth films which were characterized by scanning electron microscopy, atomic force microscopy, and optical spectroscopy. 2D diffraction gratings and sinusoidal plasmonic gratings were produced with the proposed method. Optical characterization confirmed the plasmonic activities of the resultant structures, proving the efficiency of the described method for optical applications. Thermal annealing was found to improve the surface roughness of the coating and therefore the optical properties of the plasmonic gratings.     

Factors that Influence the Formation and Stability of Thin,Cryo-EM Specimens

Poor consistency of the ice thickness from one area of a cryo-electron microscope (cryo-EM) specimen grid to another, from one grid to the next, and from one type of specimen to another, motivates a reconsideration of how to best prepare suitably thin specimens. Here we first review the three related topics of wetting, thinning, and stability against dewetting of aqueous films spread over a hydrophilic substrate. We then suggest that the importance of there being a surfactant monolayer at the air-water interface of thin, cryo-EM specimens has been largely underappreciated. In fact, a surfactant layer (of uncontrolled composition and surface pressure) can hardly be avoided during standard cryo-EM specimen preparation. We thus suggest that better control over the composition and properties of the surfactant layer may result in more reliable production of cryo-EM specimens with the desired thickness.     

Photocurrents generated by bacteriorhodopsin adsorbed on nano-black lipid membranes

Purple membranes were adsorbed on freestanding lipid bilayers, termed nano-black lipid membranes (nano-BLMs), suspending the pores of porous alumina substrates with average pore diameters of 280 nm. Nano-BLMs were obtained by first coating the upper surface of the highly ordered porous alumina substrates with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol and subsequent addition of a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and octadecylamine dissolved in n-decane onto the hydrophobic submonolayer. By means of impedance spectroscopy, the quality of the nano-BLMs was verified. The electrical parameters confirm the formation of single lipid bilayers with high membrane resistances covering the porous matrix. Adsorption of purple membranes on the nano-BLMs was followed by recording the photocurrents generated by bacteriorhodopsin upon continuous light illumination. The membrane system exhibits a very high long-term stability with the advantage that not only transient but also stationary currents are recordable. By adding the proton ionophore carbonyl cyanide-m-chlorophenylhydrazone the conductivity of the nano-BLMs increases, resulting in a higher stationary current, which proves that proton conductance occurs across the nano-BLMs.     

Biodegradable polylactide/chitosan blend membranes

Biodegradable blend membranes based on polylactide and chitosan with various compositions were prepared via a two-step processing pathway. In the first step, solutions of each component were properly mixed and cast into a gelatinous membrane, and in the second step, the obtained membrane was immersed into a mixed solution for the solvent extraction followed by a drying procedure to finally generate a well-blended membrane. An acetic acid-acetone solvent system was selected for poly(DL-lactide)/chitosan membranes, and another solvent system for poly(L-lactide)/chitosan membranes consisted of acetic acid and dimethyl sulfoxide. Some processing parameters, such as the concentration of component solutions and the composition ratio of mixed solvents and extraction solvents, were optimized by primarily considering whether the directly visible phase separation occurred during the processing procedures. Morphologies of these blend membranes were viewed using SEM. It was found that the processing parameters exerted quite notable impacts on the morphology of the membranes. The hydrophilicity of membranes was examined by measuring their water contact angle and swelling index. These blend membranes were also investigated for their miscibility using IR spectra, X-ray diffractograms, TG, DSC, and dynamic mechanical analysis methods. Although the presence of phase separation at a microscopic level was detected for these membranes, pronounced interactions between components were confirmed. The obtained results shown that some membranes prepared under optimized processing conditions had a partially miscible structure.     

Glass fibers covered with sol-gel glass as a new support for affinity chromatography columns: a review.

The search for mechanical supports for biochemically active compounds serving as immunochemical sensors has been the goal of many studies. A new compound in the form of gel fiberglass (GFG) membranes was recently developed as an example of such supports. In this review, these membranes were analyzed with respect to their use for cancer detection. The membranes are prepared from glass fibers covered with oxysilane to provide a sol-gel glass matrix. Derivatization of the support eliminates nonspecific adsorption. A thin layer of protein trapped in the gel glass during its preparation is deposited on the surface of a lattice of glass fibers. The major innovation of the membrane is its large area. External agents percolating through such a membrane contact a maximal number of molecules of the compounds trapped in the sol-gel glass. As a result, this membrane is highly effective. Each GFG column is built from a series of 20 to 30 membranes. The preparation of such columns is relatively simple, requiring only several hours. The capacity of GFG columns is high: the total amount of tumor-associated antigens (TAA) isolated by these columns from the blood of colon cancer patients reached 50% of the total protein and amounted to up to 9-12 mg/ml of serum. The main components of the isolated TAA were the soluble p66 and p51 proteins. A determination of their concentration by HPLC can be used for early cancer detection. Thus, the described method allows the easy and highly effective isolation of TAA and can be used for different goals, including cancer diagnosis. GFG supports have great potential for the isolation of various macromolecules utilizing specific ligands.     

Graphical analysis of mass and anisotropy changes observed by plasmon-waveguide resonance spectroscopy can provide useful insights into membrane protein function

Plasmon-waveguide resonance spectroscopy is a recently developed optical method that allows characterization of mass and structural changes in two-dimensionally ordered thin films (e.g., proteolipid membranes) deposited onto a sensor surface. Full analysis of these systems involves fitting theoretical curves (obtained using Maxwell's equations) to experimental spectra measured using s- and p-polarized excitation. This allows values to be obtained for refractive indices and optical extinction coefficients in these two directions, as well as a value for film thickness, thereby providing information about mass density and anisotropy changes. This is a time-consuming process that works well for simple systems in which only a single conformational event occurs, but cannot distinguish between events involving multiple conformations that proceed either sequentially or in a parallel series of events. This article describes a graphical method that can distinguish between mass density and anisotropy changes in a simpler, more rapid procedure, even for processes that proceed via multiple conformational events. This involves measurement of plasmon-waveguide resonance spectral shifts obtained upon molecular interactions occurring in deposited films with both s- and p-polarized excitation, and transforming these from an (s-p) coordinate system into a (mass-structure) coordinate system. This procedure is illustrated by data obtained upon the binding of a small peptide, penetratin, to solid-supported lipid bilayer membranes.     

Hydrocolloid coating of Xenopus laevis embryos

A novel technology for coating single cells and embryos with thin hydrocolloid (water-soluble polymer) films has been invented and patented. Coating is different from entrapment and immobilization in that the coating around the cell is thinner, comprising only a small fraction of the cell or embryo's diameter. Xenopus laevis embryos were coated with thin films of low-methoxy pectin (LMP), alginate, and iota- and kappa-carrageenans. These gums have different compositions and structures and as such created different coatings around the fertilized cells. All coated embryos appeared to develop normally, similar to noncoated embryos. Elemental detection by ICP-AES spectroscopy revealed that the embryo can control the diffusion of excess ions to which it is exposed during the coating process. The coatings delayed hatching by 18-24 h. Consequently, at hatch the embryos were at a more developed stage than their noncoated counterparts. The hydrocolloid coating reduced the thickness of the natural jelly coating (JC). With the iota-carrageenan coating, percent hatch was maximal, while with LMP it was minimal, as a result of the films' mechanical properties and thicknesses. LMP and alginate created smoother coatings than the carrageenans. Potential interactions between the coating and the natural JC are hypothesized. Overall, coatings appear to be a suitable tool for laboratories interested in performing longer-term experiments with embryos.     

Combinatorial Screening of Polymer:Fullerene Blends for Organic Solar Cells by Inkjet Printing

Polymer:fullerene blends were screened in a combinatorial approach using inkjet printing thin film libraries for photovoltaic devices. The application of inkjet printing enabled a fast and simple experimental workflow from film preparation to the study of structure‐property‐relationships with a very high material efficiency. Inkjet printing requires less material for the preparation of thin film libraries in comparison to other dispensing techniques, like spin‐coating. Two polymers (PCPDTBT, PSBTBT) and two fullerene derivatives (mono‐PCBM, bis‐PCBM) were investigated in various blend ratios, concentrations, solvent ratios, and film thicknesses. Morphological and optical properties of the inkjet printed films were investigated and compared with spin‐coated films. This study shows the principle of an experimental setup from solution preparation to film characterization for the combinatorial investigation of large polymer:fullerene libraries.     

Drying Dynamics of Solution‐Processed Perovskite Thin‐Film Photovoltaics: In Situ Characterization,Modeling, and Process Control

A key challenge for the commercialization of perovskite photovoltaics is the transfer of high‐quality spin coated perovskite thin‐films toward applying industry‐scale thin‐film deposition techniques, such as slot‐die coating, spray coating, screen printing, or inkjet printing. Due to the complexity of the formation of polycrystalline perovskite thin‐films from the precursor solution, efficient strategies for process transfer require advancing the understanding of the involved dynamic processes. This work investigates the fundamental interrelation between the drying dynamics of the precursor solution thin‐film and the quality of the blade coated polycrystalline perovskite thin‐films. Precisely defined drying conditions are established using a temperature‐stabilized drying channel purged with a laminar flow of dry air. The dedicated channel is equipped with laser reflectometry at multiple probing positions, allowing for in situ monitoring of the perovskite solution thin‐film thickness during the drying process. Based on the drying dynamics as measured at varying drying parameters, namely at varying temperature and laminar air flow velocity, a quantitative model on the drying of perovskite thin‐films is derived. This model enables process transfer to industry‐scale deposition systems beyond brute force optimization. Via this approach, homogeneous and pinhole‐free blade coated perovskite thin‐films are fabricated, demonstrating high power conversion efficiencies of up to 19.5% (17.3% stabilized) in perovskite solar cells.