Stress-dependent macromolecular crowding in the mitochondrial matrix.

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.

Inline Reaction Monitoring of Amine-Catalyzed Acetylation of Benzyl Alcohol Using a Microfluidic Stripline Nuclear Magnetic Resonance Setup.

We present an in-depth study of the acetylation of benzyl alcohol in the presence of N, N-diisopropylethylamine (DIPEA) by nuclear magnetic resonance (NMR) monitoring of the reaction from 1.5 s to several minutes. We have adapted the NMR setup to be compatible to microreactor technology, scaling down the typical sample volume of commercial NMR probes (500 µL) to a microfluidic stripline setup with 150 nL detection volume. Inline spectra are obtained to monitor the kinetics and unravel the reaction mechanism of this industrially relevant reaction. The experiments are combined with conventional 2D NMR measurements to identify the reaction products. In addition, we replace DIPEA with triethylamine and pyridine to validate the reaction mechanism for different amine catalysts. In all three acetylation reactions, we find that the acetyl ammonium ion is a key intermediate. The formation of ketene is observed during the first minutes of the reaction when tertiary amines were present. The pyridine-catalyzed reaction proceeds via a different mechanism.

Scalable 3D Nanoparticle Trap for Electron Microscopy Analysis.

Arrays of nanoscale pyramidal cages embedded in a silicon nitride membrane are fabricated with an order of magnitude miniaturization in the size of the cages compared to previous work. This becomes possible by combining the previously published wafer-scale corner lithography process with displacement Talbot lithography, including an additional resist etching step that allows the creation of masking dots with a size down to 50 nm, using a conventional 365 nm UV source. The resulting pyramidal cages have different entrance and exit openings, which allows trapping of nanoparticles within a predefined size range. The cages are arranged in a well-defined array, which guarantees traceability of individual particles during post-trapping analysis. Gold nanoparticles with a size of 25, 150, and 200 nm are used to demonstrate the trapping capability of the fabricated devices. The traceability of individual particles is demonstrated by transferring the transmission electron microscopy (TEM) transparent devices between scanning electron microscopy and TEM instruments and relocating a desired collection of particles.

A microfluidic chip with a staircase pH gradient generator, a packed column and a fraction collector for chromatofocusing of proteins.

A microfluidic device for pH gradient chromatofocusing is presented, which performs creation of a micro-column, pH gradient generation, and fraction collection in a single device. Using a sieve micro-valve, anion exchange particles were packed into a microchannel in order to realize a solid-phase absorption column. To fractionate proteins according to their isoelectric points, elution buffer solutions with a stepwise pH gradient were prepared in 16 parallel mixing reactors and flowed through the micro-column, wherein a protein mixture was previously loaded. The volume of the column is only 20 nL, hence it allows extremely low sample consumption and fast analysis compared with a conventional system. We demonstrated separation of two proteins, albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and R-Phycoerythrin (R-PE), by using a microcolumn of commercial charged polymeric particles (Source 15Q). The microfluidic device can be used as a rapid diagnostic tool to analyse crude mixtures of proteins or nucleic acids and determine adsorption/desorption characteristics of various biochemical products, which can be helpful for scientific fundamental understanding as well as instrumental in various industrial applications, especially in early stage screening and process development.

Continuous Flow 1H and 13C NMR Spectroscopy in Microfluidic Stripline NMR Chips.

Microfluidic stripline NMR technology not only allows for NMR experiments to be performed on small sample volumes in the submicroliter range, but also experiments can easily be performed in continuous flow because of the stripline's favorable geometry. In this study we demonstrate the possibility of dual-channel operation of a microfluidic stripline NMR setup showing one- and two-dimensional 1H, 13C and heteronuclear NMR experiments under continuous flow. We performed experiments on ethyl crotonate and menthol, using three different types of NMR chips aiming for straightforward microfluidic connectivity. The detection volumes are approximately 150 and 250 nL, while flow rates ranging from 0.5 µL/min to 15 µL/min have been employed. We show that in continuous flow the pulse delay is determined by the replenishment time of the detector volume, if the sample trajectory in the magnet toward NMR detector is long enough to polarize the spin systems. This can considerably speed up quantitative measurement of samples needing signal averaging. So it can be beneficial to perform continuous flow measurements in this setup for analysis of, e.g., reactive, unstable, or mass-limited compounds.

Disposable attenuated total reflection-infrared crystals from silicon wafer: a versatile approach to surface infrared spectroscopy.

Attenuated total reflection-infrared (ATR-IR) spectroscopy is increasingly used to characterize solids and liquids as well as (catalytic) chemical conversion. Here we demonstrate that a piece of silicon wafer cut by a dicing machine or cleaved manually can be used as disposable internal reflection element (IRE) without the need for polishing and laborious edge preparation. Technical aspects, fundamental differences, and pros and cons of these novel disposable IREs and commercial IREs are discussed. The use of a crystal (the Si wafer) in a disposable manner enables simultaneous preparation and analysis of substrates and application of ATR spectroscopy in high temperature processes that may lead to irreversible interaction between the crystal and the substrate. As representative application examples, the disposable IREs were used to study high temperature thermal decomposition and chemical changes of polyvinyl alcohol (PVA) in a titania (TiO(2)) matrix and assemblies of 65-450 nm thick polystyrene (PS) films.

Influence of Wettability and Geometry on Contact Electrification between Nonionic Insulators.

Contact electrification is an interfacial process in which two surfaces exchange electrical charges when they are in contact with one another. Consequently, the surfaces may gain opposite polarity, inducing an electrostatic attraction. Therefore, this principle can be exploited to generate electricity, which has been precisely done in triboelectric nanogenerators (TENGs) over the last decades. The details of the underlying mechanisms are still ill-understood, especially the influence of relative humidity (RH). Using the colloidal probe technique, we convincingly show that water plays an important role in the charge exchange process when two distinct insulators with different wettability are contacted and separated in <1 s at ambient conditions. The charging process is faster, and more charge is acquired with increasing relative humidity, also beyond RH = 40% (at which TENGs have their maximum power generation), due to the geometrical asymmetry (curved colloid surface vs planar substrate) introduced in the system. In addition, the charging time constant is determined, which is found to decrease with increasing relative humidity. Altogether, the current study adds to our understanding of how humidity levels affect the charging process between two solid surfaces, which is even enhanced up to RH = 90% as long as the curved surface is hydrophilic, paving the way for designing novel and more efficient TENGs, eco-energy harvesting devices which utilize water and solid charge interaction mechanism, self-powered sensors, and tribotronics.

Low-Variance Surface-Enhanced Raman Spectroscopy Using Confined Gold Nanoparticles over Silicon Nanocones.

Surface-enhanced Raman spectroscopy (SERS) substrates are of utmost interest in the analyte detection of biological and chemical diagnostics. This is primarily due to the ability of SERS to sensitively measure analytes present in localized hot spots of the SERS nanostructures. In this work, we present the formation of 67 ± 6 nm diameter gold nanoparticles supported by vertically aligned shell-insulated silicon nanocones for ultralow variance SERS. The nanoparticles are obtained through discrete rotation glancing angle deposition of gold in an e-beam evaporating system. The morphology is assessed through focused ion beam tomography, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. The optical properties are discussed and evaluated through reflectance measurements and finite-difference time-domain simulations. Lastly, the SERS activity is measured by benzenethiol functionalization and subsequent Raman spectroscopy in the surface scanning mode. We report a homogeneous analytical enhancement factor of 2.2 ± 0.1 × 107 (99% confidence interval for N = 400 grid spots) and made a comparison to other lithographically derived assemblies used in SERS. The strikingly low variance (4%) of our substrates facilitates its use for many potential SERS applications.

Performance evaluation of different design alternatives for microfabricated nonporous fused silica pillar columns for capillary electrochromatography.

An experimental study comparing the performance of different designs for microfabricated column structures for microchip capillary electrochromatography is presented. The work is a follow-up to our previously published modeling and simulation study on the same topic. Experiments were performed using fused silica microchips with and without octadecyltrimethoxysilane coating for nonretained and retained modes of operation, respectively. Showing the same trends as the modeling results, the foil shape produces a significant decrease in plate height with an increase of around 15% in mobile phase velocity in nonretained measurements of Coumarin 480 (C480). Measured plate heights at 1 kV/cm applied electric field were 0.77, 1.33, and 1.42 µm for foil, diamond, and hexagon, respectively. Chromatographic runs of C480 yielded minimal plate height values of 1.85 and 3.28 µm for foil and diamond, respectively. The optimization of the shape and placement of the structures appeared to have a considerable impact on the achievable performance.

Attenuated total reflection-infrared nanofluidic chip with 71 nL detection volume for in situ spectroscopic analysis of chemical reaction intermediates.

We present a micromachined silicon attenuated total reflection-infrared (ATR-IR) crystal with integrated nanofluidic glass channels which enables infrared spectroscopic studies with only 71 nL sample volume. Because of the short path length through silicon, the system allows IR spectroscopy down to 1200 cm(-1), which covers the typical fingerprint region of most organic compounds. To demonstrate proof-of-principle, the chip was used to study a Knoevenagel condensation reaction between malononitrile and p-anisaldehyde catalyzed by different concentrations of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in solvent acetonitrile. By in situ measurement, it was demonstrated for the first time that at certain concentrations of DBU, reaction intermediates become stabilized, an effect that slows down or even stops the reaction. This is thought to be caused by increased ionic character of the solvent, in which protonated DBU stabilizes the intermediates. This clearly demonstrates that infrared mechanistic studies of chemical reactions are feasible in volumes as little as 71 nL.

A supramolecular approach to enzyme immobilization in micro-channels.

A supramolecular assembly scheme is developed to enable the facile in-situ immobilization of enzymes in a microfluidic channel system. A combination of orthogonal supramolecular interactions of host (ß-cyclodextrin)-guest (adamantane) and biotin-Streptavidin (SAv) interactions are employed to generate reusable homogeneous enzyme layers in microchannels. The structural integrity and catalytic activity of the immobilized enzyme calf-intestine alkaline phosphatase (AlkPh) is demonstrated. From the kinetic analysis of a dephosphorylation reaction, the specificity constant k(cat)/K(M) for immobilized alkaline phosphatase in the channels is on the order of 10(5) M(-1) s(-1) and comparable to known literature values in other environments. These observations are ascribed to the good access of the substrate to favorably oriented enzymes across the microchannel. Therefore, this study demonstrates the great potential for adopting a supramolecular assembly scheme to immobilize enzymes in microfluidic devices.

On the pathway of photoexcited electrons: probing photon-to-electron and photon-to-phonon conversions in silicon by ATR-IR.

Photoexcitation and charge carrier thermalization inside semiconductor photocatalysts are two important steps in solar fuel production. Here, photoexcitation and charge carrier thermalization in a silicon wafer are for the first time probed by a novel, yet simple and user-friendly Attenuated Total Reflectance Infrared spectroscopy (ATR-IR) system.

In vitro and in vivo evaluation of the inflammatory response to nanoscale grooved substrates.

The immune response to an implanted biomaterial is orchestrated by macrophages. In this study various nanogrooved patterns were created by using laser interference lithography and reactive ion etching. The created nanogrooves mimic the natural extracellular matrix environment. Macrophage cell culture demonstrated that interleukin 1ß and TNF-α cytokine production were upregulated on nanogrooved substrates. In vivo subcutaneous implantation in a validated mouse cage model for 14 days demonstrated that nanogrooves enhanced and guided cell adhesion, and few multinucleated cells were formed. In agreement with the in vitro results, cytokine production was found to be nanogroove dependent, as interleukin 1ß, TNF-α, TGF-ß and osteopontin became upregulated. The results indicate that biomaterial surface texturing, especially at the nanometric scale, can be used to control macrophage activation to induce a wound healing response, rather than a profound inflammatory response. From the Clinical Editor: The authors investigate various nano-grooved patterns that mimic the natural extracellular matrix environment and demonstrate (both in macrophage cultures and in vivo) that interleukin 1ß and TNF-α cytokine production is dependent upon surface texturing at the nanometric scale. They propose that modified surfaces may trigger macrophage activation to promote a wound healing response.

Triboelectric Charging of Particles, an Ongoing Matter: From the Early Onset of Planet Formation to Assembling Crystals.

Triboelectrification is the spontaneous charging of two bodies when released from contact. Even though its manifestation is commonplace, in for instance triboelectric nanogenerators, scientists find the tribocharging mechanism a mystery. The primary aim of this mini-review is to provide an overview of different tribocharging concepts that have been applied to study and realize the formation of ordered stable structures using different objects on various length scales. Relevance spans from materials to planet formations. Especially, dry assembly methods of particles of different shapes based on tribocharging to obtain crystal structures or monolayers are considered. In addition, the current technology employed to examine tribocharging in (semi)dry environments is discussed as well as the relevant forces playing a role in the assembly process. In brief, this mini-review is expected to provide a better understanding of tribocharging in assembling objects on the nano- and micrometer scales.

Large Dense Periodic Arrays of Vertically Aligned Sharp Silicon Nanocones.

Convex cylindrical silicon nanostructures, also referred to as silicon nanocones, find their value in many applications ranging from photovoltaics to nanofluidics, nanophotonics, and nanoelectronic applications. To fabricate silicon nanocones, both bottom-up and top-down methods can be used. The top-down method presented in this work relies on pre-shaping of silicon nanowires by ion beam etching followed by self-limited thermal oxidation. The combination of pre-shaping and oxidation obtains high-density, high aspect ratio, periodic, and vertically aligned sharp single-crystalline silicon nanocones at the wafer-scale. The homogeneity of the presented nanocones is unprecedented and may give rise to applications where numerical modeling and experiments are combined without assumptions about morphology of the nanocone. The silicon nanocones are organized in a square periodic lattice, with 250 nm pitch giving arrays containing 1.6 billion structures per square centimeter. The nanocone arrays were several mm2 in size and located centimeters apart across a 100-mm-diameter single-crystalline silicon (100) substrate. For single nanocones, tip radii of curvature < 3 nm were measured. The silicon nanocones were vertically aligned, baring a height variation of < 5 nm (< 1%) for seven adjacent nanocones, whereas the height inhomogeneity is < 80 nm (< 16%) across the full wafer scale. The height inhomogeneity can be explained by inhomogeneity present in the radii of the initial columnar polymer mask. The presented method might also be applicable to silicon micro- and nanowires derived through other top-down or bottom-up methods because of the combination of ion beam etching pre-shaping and thermal oxidation sharpening. A novel method is presented where argon ion beam etching and thermal oxidation sharpening are combined to tailor a high-density single-crystalline silicon nanowire array into a vertically aligned single-crystalline silicon nanocones array with < 3 nm apex radius of curvature tips, at the wafer scale.

Enhanced Photocatalytic Hydrogen Evolution from Water Splitting on Ta2O5/SrZrO3 Heterostructures Decorated with CuxO/RuO2 Cocatalysts.

Photocatalytic H2 generation by water splitting is a promising alternative for producing renewable fuels. This work synthesized a new type of Ta2O5/SrZrO3 heterostructure with Ru and Cu (RuO2/CuxO/Ta2O5/SrZrO3) using solid-state chemistry methods to achieve a high H2 production of 5164 µmol g-1 h-1 under simulated solar light, 39 times higher than that produced using SrZrO3. The heterostructure performance is compared with other Ta2O5/SrZrO3 heterostructure compositions loaded with RuO2, CuxO, or Pt. CuxO is used to showcase the usage of less costly cocatalysts to produce H2. The photocatalytic activity toward H2 by the RuO2/CuxO/Ta2O5/SrZrO3 heterostructure remains the highest, followed by RuO2/Ta2O5/SrZrO3 > CuxO/Ta2O5/SrZrO3 > Pt/Ta2O5/SrZrO3 > Ta2O5/SrZrO3 > SrZrO3. Band gap tunability and high optical absorbance in the visible region are more prominent for the heterostructures containing cocatalysts (RuO2 or CuxO) and are even higher for the binary catalyst (RuO2/CuxO). The presence of the binary catalyst is observed to impact the charge carrier transport in Ta2O5/SrZrO3, improving the solar to hydrogen conversion efficiency. The results represent a valuable contribution to the design of SrZrO3-based heterostructures for photocatalytic H2 production by solar water splitting.

Self-Aligned Crystallographic Multiplication of Nanoscale Silicon Wedges for High-Density Fabrication of 3D Nanodevices.

High-density arrays of silicon wedges bound by {111} planes on silicon (100) wafers have been created by combining convex corner lithography on a silicon dioxide hard mask with anisotropic, crystallographic etching in a repetitive, self-aligned multiplication procedure. A mean pitch of around 30 nm has been achieved, based on an initial pitch of ∼120 nm obtained through displacement Talbot lithography. The typical resolution of the convex corner lithography was reduced to the sub-10 nm range by employing an 8 nm silicon dioxide mask layer (measured on the {111} planes). Nanogaps of 6 nm and freestanding silicon dioxide flaps as thin as 1-2 nm can be obtained when etching the silicon at the exposed apices of the wedges. To enable the repetitive procedure, it was necessary to protect the concave corners between the wedges through "concave" corner lithography. The produced high-density arrays of wedges offer a promising template for the fabrication of large arrays of nanodevices in various domains with relevant details in the sub-10 nm range.

Novel shape and placement definitions with retention modeling for solid microfabricated pillar columns for CEC and HPLC.

A novel design approach for optimizing the shape of microfabricated pillar columns for LC is presented. 2-D flow simulations are performed with a focus on electrokinetically driven flow, in order to evaluate the performance of the new method. The proposed foil shape is compared with geometrical shapes known from the literature, for various arrangements. It yields a much more uniform velocity field distribution and a decrease in plate height values up to 25%. In addition to shape optimization, a new method for spatial arrangement of structures is presented. With the aim of conserving the hydrodynamic balance, the axial spacing of the pillars is adjusted according to the proposed equivalent width approach. When compared with a fixed interpillar spacing in all directions, it increases the flow uniformity and results in an 18% lower plate height. A new direct simulation approach is implemented to model both flow field and retention for solid microfabricated pillar structures in the 2-D domain. This model, which defines retention as inward/outward fluxes through the wall surfaces as first order reactions, enables monitoring of the time-dependent process and an evaluation of the parameters affecting performance. The meaning of the obtained results in a practical setting, with limitations in photolithography and microfabrication, will be highlighted.

A brush-gel/metal-nanoparticle hybrid film as an efficient supported catalyst in glass microreactors.

A polymer-brush-based material was applied for the formation and in situ immobilization of silver and palladium nanoparticles, as a catalytic coating on the inner wall of glass microreactors. The brush film was grown directly on the microchannel interior by means of atom-transfer radical polymerization (ATRP), which allows control over the polymer film thickness and therefore permits the tuning of the number of nanoparticles formed on the channel walls. The wide applicability of the catalytic devices is demonstrated for the reduction of 4-nitrophenol and for the Heck reaction.

Wafer-scale 3D shaping of high aspect ratio structures by multistep plasma etching and corner lithography.

The current progress of system miniaturization relies extensively on the development of 3D machining techniques to increase the areal structure density. In this work, a wafer-scale out-of-plane 3D silicon (Si) shaping technology is reported, which combines a multistep plasma etching process with corner lithography. The multistep plasma etching procedure results in high aspect ratio structures with stacked semicircles etched deep into the sidewall and thereby introduces corners with a proper geometry for the subsequent corner lithography. Due to the geometrical contrast between the gaps and sidewall, residues are left only inside the gaps and form an inversion mask inside the semicircles. Using this mask, octahedra and donuts can be etched in a repeated manner into Si over the full wafer area, which demonstrates the potential of this technology for constructing high-density 3D structures with good dimensional control in the bulk of Si wafers.