Development of a DsRed-expressing HepaRG cell line for real-time monitoring of hepatocyte-like cell differentiation by fluorescence imaging, with application in screening of novel geometric microstructured cell growth substrates.

The bipotent nature of the HepaRG cell line is a unique property among human hepatoma-derived cells. Cell treatment with specific differentiation inducers results in a mixture of hepatocyte- and biliary-like cells, accompanied by upregulation of liver-specific proteins, drug metabolizing enzymes, transcription regulators, membrane receptors or innate immune response effectors. These features make the HepaRG cells a suitable and handy replacement for primary hepatocytes, to study hepatic functions in vitro. However, cell differentiation is a long, variable process, requiring special culture conditions, while the resulting mixed cell populations is usually a major drawback. This process can potentially be controlled by interface characteristics, such as substrate topography. To screen for such novel substrates, we have first developed a new HepaRG cell line, designated as HepaRGDsRed, expressing the reporter gene DsRed. The fluorescent protein was expressed in hepatocyte- and not biliary-like cells, in a differentiation dependent-manner. We have further used replicated microstructured gradients of polydimethylsiloxane (PDMS) that allow three-dimensional manipulation in vitro, to monitor HepaRGDsRed differentiation in real time. We demonstrate that this approach enables the controlled assembly of viable hepatocyte-like cells for functional studies, which can be maintained in culture without loss of differentiation. The regulated expression of the DsRed reporter proved a valuable tool not only for rapid screening of novel cell growth substrates favoring cell differentiation, but also, to enrich the hepatocyte-like cell population by fluorescence-activated cell sorting to investigate liver-specific processes in vitro.

Molecular dimensions and surface diffusion assisted mechanically robust slippery perfluoropolyether impregnated mesoporous alumina interfaces.

Accomplishing mechanically robust omniphobic surfaces is a long-existing challenge, and can potentially find applications in bioengineering, tribology and paint industries. Slippery liquid impregnated mesoporous α-Al2O3 interfaces are achieved with water, alkanes, water based and oil based high viscosity acrylic paints. Incredibly high abrasion-resistance (wear coefficients ≤10-8 mm3 N-1 m-1) and ultra-low friction coefficients (≥0.025) are attained, attributing to the hard alumina matrix and continuous replenishment of perfluoropolyether aided by capillarity and surface diffusion processes. A variety of impregnating liquids employed suggest that large molecules, faster surface diffusion and lowest evaporation rate generate the rare combination of high wear-resistance and omniphobicity. It is noteworthy that these novel liquid impregnated Al2O3 composites exhibit outstanding load bearing capacity up to 350 MPa; three orders of magnitude higher than achievable by the state of the art omniphobic surfaces. Further, our developed thermodynamic calculations suggest that the relative thermodynamic stability of liquid impregnated composites is linearly proportional to the spreading coefficient (S) of the impregnating liquid with the matrix material and is an important tool for the selection of an appropriate matrix material for a given liquid.

Microstructure and ferroelectricity of BaTiO3 thin films on Si for integrated photonics.

Significant progress has been made in integrating novel materials into silicon photonic structures in order to extend the functionality of photonic circuits. One of these promising optical materials is BaTiO3 or barium titanate (BTO) that exhibits a very large Pockels coefficient as required for high-speed light modulators. However, all previous demonstrations show a noticable reduction of the Pockels effect in BTO thin films deposited on silicon substrates compared to BTO bulk crystals. Here, we report on the strong dependence of the Pockels effect in BTO thin films on their microstructure, and provide guidelines on how to engineer thin films with strong electro-optic response. We employ several deposition methods such as molecular beam epitaxy and chemical vapor deposition to realize BTO thin films with different morphology and crystalline structure. While a linear electro-optic response is present even in porous, polycrystalline BTO thin films with an effective Pockels coefficient r eff = 6 pm V-1, it is maximized for dense, tetragonal, epitaxial BTO films (r eff = 140 pm V-1). By identifying the key structural predictors of electro-optic response in BTO/Si, we provide a roadmap to fully exploit the linear electro-optic effect in novel hybrid oxide/semiconductor nanophotonic devices.

Room Temperature Direct Electron Beam Lithography in a Condensed Copper Carboxylate.

High-resolution metallic nanostructures can be fabricated with multistep processes, such as electron beam lithography or ice lithography. The gas-assisted direct-write technique known as focused electron beam induced deposition (FEBID) is more versatile than the other candidates. However, it suffers from low throughput. This work presents the combined approach of FEBID and the above-mentioned lithography techniques: direct electron beam lithography (D-EBL). A low-volatility copper precursor is locally condensed onto a room temperature substrate and acts as a positive tone resist. A focused electron beam then directly irradiates the desired patterns, leading to local molecule dissociation. By rinsing or sublimation, the non-irradiated precursor is removed, leaving copper-containing structures. Deposits were formed with drastically enhanced growth rates than FEBID, and their composition was found to be comparable to gas-assisted FEBID structures. The influence of electron scattering within the substrate as well as implementing a post-purification protocol were studied. The latter led to the agglomeration of high-purity copper crystals. We present this as a new approach to electron beam-induced fabrication of metallic nanostructures without the need for cryogenic or hot substrates. D-EBL promises fast and easy fabrication results.

Epitaxial Growth of Silicon on Silicon Wafers by Direct Laser Melting.

Additive manufacturing (AM) of brittle materials remains challenging, as they are prone to cracking due to the steep thermal gradients present during melting and cooling after laser exposition. Silicon is an ideal brittle material for study since most of the physical properties of single-element materials can be found in the literature and high-purity silicon powders are readily available. Direct laser melting (DLM) of silicon powder was performed to establish the conditions under which cracks occur and to understand how the solidification front impacts the final microstructure. Through careful control of process conditions, paying special attention to thermal gradients and the growth velocity, epitaxial pillars free of cracks could be grown to a length of several millimeters.

Gold nanoring arrays from responsive block copolymer templates.

We report a pH-mediated synthetic route for the production of ordered and size-tuneable arrays of gold nanorings using responsive block copolymer micelles as templates.

Perfluoropolyether-Impregnated Mesoporous Alumina Composites Overcome the Dewetting-Tribological Properties Trade-Off.

Conventional omniphobic surfaces suffer from wear-sensitivity due to soft apolar coatings or substrates and protruding surface features that are eroded even for mild abrasion treatments, leading to the loss of dewetting properties after wear. Evidently, there was a trade-off between dewetting and tribological properties. Here, we show the establishment of self-healing slippery properties post severe abrasion by utilizing perfluoropolyether-impregnated mesoporous Al2O3 (MPA) composites. The hard polar alumina matrix provides the optimal tribological properties, and the liquid lubricant in the porous network contributes to both tribological and self-healing dewetting properties. These composites sustained normal pressures up to 350 MPa during reciprocating sliding contacts. The severely abraded surfaces are capable of self-replenishing in ambient environment, driven by capillarity and surface diffusion processes, and regained their slippery properties toward water and hexadecane after 15 h of self-healing. Eventually, a dewetting-tribology diagram has been introduced to show different regimes, namely-optimal slippery properties, optimal tribological properties, and a mixed regime). We found out that the microstructural expression [Formula: see text] is a robust guiding tool to predict the regime of interest. This dewetting-tribological diagram may be marked as an inception to designing abrasion-resistant slippery liquid impregnated composites for overcoming the dewetting tribological properties trade-off. Such surfaces may potentially find applications in paint industries and as anti-icing surfaces.

Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture.

For the observation of single molecule dynamics with fluorescence fluctuation spectroscopy (FFS) very low fluorophore concentrations are necessary. For in vitro measurements, this requirement is easy to fulfill. In biology however, micromolar concentrations are often encountered and may pose a real challenge to conventional FFS methods based on confocal instrumentation. We show a higher confinement of the sampling volume in the near-field of sub-wavelength sized apertures in a thin gold film. The gold apertures have been measured and characterized with fluorescence correlation spectroscopy (FCS), indicating light confinement beyond the far-field diffraction limit. We measured a reduction of the effective sampling volume by an order of magnitude compared to confocal instrumentation.

Selective growth of titanium dioxide by low-temperature chemical vapor deposition.

A key factor in engineering integrated optical devices such as electro-optic switches or waveguides is the patterning of thin films into specific geometries. In particular for functional oxides, etching processes are usually developed to a much lower extent than for silicon or silicon dioxide; therefore, selective area deposition techniques are of high interest for these materials. We report the selective area deposition of titanium dioxide using titanium isopropoxide and water in a high-vacuum chemical vapor deposition (HV-CVD) process at a substrate temperature of 225 °C. Here­contrary to conventional thermal CVD processes­only hydrolysis of the precursor on the surface drives the film growth as the thermal energy is not sufficient to thermally decompose the precursor. Local modification of the substrate surface energy by perfluoroalkylsilanization leads to a reduced surface residence time of the precursors and, consequently, to lower reaction rate and a prolonged incubation period before nucleation occurs, hence, enabling selective area growth. We discuss the dependence of the incubation time and the selectivity of the deposition process on the presence of the perfluoroalkylsilanization layer and on the precursor impinging rates­with selectivity, we refer to the difference of desired material deposition, before nucleation occurs in the undesired regions. The highest measured selectivity reached (99 ± 5) nm, a factor of 3 superior than previously reported in an atomic layer deposition process using the same chemistry. Furthermore, resolution of the obtained patterns will be discussed and illustrated.

Combinatorial Characterization of TiO2 Chemical Vapor Deposition Utilizing Titanium Isopropoxide.

The combinatorial characterization of the growth kinetics in chemical vapor deposition processes is challenging because precise information about the local precursor flow is usually difficult to access. In consequence, combinatorial chemical vapor deposition techniques are utilized more to study functional properties of thin films as a function of chemical composition, growth rate or crystallinity than to study the growth process itself. We present an experimental procedure which allows the combinatorial study of precursor surface kinetics during the film growth using high vacuum chemical vapor deposition. As consequence of the high vacuum environment, the precursor transport takes place in the molecular flow regime, which allows predicting and modifying precursor impinging rates on the substrate with comparatively little experimental effort. In this contribution, we study the surface kinetics of titanium dioxide formation using titanium tetraisopropoxide as precursor molecule over a large parameter range. We discuss precursor flux and temperature dependent morphology, crystallinity, growth rates, and precursor deposition efficiency. We conclude that the surface reaction of the adsorbed precursor molecules comprises a higher order reaction component with respect to precursor surface coverage.

Surface 3D Micro Free Forms: Multifunctional Microstructured Mesoporous α-Alumina by in Situ Slip Casting Using Excimer Laser Ablated Polycarbonate Molds.

Ceramic surface microstructuring is a rapidly growing field with a variety of applications in tribology, wetting, biology, and so on. However, there are limitations to large-area microstructuring and fabrication of three-dimensional (3D) micro free forms. Here, we present a route to obtain intricate surface structures through in situ slip casting using polydimethylsiloxane (PDMS) negative molds which are replicated from excimer laser ablated polycarbonate (PC) master molds. PC sheets are ablated with a nanosecond KrF (λ = 248 nm) excimer laser mask projection system to obtain micron-scale 3D surface features over a large area of up to 3 m(2). Complex surface structures that include 3D free forms such as 3D topography of Switzerland, shallow structures such as diffractive optical elements (60 nm step) and conical micropillars have been obtained. The samples are defect-free produced with thicknesses of up to 10 mm and 120 mm diameter. The drying process of the slip cast alumina slurry takes place as a one-dimensional process, through surface evaporation and water permeation through the PDMS membrane. This allows homogeneous one-dimensional shrinkage during the drying process, independent of the sample's lateral dimensions. A linear mass diffusion model has been proposed to predict and explain the drying process of these ceramic colloidal suspensions. The calculated drying time is linearly proportional to the height of the slurry and the thickness of the negatively structured PDMS and is validated by the experimental results. An experimentally observed optimum Sylgard PDMS thickness range of ∼400 µm to 1 mm has achieved the best quality microstructured green compacts. Further, the model predicts that the drying time is independent of the microstructured areas and was validated using experimental observations carried out with microstructured areas of 300 mm(2), 1200 mm(2), and 120 cm(2). Therefore, in principle, the structures can be further replicated in areas up to 3 m(2) with the same drying time for the same slurry height. The surface-structured ceramics display interesting wetting properties, for example, eicosane-coated mesoporous microstructured alumina shows superhydrophobic behavior. Additionally, ceramic bulk samples could be further used as second-generation very hard and low-wear molds for further microfabrication.

Hierarchical positioning of gold nanoparticles into periodic arrays using block copolymer nanoring templates.

We report a simple and versatile self-assembly method for controlling the placement of functional gold nanoparticles on silicon substrates using micellar templates. The hierarchical positioning of gold nanoparticles is achieved in one-step during the spontaneous phase inversion of spherical poly(styrene)-block-poly(2-vinylpyridine) copolymer micelles into nanoring structures. The placement is mainly driven by the establishment of electrostatic interactions between the nanoparticle ligands and the pyridine groups exposed at the interface. In particular, we show the formation of ordered arrangements of single gold nanoparticles or nanoparticle clusters and demonstrate that their morphologies, densities and periodicities can be tuned by simply varying the initial block copolymer molecular weight or the deposition conditions. Besides gold nanoparticles, the method can be used for controlling the assembly of a large variety of nanoscale building blocks, thus opening an attractive pathway for generating functional hybrid surfaces with periodic nanopatterns.

Determination of local refractive index variations in thin films by heterodyne interferometric scanning near-field optical microscopy.

We report on a heterodyne interferometric scanning near-field optical microscope developed for characterizing, at the nanometric scale, refractive index variations in thin films. An optical lateral resolution of 80 nm (lambda/19) and a precision smaller than 10(-4) on the refractive index difference have been achieved. This setup is suitable for a wide set of thin films, ranging from periodic to heterogeneous samples, and turns out to be a very promising tool for determining the optical homogeneity of thin films developed for nanophotonics applications.

Water wetting transition parameters of perfluorinated substrates with periodically distributed flat-top microscale obstacles.

Superhydrophobicity is obtained on photolithographically structured silicon surfaces consisting of flat-top pillars after a perfluorosilanization treatment. Systematic static contact angle measurements were carried out on these surfaces as a function of pillar parameters that geometrically determine the surface roughness, including pillar height, diameter, top perimeter, overall filling factor, and disposition. In line with thermodynamics models, two regimes of static contact angles are observed varying each parameter independently: the "Cassie" regime, in which the water drop sits suspended on top of the pillars (referred to as composite), corresponding to experimental contact angles greater than 140-150 degrees, and the "Wenzel" regime, in which water completely wets the asperities (referred to as wetted), corresponding to lower experimental contact angles. A transition between the Cassie and Wenzel regimes corresponds to a set of well-defined parameters. By smoothly depositing water drops on the surfaces, this transition is observed for surface parameter values far from the calculated ones for the thermodynamic transition, therefore offering evidence for the existence of metastable composite states. For all studied parameters, the position of the experimental transition correlates well with a rough estimation of the energy barrier to be overcome from a composite metastable state in order to reach the thermodynamically favored Wenzel state. This energy barrier is estimated as the surface energy variation between the Cassie state and the hypothetical composite state with complete filling of the surface asperities by water, keeping the contact angle constant.

Reversible immobilization of peptides: surface modification and in situ detection by attenuated total reflection FTIR spectroscopy.

A generic method is described for the reversible immobilization of polyhistidine-bearing polypeptides and proteins on attenuated total reflecting (ATR) sensor surfaces for the detection of biomolecular interactions by FTIR spectroscopy. Nitrilotriacetic acid (NTA) groups are covalently attached to self-assembled monolayers of either thioalkanes on gold films or mercaptosilanes on silicon dioxide films deposited on germanium internal reflection elements. Complex formation between Ni2+ ions and NTA groups activates the ATR sensor surface for the selective binding of polyhistidine sequences. This approach not only allows a stable and reversible immobilization of histidine-tagged peptides (His-peptides) but also simultaneously allows the direct in situ quantification of surface-adsorbed molecules from their specific FTIR spectral bands. The surface concentrations of both NTA and His-peptide on silanized surfaces were determined to be 1.1 and 0.4 molecules nm-2, respectively, which means that the surface is densely covered. A comparison of experimental FTIR spectra with simulated spectra reveals a surface-enhancement effect of one order of magnitude for the gold surfaces. With the presented sensor surfaces, new ways are opened up to investigate, in situ and with high sensitivity and reproducibility, protein-ligand, protein-protein, protein-DNA interactions, and DNA hybridization by ATR-FTIR spectroscopy.