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.

Evaluation of Gas Chromatography Columns with Radially Elongated Pillars as Second-Dimension Columns in Comprehensive Two-Dimensional Gas Chromatography.

The present study investigated the use of a dedicated gas chromatography (GC) column (L = 70 cm, 75 µm deep, and 6.195 mm wide) with radially elongated pillars (REPs) as the second column in a comprehensive two-dimensional gas chromatography (GC × µGC) system. Three stationary phases [apolar polydimethylsiloxane (PDMS), medium polar room-temperature ionic liquid (RTIL) based on monocationic phosphonium, and polar polyethylene glycol (PEG-1000)] have been coated using the static method at constant pressure or using an original vacuum pressure program (VPP) from 400 to 4 mbar. The best efficiency reached up to N = 62,000 theoretical plates for a film thickness of 47 nm at 100 °C for an iso-octane peak (k = 0.16) at an optimal flow rate of 4.8 mL/min. The use of the VPP improved the efficiency by approximately 15%. Efficiencies up to 28,000 and 47,000 were obtained for PEG-1000 and RTIL, respectively. A temperature-programmed separation of a mixture of 11 volatile compounds on a PDMS-coated chip was obtained in less than 36 s. The PDMS-, PEG-1000-, and RTIL-coated chips were tested as the second column using a microfluidic reverse fill/flush flow modulator in a GC × µGC system. The REP columns were highly compatible with the operating conditions in terms of flow rate and with more than 30,000 plates for the iso-octane peak. Moreover, a commercial solvent called white spirit containing alkanes and aromatic compounds was injected in three sets of columns in normal and reverse modes, demonstrating the great potential of the chip as a second-dimension separation column.

Wafer-Scale Particle Assembly in Connected and Isolated Micromachined Pockets via PDMS Rubbing.

The present contribution reports on a study aiming to find the most suitable rubbing method for filling arrays of separated and interconnected micromachined pockets with individual microspheres on rigid, uncoated silicon substrates without breaking the particles or damaging the substrate. The explored dry rubbing methods generally yielded unsatisfactory results, marked by very large percentages of empty pockets and misplaced particles. On the other hand, the combination of wet rubbing with a patterned rubbing tool provided excellent results (typically <1% of empty pockets and <5% of misplaced particles). The wet method also did not leave any damage marks on the silicon substrate or the particles. When the pockets were aligned in linear grooves, markedly the best results were obtained when the ridge pattern of the rubbing tool was moved under a 45° angle with respect to the direction of the grooves. The method was tested for both silica and polystyrene particles. The proposed assembly method can be used in the production of medical devices, antireflective coatings, and microfluidic devices with applications in chemical analysis and/or catalysis.

Self-organization of agitated microspheres on various substrates.

The vibration dynamics of relatively large granular grains is extensively treated in the literature, but comparable studies on the self-assembly of smaller agitated beads are lacking. In this work, we investigate how the particle properties and the properties of the underlying substrate surface affect the dynamics and self-organization of horizontally agitated monodisperse microspheres with diameters between 3 and 10 µm. Upon agitation, the agglomerated hydrophilic silica particles locally leave traces of particle monolayers as they move across the flat uncoated and fluorocarbon-coated silicon substrates. However, on the micromachined silicon tray with relatively large surface roughness, the agitated silica agglomerates form segregated bands reminiscent of earlier studies on granular suspensions or Faraday heaps. On the other hand, the less agglomerated hydrophobic polystyrene particles form densely occupied monolayer arrangements regardless of the underlying substrate. We explain the observations by considering the relevant adhesion and friction forces between particles and underlying substrates as well as those among the particles themselves. Interestingly, for both types of microspheres, large areas of the fluorocarbon-coated substrates are covered with densely occupied particle monolayers. By qualitatively examining the morphology of the self-organized particle monolayers using the Voronoi approach, it is understood that these monolayers are highly disordered, i.e., multiple symmetries coexist in the self-organized monolayers. However, more structured symmetries are identified in the monolayers of the agitated polystyrene microspheres on all the substrates, albeit not all precisely positioned on a hexagonal lattice. On the other hand, both the silica and polystyrene monolayers on the bare silicon substrates transition into less disordered structures as time progresses. Using Kelvin probe force microscopy measurements, we show that due to the tribocharging phenomenon, the formation of particle monolayers is promoted on the fluorocarbon surface, i.e., a local electrostatic attraction exists between the particle and the substrate.

A 3D polydimethylsiloxane microhourglass-shaped channel array made by reflowing photoresist structures for engineering a blood capillary network.

This paper describes an innovative yet straightforward fabrication technique to create three-dimensional microstructures with controllable tapered geometries by combining conventional photolithography and thermal reflow of photoresist. Positive photoresist-based microchannel structures with varying width-to-length ratios were reflowed after their fabrication to generate three-dimensional funnel structures with varying curvatures. A polydimethylsiloxane hourglass-shaped microchannel array was next cast on these photoresist structures, and primary human lung microvascular endothelial cells were cultured in the device to engineer an artificial capillary network. Our work demonstrates that this cost-effective and straightforward fabrication technique has great potential in engineering three-dimensional microstructures for biomedical and biotechnological applications such as blood vessel regeneration strategies, drug screening for vascular diseases, microcolumns for bioseparation, and other fluid dynamic studies at microscale.

Spatial Segregation of Microspheres by Rubbing-Induced Triboelectrification on Patterned Surfaces.

Particle (monolayer) assembly is essential to various scientific and industrial applications, such as the fabrication of photonic crystals, optical sensors, and surface coatings. Several methods, including rubbing, have been developed for this purpose. Here, we report on the serendipitous observation that microparticles preferentially partition onto the fluorocarbon-coated parts of patterned silicon and borosilicate glass wafers when rubbed with poly(dimethylsiloxane) slabs. To explore the extent of this effect, we varied the geometry of the pattern, the substrate material, the ambient humidity, and the material and size of the particles. Partitioning coefficients amounted up to a factor of 12 on silicon wafers and even ran in the 100s on borosilicate glass wafers at zero humidity. Using Kelvin probe force microscopy, the observations can be explained by triboelectrification, inducing a strong electrostatic attraction between the particles and the fluorocarbon zones, while the interaction with the noncoated zones is insignificant or even weakly repulsive.

Systematic Investigation of Insulin Fibrillation on a Chip.

A microfluidic protein aggregation device (microPAD) that allows the user to perform a series of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific conditions. Parallel processes of metering reagents, stepwise concentration gradient generation, and mixing are achieved simultaneously by pneumatic valves. Fibrillation of bovine insulin was selected to test the device. The effect of insulin and sodium chloride (NaCl) concentration on the formation of fibrillar structures was studied by observing the growth rate of partially folded protein, using the fluorescent marker Thioflavin-T. Moreover, dual gradients of different NaCl and hydrochloric acid (HCl) concentrations were formed, to investigate their interactive roles in the formation of insulin fibrils and spherulites. The chip-system provides a bird's eye view on protein aggregation, including an overview of the factors that affect the process and their interactions. This microfluidic platform is potentially useful for rapid analysis of the fibrillation of proteins associated with many misfolding-based diseases, such as quantitative and qualitative studies on amyloid growth.

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.

Massively parallel sequencing techniques for forensics: A review.

DNA sequencing, starting with Sanger's chain termination method in 1977 and evolving into the next generation sequencing (NGS) techniques of today that employ massively parallel sequencing (MPS), has become essential in application areas such as biotechnology, virology, and medical diagnostics. Reflected by the growing number of articles published over the last 2-3 years, these techniques have also gained attention in the forensic field. This review contains a brief description of first, second, and third generation sequencing techniques, and focuses on the recent developments in human DNA analysis applicable in the forensic field. Relevance to the forensic analysis is that besides generation of standard STR-profiles, DNA repeats can also be sequenced to look for polymorphisms. Furthermore, additional SNPs can be sequenced to acquire information on ancestry, paternity or phenotype. The current MPS systems are also very helpful in cases where only a limited amount of DNA or highly degraded DNA has been secured from a crime scene. If enough autosomal DNA is not present, mitochondrial DNA can be sequenced for maternal lineage analysis. These developments clearly demonstrate that the use of NGS will grow into an indispensable tool for forensic science.

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.

Chip-Based Multicapillary Column with Maximal Interconnectivity to Combine Maximum Efficiency and Maximum Loadability.

On the basis of our previous work on the design of pillar array columns for liquid chromatography, we report on a new pillar array design for high-efficiency, high volumetric loadability gas chromatography columns. The proposed pillar array configuration leads to a column design which can either be considered as a packed bed with perfectly ordered and uniform flow paths or as multicapillary columns (8 parallel tracks) with a maximal interconnectivity between the flow paths to avoid the so-called polydispersity effect (dispersion arising from the inevitable differences in migration velocity between parallel flow paths). Despite our relative inexperience with column coating, and most probably (not supported by data) suffering from the same problem of stationary phase pooling in the right-angled corners of the flow-through channels as other chip-based GC devices, the efficiencies obtained in a L = 70 cm long and 75 µm deep and 6.195 mm wide chip for, respectively, quasi-unretained and retained components (k = 7) went up to N = 60 000 and 12 500 under isothermal conditions using H2 as carrier gas and a downstream restriction. Under programmed temperature conditions (Ti = 80 °C, Tf = 175 °C at 30 °C/min, and a H2 flow of 0.4 mL/min), a peak capacity of 170 was obtained in 3.6 min. For retained compounds, the optimal flow rate is found to be on the order of 0.4 mL/min, achieved at an operating pressure of 2.3 bar. Intrinsically, the column combines the efficiency of a 75 µm capillary with the volumetric loadability of a 240 µm capillary.

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.

Electrolysis-Driven and Pressure-Controlled Diffusive Growth of Successive Bubbles on Microstructured Surfaces.

Control over the bubble growth rates forming on the electrodes of water-splitting cells or chemical reactors is critical with respect to the attainment of higher energy efficiencies within these devices. This study focuses on the diffusion-driven growth dynamics of a succession of H2 bubbles generated at a flat silicon electrode substrate. Controlled nucleation is achieved by means of a single nucleation site consisting of a hydrophobic micropit etched within a micrometer-sized pillar. In our experimental configuration of constant-current electrolysis, we identify gas depletion from (i) previous bubbles in the succession, (ii) unwanted bubbles forming on the sidewalls, and (iii) the mere presence of the circular cavity where the electrode is being held. The impact of these effects on bubble growth is discussed with support from numerical simulations. The time evolution of the dimensionless bubble growth coefficient, which is a measure of the overall growth rate of a particular bubble, of electrolysis-generated bubbles is compared to that of CO2 bubbles growing on a similar surface in the presence of a supersaturated solution of carbonated water. For electrolytic bubbles and under the range of current densities considered here (5-15 A/m2), it is observed that H2 bubble successions at large gas-evolving substrates first experience a stagnation regime, followed by a fast increase in the growth coefficient before a steady state is reached. This clearly contradicts the common assumption that constant current densities must yield time-invariant growth rates. Conversely, for the case of CO2 bubbles, the growth coefficient successively decreases for every subsequent bubble as a result of the persistent depletion of dissolved CO2.

A microfluidic device for the batch adsorption of a protein on adsorbent particles.

A microfluidic platform or "microfluidic batch adsorption device" is presented, which performs two sets of 9 parallel protein incubations with/without adsorbent particles to achieve an adsorption isotherm of a protein in a single experiment. The stepwise concentration gradient of a target protein was created by the integration of microvalves into the device. The nanoliter-scale reactor (41 nl) allows about 5000 times reduction of sample consumption and fast analysis compared with a conventional 96 well plate. The integration of two sets of parallel reactors as reference reactors and adsorption reactors, respectively, in a single microfluidic format has many advantages, such as the exclusion of the influence of undesired experimental fluctuations, and the possibility of real-time tracing of adsorption processes. We performed batch adsorption of albumin-fluorescein isothiocyanate conjugate (FITC-BSA) on polymeric particles (Source 15Q) to obtain an adsorption isotherm. The obtained on-chip parameters maximum adsorption amount (Qmax) and adsorption constant (Keq) were 0.33 ± 0.03 ng per particle and 0.97 ± 0.22 L g-1, respectively, which are in good agreement with off-chip values (Qmax = 0.34 ± 0.01 ng per particle and Keq = 0.81 ± 0.10 L g-1). On-chip adsorption isotherms of FITC-BSA at various concentrations of sodium chloride (NaCl) were measured to evaluate the effect of this salt on the adsorption capability of Source 15Q. The microfluidic device serves as a new analytical tool, useful in biotechnological and industrial applications, where the adsorption behavior of (bio)molecules on commercial adsorbent particles plays critical roles, such as protein separation and purification, detection of analytes and biomarkers, and solid-phase immunoassays.

Characterization of opto-electrical enhancement of tandem photoelectrochemical cells by using photoconductive-AFM.

Solar-to-hydrogen conversion by water splitting in photoelectrochemical cells (PECs) is a promising approach to alleviate problems associated with intermittency in solar energy supply and demand. Several interfacial resistances in photoelectrodes limit the performance of such cells, while the properties of interfaces are not easy to analyze in situ. We applied photoconductive-AFM to analyze the performance of WO3/p+n Si photoanodes, containing an ultra-thin metal interface of either Au or Pt. The Au interface consisted of Au nanoparticles with well-ordered interspacing, while Pt was present in the form of a continuous film. Photoconductive-AFM data show that upon illumination significantly larger currents are measured for the WO3/p+n Si anode equipped with the Au interface, as compared to the WO3/p+n Si anode with the Pt interface, in agreement with the better performance of the former electrode in a photoelectrochemical cell. The remarkable performance of the Au-containing electrode is proposed to be the result of favorable electron-hole recombination rates induced by the Au nanoparticles in a plasmon resonance excited state.

In-line sample concentration by evaporation through porous hollow fibers and micromachined membranes embedded in microfluidic devices.

Two types of microfluidic systems, a porous hollow fiber and a thin supported membrane with an array of micromachined holes, are investigated for concentrating mass-limited analyte samples. Water evaporation is driven by the partial pressure difference across the hydrophobic membrane, induced by dry sweeping gas on the permeate side. An analytical model permitting clarification of the contribution of design and process parameters on acquisition of concentrated solution and prediction of achievable concentration factors is presented. Concentrating an exemplary solution utilizing the two systems has been studied at different experimental conditions to validate the model. The results show that the hollow fiber gives controllable concentration factors of more than 10. For the micromachined membrane concentrator concentration factors of 6-8 were achieved, at much lower flow rates than predicted by the model. Because of the asymptotic dependence of concentration factor on flow rate, accurate control of the liquid feed is extremely critical in the flow rate range where high concentration factors are obtained, and the smallest variations in liquid flow rate may easily lead to supersaturation and deposition of solutes in the pores. This changes membrane porosity in an unpredictable way and limits the maximum attainable concentration factor.

In situ measurement of the transversal dispersion in ordered and disordered two-dimensional pillar beds for liquid chromatography.

Using a fully transparent micropillar array chip and an optical "injection" setup capable of writing pulsed and continuous patterns into the flow by uncaging a fluorescent dye, highly detailed measurements of the transversal dispersion process in two-dimensional (2D) chromatographic beds could be made. With the use of water-glycerol mobile phase spanning a wide range of viscosities, the obtained data cover a broad range of reduced velocities (0 < ν < 3000) and show a clear leveling-off of the transversal dispersion coefficient at large reduced velocities. With dependence on the packing density, this leveling-off occurs at a value of about Dtrans/Dmol = 10 (ε = 0.4), Dtrans/Dmol = 4 (ε = 0.6) and Dtrans/Dmol = 2.5 (ε = 0.8). Another interesting observation that could be made is that the effect of the bed order on the observed transversal dispersion process is relatively small. The observed leveling-off in the relation between the measured Dtrans values and the reduced liquid velocity furthermore clearly invalidates the classical Galton-board model, predicting a linear increase of Dtrans with the reduced velocity. On the other hand, it corroborates a recently proposed series-connection transport model for Dtrans in 2D porous media.

Integration of uniform porous shell layers in very long pillar array columns using electrochemical anodization for liquid chromatography.

Electrochemical anodization has been applied to grow porous shell layers of 300 nm (30 nm pores) in 5 µm diameter pillar array columns (PACs) with a spacing of 2.5 µm. Using turn structures preceded and followed by the flow distributor structures recently introduced by our group and filled with radially elongated pillars, columns with quasi unlimited channel lengths could be conceived. The uniformity of the porous PAC was assessed by determining local plate heights along the channel, which appeared to be constant. Minimal (absolute) plate heights (H) between 4 and 6 µm were obtained at optimal flow rates when imposing increasing retention factors. Upon measuring the surface area involved in chromatographic retention as an indicator of the available surface area, an increase in the surface area by a factor of about 30 compared to that of non-anodized pillars was found. On reconfiguring a commercial HPLC instrument to enable on-chip injections, 90% of the performance (expressed in theoretical plates) could be maintained for a 1 m column, while for a 25 cm column severe losses were still observed. As the corresponding pressure drop for optimal operation of retained components is on the order of 10 bar per m only, portable and cheaper HPLC devices with high efficiencies become realistically conceivable.

Enhanced Performance of an Acoustofluidic Device by Integrating Temperature Control.

Acoustofluidics is an emerging research field wherein either mixing or (bio)-particle separation is conducted. High-power acoustic streaming can produce more intense and rapid flow patterns, leading to faster and more efficient liquid mixing. However, without cooling, the temperature of the piezoelectric element that is used to supply acoustic power to the fluid could rise above 50% of the Curie point of the piezomaterial, thereby accelerating its aging degradation. In addition, the supply of excessive heat to a liquid may lead to irreproducible streaming effects and gas bubble formation. To control these phenomena, in this paper, we present a feedback temperature control system integrated into an acoustofluidic setup using bulk acoustic waves (BAWs) to elevate mass transfer and manipulation of particles. The system performance was tested by measuring mixing efficiency and determining the average velocity magnitude of acoustic streaming. The results show that the integrated temperature control system keeps the temperature at the set point even at high acoustic powers and improves the reproducibility of the acoustofluidic setup performance when the applied voltage is as high as 200 V.