Search

We demonstrate an automated microfluidic nuclear magnetic resonance (NMR) system that quantitatively characterizes protein-ligand interactions without user intervention and with minimal sample needs through protein-detected heteronuclear 2D NMR spectroscopy. Quantitation of protein-ligand interactions is of fundamental importance to the understanding of signaling and other life processes. As is well-known, NMR provides rich information both on the thermodynamics of binding and on the binding site. However, the required titrations are laborious and tend to require large amounts of sample, which are not always available. The present work shows how the analytical power of NMR detection can be brought in line with the trend of miniaturization and automation in life science workflows.

It is challenging to investigate fast supramolecular processes due to the lack of appropriate characterization methods with high structural resolution. In thisstudy, microfluidic nuclear magnetic resonance (μFNMR) spectroscopy was employed to monitor the kinetics of threading and dethreading of a pseudo[6]rotaxane system. By employing high time resolution μF-NMR, 1H and two-dimensional (2D) rotating frame nuclear Overhauser effect spectroscopy (ROESY) NMR spectra were recorded at any timepoint within 1.5 s after the onset of the process. This technique enabled the successful identification of kinetic intermediates and rate-determining steps,usually impossible to determine by other spectroscopic techniques. Thus, our study demonstrates the capability of μF-NMR in investigating the mechanismof complex supramolecular systems.

Hyperpolarized nuclear magnetic resonance and lab-on-a-chip microfluidics are two dynamic, but until recently quite distinct, fields of research. Recent developments in both areas increased their synergistic overlap. By microfluidic integration, many complex experimental steps can be brought together onto a single platform. Microfluidic devices are therefore increasingly finding applications in medical diagnostics, forensic analysis, and biomedical research. In particular, they provide novel and powerful ways to culture cells, cell aggregates, and even functional models of entire organs. Nuclear magnetic resonance is a non-invasive, high-resolution spectroscopic technique which allows real-time process monitoring with chemical specificity. It is ideally suited for observing metabolic and other biological and chemical processes in microfluidic systems. However, its intrinsically low sensitivity has limited its application. Recent advances in nuclear hyperpolarization techniques may change this: under special circumstances, it is possible to enhance NMR signals by up to 5 orders of magnitude, which dramatically extends the utility of NMR in the context of microfluidic systems. At the same time, hyperpolarization requires complex chemical and/or physical manipulations, which in turn may benefit from microfluidic implementation. In fact, many hyperpolarization methodologies rely on processes that are more efficient at the micro-scale, such as molecular diffusion, penetration electromagnetic radiation into the sample, or restricted molecular mobility on a surface. In this review we examine the confluence between the fields of hyperpolarization-enhanced NMR and microfluidics, and assess how these areas of research have mutually benefited one another, and will continue to do so., Comment: 63 pages, 21 Figures

In this work we demonstrate the use of microfluidic NMR for in situ culture and quantitative analysis of metabolism in hepatocellular carcinoma (HCC) cell lines. A hydrothermal heating system is used to enable continuous in situ NMR observation of HCC cell culture over a 24 h incubation period. This technique is nondestructive, non-invasive and can measure millimolar concentrations at microlitre volumes, within a few minutes and in precisely controlled culture conditions. This is sufficient to observe changes in primary energy metabolism, using around 500–3500 cells per device, and with a time resolution of 17 min. The ability to observe intracellular responses in a time-resolved manner provides a more detailed view of a biological system and how it reacts to stimuli. This capability will allow detailed metabolomic studies of cell-culture based cancer models, enabling quantification of metabolic reporgramming, the metabolic tumor microenvironment, and the metabolic interplay between cancer- and immune cells.

The theory of nuclear spin relaxation in a liquid permeating a solid structure of irregular geometry is examined. The effects of restricted diffusion and the demagnetizing field generated by an inhomogeneous distribution of magnetic susceptibility in the system are explored. A framework comprising Brownian Dynamics, average Hamiltonian theory, and Liouville-space spin dynamics is proposed for simulating nuclear spin relaxation in 3D models of random structures obtained from CT scans of actual samples. Simulations results are compared with experimental data. An analytical solution valid within approximation is also reported.

Microfluidic systems hold great potential for the study of live microscopic cultures of cells, tissue samples, and small organisms. Integration of hyperpolarisation would enable quantitative studies of metabolism in such volume limited systems by high-resolution NMR spectroscopy. We demonstrate, for the first time, the integrated generation and detection of a hyperpolarised metabolite on a microfluidic chip. The metabolite 1-$^{13}$C-fumarate is produced in a nuclear hyperpolarised form by (i) introducing para-enriched hydrogen into the solution by diffusion through a polymer membrane, (ii) reaction with a substrate in the presence of a ruthenium-based catalyst, and (iii) conversion of the singlet-polarised reaction product into a magnetised form by the application of a radiofrequency pulse sequence, all on the same microfluidic chip. The microfluidic device delivers a continuous flow of hyperpolarised material at the 2.5 $\mu\text{L}/\text{min}$ scale, with a polarisation level of 4%. We demonstrate two methods for mitigating singlet-triplet mixing effects which otherwise reduce the achieved polarisation level.

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu-detoxification and Cu delivery for cytochrome c oxidase (cbb3 -Cox) assembly depend on two distinct Cu-exporting P1B -type ATPases. The low-affinity CopA is suggested to export excess Cu and the high-affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb3 -Cox biogenesis. In most organisms, CopA-like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI-like ATPases is unknown. Here we identified a CopZ-like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox-sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu-sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb3 -Cox activity is reduced, suggesting that CopZ not only supplies Cu to P1B -type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI-CopZ protein complex in native R. capsulatus membranes.

Quantitatively predicting the reactivity of dynamic covalent reaction is essential to understand and rationally design complex structures and reaction networks. Herein, the reactivity of aldehydes and amines in various rapid imine formation in aqueous solution by microfluidic NMR spectroscopy was quantified. Investigation of reaction kinetics allowed to quantify the forward rate constants k+ by an empirical equation, of which three independent parameters were introduced as reactivity parameters of aldehydes (SE , E) and amines (N). Furthermore, these reactivity parameters were successfully used to predict the unknown forward rate constants of imine formation. Finally, two competitive reaction networks were rationally designed based on the proposed reactivity parameters. Our work has demonstrated the capability of microfluidic NMR spectroscopy in quantifying the kinetics of label-free chemical reactions, especially rapid reactions that are complete in minutes.

We present a microfluidic platform that allows in operando nuclear magnetic resonance (NMR) observation of serial mixing experiments. Gradually adding one reagent to another is a fundamental experimental modality, widely used to quantify equilibrium constants, for titrations, and in chemical kinetics studies. NMR provides a non-invasive means to quantify concentrations and to follow structural changes at the molecular level as a function of exchanged volume. Using active pneumatic valving on the microfluidic device directly inside an NMR spectrometer equipped with a transmission-line NMR microprobe, the system allows injection of aliquots and in situ mixing in a sample volume of less than 10 μL.

The superior mass sensitivity of microcoil technology in nuclear magnetic resonance (NMR) spectroscopy provides potential for the analysis of extremely small-mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the microcoil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic microcoil production methods are needed. One method with great potential is 5-axis computer numerical control (CNC) micromilling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision of >25 μm (making the machining of common spiral microcoils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 μm precision was used to produce working planar microcoils, microstrips, and novel microsensor designs, with some tested on the NMR in less than 24 h after the start of the design process. Sample wells could be built into the microsensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 μm between the sample and the sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micromilling as a versatile tool for the rapid prototyping of NMR microsensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted-tube resonator was produced that allowed for 2D 1H–13C NMR of D. magna neonates and exhibited 1H sensitivity (nLODω600 = 1.49 nmol s1/2) close to that of double strip lines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

PccA and SenC are periplasmic copper chaperones required for the biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox) in Rhodobacter capsulatus at physiological Cu concentrations. However, both proteins are dispensable for cbb3-Cox assembly when the external Cu concentration is high. PccA and SenC bind Cu using Met and His residues and Cys and His residues as ligands, respectively, and both proteins form a complex during cbb3-Cox biogenesis. SenC also interacts directly with cbb3-Cox, as shown by chemical cross-linking. Here we determined the periplasmic concentrations of both proteins in vivo and analyzed their Cu binding stoichiometries and their Cu(I) and Cu(II) binding affinity constants (KD) in vitro. Our data show that both proteins bind a single Cu atom with high affinity. In vitro Cu transfer assays demonstrate Cu transfer both from PccA to SenC and from SenC to PccA at similar levels. We conclude that PccA and SenC constitute a Cu relay system that facilitates Cu delivery to cbb3-Cox.

NMR offers many possibilities in chemical analysis, structural investigations, and medical diagnostics. Although it is broadly used, one of NMR spectroscopies main drawbacks is low sensitivity. Hyperpolarization techniques enhance NMR signals by more than four orders of magnitude allowing the design of new contrast agents. Parahydrogen induced polarization that utilizes the para-hydrogen's singlet state to create enhanced signals is of particular interest since it allows to produce molecular imaging agents within seconds. Herein, we present a strategy for signal enhancement of the carbonyl 13 C in amino acids by using parahydrogen, as demonstrated for glycine and alanine. Importantly, the hyperpolarization step is carried out in water and chemically unmodified canonical amino acids are obtained. Our approach thus offers a high degree of biocompatibility, which is crucial for further application. The rapid sample hyperpolarization (within seconds) may enable the continuous production of biologically useful probes, such as metabolic contrast agents or probes for structural biology.

We show that high-resolution NMR can reach picomole sensitivity for micromolar concentrations of analyte by combining parahydrogen induced hyperpolarisation (PHIP)with a high-sensitivity transmission line micro-detector. The para-enriched hydrogen gas is introduced into solution by diffusion through a membrane integrated into a microfluidic chip. NMR microdetectors, operating with sample volumes of a few $\mu$L or less, benefit from a favourable scaling of mass sensitivity. However, the small volumes make it very difficult to detect species present at less than millimolar concentrations in microfluidic NMR systems. In view of overcoming this limitation, we implement parahydrogen-induced polarisation (PHIP) on a microfluidic device with 2.5~$\mathrm{\mu L}$ detection volume. Integrating the hydrogenation reaction into the chip minimises polarisation losses to spin-lattice relaxation, allowing the detection of picomoles of substance. This corresponds to a concentration limit of detection of better than $\mathrm{1\,\mu M\,\sqrt{s}}$, unprecedented at this sample volume. The stability and sensitivity of the system allows quantitative characterisation of the signal dependence on flow rates and other reaction parameters and permits homo- and heteronuclear 2D NMR experiments at natural $^{13}\mathrm{C}$ abundance., Comment: 27 pages, 8 figures

Microfluidic NMR spectroscopy can probe chemical and bio-chemical processes non-invasively in a tightly controlled environment. We present a dual-channel modular probe assembly for high efficiency microfluidic NMR spectroscopy and imaging. It is compatible with a wide range of microfluidic devices, without constraining the fluidic design. It collects NMR signals from a designated sample volume on the device with high sensitivity and resolution. Modular design allows adapting the detector geometry to different experimental conditions with minimal cost, by using the same probe base. The complete probe can be built from easily available parts. The probe body mainly consists of prefabricated aluminium profiles, while the probe circuit and detector are made from printed circuit boards. We demonstrate a double resonance HX probe with a limit of detection of 1.4nmols−1/2 for protons at 600 MHz, resolution of 3.35 Hz, and excellent B1 homogeneity. We have successfully acquired 1H-13 C and 1H-15N heteronuclear correlation spectra (HSQC), including a 1H-15N HSQC spectrum of 1mM 15N labeled ubiquitin in 2.5μl of sample volume.

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu-detoxification and Cu delivery for cytochrome c oxidase (cbb(3)-Cox) assembly depend on two distinct Cu-exporting P(1B)-type ATPases. The low-affinity CopA is suggested to export excess Cu and the high-affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb(3)-Cox biogenesis. In most organisms, CopA-like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI-like ATPases is unknown. Here we identified a CopZ-like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox-sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu-sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb(3)-Cox activity is reduced, suggesting that CopZ not only supplies Cu to P(1B)-type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI-CopZ protein complex in native R. capsulatus membranes.

Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, such as respiratory heme-copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In this study, we analyzed Cu delivery to the cbb3 -type cytochrome c oxidase (cbb3 -Cox) of Rhodobacter capsulatus. We identified the PCuA C-like periplasmic chaperone PccA and analyzed its contribution to cbb3 -Cox assembly. Our data demonstrate that PccA is a Cu-binding protein with a preference for Cu(I), which is required for efficient cbb3 -Cox assembly, in particular, at low Cu concentrations. By using in vivo and in vitro cross-linking, we show that PccA forms a complex with the Sco1-homologue SenC. This complex is stabilized in the absence of the cbb3 -Cox-specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. These data demonstrate that the interplay between PccA and SenC not only is required for Cu delivery during cbb3 -Cox assembly but also regulates Cu homeostasis in R. capsulatus.

A compact microfluidic device for perfusion culture of mammalian cells under in situ metabolomic observation by NMR spectroscopy is presented. The chip is made from poly(methyl methacrylate) (PMMA), and uses a poly(dimethyl siloxane) (PDMS) membrane to allow gas exchange. It is integrated with a generic micro-NMR detector developed recently by our group [J. Magn. Reson., 2016, 262, 73-80]. While PMMA is an excellent material in the context of NMR, PDMS is known to produce strong background signals. To mitigate this, the device keeps the PDMS away from the detection area. The oxygen permeation into the device is quantified using a flow chemistry approach. A solution of glucose is mixed on the chip with a solution of glucose oxidase, before flowing through the gas exchanger. The resulting concentration of gluconate is measured by (1)H NMR spectroscopy as a function of flow rate. An oxygen equilibration rate constant of 2.4 s(-1) is found for the device, which is easily sufficient to maintain normoxic conditions in a cell culture at low perfusion flow rates.

Integration of high-resolution nuclear magnetic resonance (NMR) spectroscopy with microfluidic lab-on-a-chip devices is challenging due to limited sensitivity and line broadening caused by magnetic susceptibility inhomogeneities. We present a novel double-stripline NMR probe head that accommodates planar microfluidic devices, and obtains the NMR spectrum from a rectangular sample chamber on the chip with a volume of 2 ??l. Finite element analysis was used to jointly optimise the detector and sample volume geometry for sensitivity and RF homogeneity. A prototype of the optimised design has been built, and its properties have been characterised experimentally. The performance in terms of sensitivity and RF homogeneity closely agrees with the numerical predictions. The system reaches a mass limit of detection of 1.57 nmol View the MathML sources, comparing very favourably with other micro-NMR systems. The spectral resolution of this chip/probe system is better than 1.75 Hz at a magnetic field of 7 T, with excellent line shape.

A generic approach is presented that allows high-resolution NMR spectroscopy of water/oil droplet emulsions in microfluidic devices. Microfluidic NMR spectroscopy has recently made significant advances due to the design of micro-detector systems and their successful integration with microfluidic devices. Obtaining NMR spectra of droplet suspensions, however, is complicated by the inevitable differences in magnetic susceptibility between the chip material, the continuous phase, and the droplet phases. This leads to broadening of the NMR resonance lines and results in loss of spectral resolution. We have mitigated the susceptibility difference between the continuous (oil) phase and the chip material by incorporating appropriately designed air-filled structures into the chip. The susceptibilities of the continuous and droplet (aqueous) phases have been matched by doping the droplet phase with a Eu3+ complex. Our results demonstrate that this leads to a proton line width in the droplet phase of about 3 Hz, enabling high-resolution NMR techniques.

PccA and SenC are periplasmic copper chaperones required for the biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox) in Rhodobacter capsulatus at physiological Cu concentrations. However, both proteins are dispensable for cbb3-Cox assembly when the external Cu concentration is high. PccA and SenC bind Cu using Met and His residues and Cys and His residues as ligands, respectively, and both proteins form a complex during cbb3-Cox biogenesis. SenC also interacts directly with cbb3-Cox, as shown by chemical cross-linking. Here we determined the periplasmic concentrations of both proteins in vivo and analyzed their Cu binding stoichiometries and their Cu(I) and Cu(II) binding affinity constants (KD) in vitro. Our data show that both proteins bind a single Cu atom with high affinity. In vitro Cu transfer assays demonstrate Cu transfer both from PccA to SenC and from SenC to PccA at similar levels. We conclude that PccA and SenC constitute a Cu relay system that facilitates Cu delivery to cbb3-Cox.

Improved knowledge of the electrode-tissue impedance will be useful in optimizing the clinical protocols and resulting efficacy of the existing and emerging approaches to spinal cord stimulation. Toward that end, the complex impedance (amplitude and phase) of in vivo ovine spinal cord tissue was measured at the electrode-pial subdural surface interface from 5 Hz to 1 MHz, and with the bi-polar electrodes oriented both parallel and perpendicular to the rostral-caudal axis of the spinal cord. At stimulation frequencies above 10 kHz, most of the impedance then becomes resistive in nature and the phase diference between the stimulation signal and the resulting current drops to ≈ 10˚, thus maximizing power transfer to the tissues. Also, at these higher frequencies, the current pulse maintains significantly greater fidelity to the shape of the stimulation signal applied across the electrodes. Lastly, there were lower impedances associated with parallel as opposed to perpendicular orientation of the electrodes, thus reflecting the effects of fiber orientation within the spinal cord. Impedance differences of this kind have not been reported with epidural stimulation because of the electrical shunting effects of the intervening layer of relatively high conductivity cerebrospinal fluid. These observations provide a quantitative basis for improved models of spinal cord stimulation and suggest certain advantages for direct intradural stimulation relative to the standard epidural approaches. (Some figures in this article are in colour only in the electronic version)

Micro-MRI (Magnetic Resonance Imaging) and NMR (Nuclear Magnetic Resonance) spectroscopy are used to image and study metabolic activity of precision-cut liver slices (PCLS) on a microfluidic perfusion device. The imaging experiments were performed under static medium conditions while metabolic activity was monitored under continuous flow of medium. LDH (Lactate dehydrogenase) leakage assay was performed to assess the tissue viability on different days. MR images having ~30 (μm)2 in-plane resolution are recorded. This method could be used to culture and monitor PCLS non-invasively.

Microfluidic-NMR spectroscopy has been extended to study the kinetics in supramolecular chemistry and molecular assembly. Kinetics of a multicomponent host-guest supramolecular system containing viologen derivatives, β-cyclodextrins and cucurbit [7]urils are studied by a PMMA based microfluidic chip combined with a dedicated transmission line probe for NMR detection. By combining microfluidic technology with NMR spectroscopy, the amount of material required for a full kinetic study could be minimized. This is crucial in supramolecular chemistry, which often involves highly sophisticated and synthetically costly building blocks. The small size of the microfluidic structure is crucial in bringing the time scale for kinetic monitoring down to seconds. At the same time, the transmission line NMR probe provides sufficient sensitivity to work at low (2 mM) concentrations.

Simulation of the nonlinear mechanical response of materials with explicit representation of microstructural features is extremely challenging. These models typically involve a very large number of degrees of freedom, and are prone to convergence difficulties when searching for roots to nonlinear equilibrium equations. We focus on an idealized material model that is motivated by the microstructure of synthetic nacre: individual ‘bricks’ (representing ceramic platelets) interact through nonlinear cohesive springs (representing a small volume fraction of polymer that bonds the platelets). The model simulates composite fracture through rupture of the cohesive springs. The problem is cast in terms of energy minimization and is essentially described by ‘nearest neighbor’ interactions. The principal focus of this paper is to illustrate the computational gains achievable by the strategic marriage of robust, global Monte Carlo minimization algorithms to the graphics processing unit architecture, and to describe how they were realized on the Nvidia GPU. Results comparing the computation times for graphics processing unit and central processing unit implementations demonstrate that a new adaptive version of the simulated annealing algorithm yields a speedup of approximately 5 times, whereas the graphics processing unit implementation yields a speed-up of about 16 times over conventional four-core central processing unit implementations. The resulting speed enhancement for adaptive graphics processing unit minimization of a factor of 80 enables a far broader range of simulations than has previously been possible. Simulations involving as many as 300,000 bricks can be performed in hours, as compared to weeks required by central processing unit implementation. Many aspects of this approach are translatable to other physical problems involving energy minimization in systems with large numbers of degrees of freedom.

© 2015 Elsevier Ltd. Stiff ceramic platelets (or bricks) that are aligned and bonded to a second ductile phase with low volume fraction (mortar) are a promising pathway to produce stiff, high-toughness composites. For certain ranges of constituent properties, including those of some synthetic analogs to nacre, one can demonstrate that the deformation is dominated by relative brick motions. This paper describes simulations of fracture that explicitly track the motions of individual rigid bricks in an idealized microstructure; cohesive tractions acting between the bricks introduce elastic, plastic and rupture behaviors. Results are presented for the stresses and damage near macroscopic cracks with different brick orientations relative to the loading orientation. The anisotropic macroscopic initiation toughness is computed for small-scale yielding conditions and is shown to be independent of specimen geometry and loading configuration. The results are shown to be in agreement with previously published experiments on synthetic nacre.

The Stern–Gerlach experiment is a seminal experiment in quantum physics, involving the interaction between a particle with spin and an applied magnetic field gradient. A recent article [Wennerström et al., Phys. Chem. Chem. Phys., 2012, 14, 1677–1684] claimed that a full understanding of the Stern–Gerlach experiment can only be attained if transverse spin relaxation is taken into account, generated by fluctuating magnetic fields originating in the magnetic materials which generate the field gradient. This interpretation is contrary to the standard quantum description of the Stern–Gerlach experiment, which requires no dissipative effects. We present simulations of conventional quantum dynamics in the Stern–Gerlach experiment, using extended Wigner functions to describe the propagation of the quantum state in space and time. No relaxation effects are required to reproduce the qualitative experimental behaviour. We also present simulations of quantum dynamics in the Rabi experiment, in which an applied radiofrequency field induces spin transitions in the particle wave.

Spinal cord stimulation currently relies on extradural electrode arrays that are separated from the spinal cord surface by a highly conducting layer of cerebrospinal fluid. It has recently been suggested that intradural placement of the electrodes in direct contact with the pial surface could greatly enhance the specificity and efficiency of stimulation. The present computational study aims at quantifying and comparing the electrical current distributions as well as the spatial recruitment profiles resulting from extra- and intra-dural electrode arrangements. The electrical potential distribution is calculated using a 3D finite element model of the human thoracic spinal canal. The likely recruitment areas are then obtained using the potential as input to an equivalent circuit model of the pre-threshold axonal response. The results show that the current threshold to recruitment of axons in the dorsal column is more than an order of magnitude smaller for intradural than extradural stimulation. Intradural placement of the electrodes also leads to much higher contrast between the stimulation thresholds for the dorsal root entry zone and the dorsal column, allowing better focusing of the stimulus.

High-resolution proton NMR spectroscopy is well-established as a tool for metabolomic analysis of biological fluids at the macro scale. Its full potential has, however, not been realised yet in the context of microfluidic devices. While microfabricated NMR detectors offer substantial gains in sensitivity, limited spectral resolution resulting from mismatches in the magnetic susceptibility of the sample fluid and the chip material remains a major hurdle. In this contribution, we show that susceptibility broadening can be avoided even in the presence of substantial mismatch by including suitably shaped compensation structures into the chip design. An efficient algorithm for the calculation of field maps from arbitrary chip layouts based on Gaussian quadrature is used to optimise the shape of the compensation structure to ensure a flat field distribution inside the sample area. Previously, the complexity of microfluidic NMR systems has been restricted to simple capillaries to avoid susceptibility broadening. The structural shimming approach introduced here can be adapted to virtually any shape of sample chamber and surrounding fluidic network, thereby greatly expanding the design space and enabling true lab-on-a-chip systems suitable for high-resolution NMR detection.

Cytochromes (cyts) are ubiquitous heme containing proteins that are key components of energy transduction pathways. They participate in a wide variety of electron transfer reactions, which are essential for cellular processes responsible for chemical energy (ATP) production. The cbb3-type cyt c oxidase (cbb3-Cox) provides an excellent model to study biogenesis of membrane-integral, oligomeric cyt c complexes. Its subunits contain three hemes c, two hemes b and a copper (CuB) atom as cofactors that use distinct insertion processes. In cyts c, heme b is covalently ligated (referred to as heme c) via a complex maturation process that involves in some species up to nine components (Ccm-System I). In addition to the cyts c, many cyt c containing complexes carry other cofactors, and insertion of these cofactors requires additional biogenesis components besides the Ccm-system I. In the case of cbb3-Cox, the mechanisms underlying incorporation of hemes b into the catalytic subunit are not well understood. However, remarkable progress was achieved recently on how the single CuB atom at the catalytic heart of this heme-copper oxidase is acquired. Finally, insertion of the cofactors must be temporally and spatially coordinated with the assembly of the subunits in order to yield a functional cbb3-Cox enzyme. In this chapter, we discuss the biogenesis of cbb3-Cox from the insertion of its catalytic heme-copper (CuB) center and maturation of its c-type cyts to the assembly of its mature subunits, mainly focusing on studies carried out with the anoxygenic phototrophic bacterium Rhodobacter capsulatus.

We describe a novel spinal cord (SC) stimulator that is designed to overcome a major shortcoming of existing stimulator devices: their restricted capacity to selectively activate targeted axons within the dorsal columns. This device overcomes that limitation by delivering electrical stimuli directly to the pial surface of the SC. Our goal in testing this device was to measure its ability to physiologically activate the SC and examine its capacity to modulate somatosensory evoked potentials (SSEPs) triggered by peripheral stimulation. In this acute study on adult sheep (n = 7), local field potentials were recorded from a grid placed in the subdural space of the right hemisphere during electrical stimulation of the left tibial nerve and the spinal cord. Large amplitude SSEPs (>200 µV) in response to SC stimulation were consistently obtained at stimulation strengths well below the thresholds inducing neural injury. Moreover, stimulation of the dorsal columns with signals employed routinely by devices in standard clinical use, e.g., 50 Hz, 0.2 ms pulse width, produced long-lasting changes (>4.5 h) in the SSEP patterns produced by subsequent tibial nerve stimulation. The results of these acute experiments demonstrate that this device can be safely secured to the SC surface and effectively activate somatosensory pathways.

Wireless signal transmission will play a critical role in developing reliable subdural spinal cord stimulation systems. We have developed an approach to inductively coupling signals across the epidural spacing between the pial and epidural surfaces. The major design constraints include tolerance of coil misalignments from spinal cord geometries in addition to reasonable power efficiencies within the expected range of movement. The design of the primary side as a driving circuit is simplified by several turns of commercial magnetic wire, whereas the implanted secondary side is implemented in a micro-planar spiral coil tuned to a resonant frequency of 1.6 MHz. We present the results of wireless inductive coupling experiments that demonstrate the ability to transmit and receive a frequency modulated 1.6 MHz carrier signal between primary and secondary coil antennae scaled to 10 mm. Power delivery is in the range of 400 mW at a link efficiency of 32 % for strong coupling (coil separations of 0.5 mm ) and in the range of 70 mW at 4 % efficiency for weak coupling (coil separations of 10 mm).

Inductively coupled planar resonators offer convenient integration of high-resolution NMR spectroscopy with microfluidic lab-on-a-chip devices. Planar spiral resonators are fabricated lithographically either by gold electroplating or by etching Cu laminated with polyimide. Their performance is characterized by NMR imaging as well as spectroscopy. A single-scan limit of detection LOD(t) = 0.95 nmol s(1/2) was obtained from sample volumes around 1 μL. The sensitivity of this approach is similar to that obtained by microstripline and microslot probes.

We have designed, built and tested a novel device for placing intradural neurmodulator implants directly on the pial surface of the spinal cord. This applier tool is designed for ergonomic handling of delicate electro-mechanical devices such as the Iowa-Patch™ spinal cord stimulator implant, which is aimed at overcoming certain shortcomings in the performance of standard epidural stimulator devices. The applier is approximately 14 cm long, 6 mm in diameter, made of stainless steel components, and has simple and reliable mechanisms for the attachment and release of the implant from it. We describe the design of the device, details of its construction, and its performance during in vivo testing of somatosensory evoked potentials in an ovine model of intradural spinal cord stimulation.

Thermomechanical properties of poly(acrylic acid-co-acrylamide) hydrogels have been measured for a range of gels while systematically varying the acrylamide/acrylic acid ratio. The gels have been equilibrated with a buffer solution at constant pH and salinity. The gels were characterized in terms of their equilibrium swelling ratio, elastic modulus, and electrochemical potential. The results are in quantitative agreement with the predictions from a recently published thermodynamic field theory.

This paper describes a micromechanical analysis of the uniaxial response of composites comprising elastic platelets (bricks) bonded together with thin elastic perfectly plastic layers (mortar). The model yields closed-form results for the spatial variation of displacements in the bricks as a function of constituent properties, which can be used to calculate the effective properties of the composite, including elastic modulus, strength and work-to-failure. Regime maps are presented which indicate critical stresses for failure of the bricks and mortar as a function of constituent properties and brick architecture. The solution illustrates trade-offs between elastic modulus, strength and dissipated work that are a result of transitions between various failure mechanisms associated with brick rupture and rupture of the interfaces. Detailed scaling relationships are presented with the goal of providing material developers with a straightforward means to identify synthesis targets that balance competing mechanical behaviors and optimize material response. Ashby maps are presented to compare potential brick and mortar composites with existing materials, and identify future directions for material development. (C) 2012 Elsevier Ltd. All rights reserved

Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, like respiratory heme-copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In the current work we analyzed Cu delivery to the cbb3-type cytochrome c oxidase (cbb3-Cox) of Rhodobacter capsulatus. We identified the PCuAC-like periplasmic chaperone PccA and analyzed its contribution to cbb3-Cox assembly. Our data demonstrate that PccA is a Cu-binding protein with a preference for Cu(I), which is required for efficient cbb3-Cox assembly, in particular at low Cu concentrations. By using in vivo and in vitro crosslinking we show that PccA forms a complex with the Sco1-homologue SenC. This complex is stabilized in the absence of the cbb3-Cox specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. These data demonstrate that the interplay between PccA and SenC is not only required for Cu delivery during cbb3-Cox assembly, but that it also regulates Cu homeostasis in R. capsulatus.

© 2015 Published by Elsevier Ltd. This paper examines the effect of non-uniform microstructures on the macroscopic fracture properties of idealized brick and mortar composites, which consist of rigid bricks bonded with elastic-plastic mortar that ruptures at finite strain. A simulation tool that harnesses the parallel processing power of graphics processing units (GPUs) was used to simulate fracture in virtual specimens, whose microstructures were generated by sampling a probability distribution of brick sizes. In the simulations, crack advance is a natural outcome of local ruptures in the cohesive zones bonding the bricks: the macroscopic initiation toughness for small-scale yielding is inferred by correlating the critical load needed to advance a pre-defined crack with an associated far-field energy release rate. Quantitative connections between the statistical parameters defining heterogeneous brick distributions and the statistics of initiation toughness are presented. The nature of crack tip damage and stresses ahead of the crack tip are illustrated as a function of brick size variability. The results offer quantitative insights that can be used to identify microstructural targets for process development, notably specific brick size distributions that still provide macroscopic toughening.

We present an experimental study of the energetics of repulsion between end-grafted fragments of double-stranded DNA. The absorption isotherm of thiolated DNA fragments has been measured as a function of DNA chain length as well as the salinity of the surrounding solution. The results are consistent with a simple excluded-volume model of the interaction between neighboring DNA strands.

The streaming potential through poly(acrylic acid)-co-acrylamide polymer hydrogel (poly-AA-co-AM) membranes has been measured as a function of gel composition and ionic strength of the solvent. Polyelectrolyte hydrogels have important potential applications as mechanical transducers. The current work aims at providing a quantitative basis to understanding the relationship between gel composition and structure and the electromechanical coupling properties. The streaming potential increases in magnitude with the spatial density of negative charges in the gel, up to a saturation point where the density of charges on the polymer backbone becomes comparable to the salt concentration of the equilibrating solution. A simple theory of coupled charge and solvent transport based on the model proposed by de Gennes et al. [Europhys. Lett. 2000, 50, 513] quantitatively accounts for the observed effects.

We report an indentation method to quantify the electromechanical coupling in polyelectrolyte gels (PGs). PGs produce electric fields in response to mechanical stress and are therefore promising for mechanical sensor applications. The method exposes thin gel samples to well-defined pressure distributions through a spherical indentor, while the electrical response is measured with an array of platinum electrodes embedded in the support. A series of copolymer gels of acrylamide and acrylic acid were synthesized and equilibrated at a fixed pH, leading to samples with systematically varying spatial densities of both charged groups and cross-links. They were characterized by measuring the potential difference between the gel and the equilibrating solution (Donnan potential) as well as their electromechanical coupling through the indentation method. The electromechanical coupling was found to be proportional to the Donnan potential, while the latter is a universal function of the spatial density of ionizable groups in the gel, irrespective of the cross-link density.

Frequency-specific components that passively control the flow in a channel in an analogous manner to that of the resistors, capacitors and diodes of an electronic circuit could eliminate the need to exert active control in microfluidic circuits with bulky external pumps. A fundamental challenge in the design of microfluidic devices lies in the need to control the transport of fluid according to complex patterns in space and time, and with sufficient accuracy. Although strategies based on externally actuated valves have enabled marked breakthroughs in chip-based analysis1,2,3,4,5, this requires significant off-chip hardware, such as vacuum pumps and switching solenoids, which strongly tethers such devices to laboratory environments6,7,8,9,10. Severing the microfluidic chip from this off-chip hardware would enable a new generation of devices that place the power of microfluidics in a broader range of disciplines. For example, complete on-chip flow control would empower highly portable microfluidic tools for diagnostics, forensics, environmental analysis and food safety, and be particularly useful in field settings where infrastructure is limited. Here, we demonstrate an elegantly simple strategy for flow control: fluidic networks with embedded deformable features are shown to transport fluid selectively in response to the frequency of a time-modulated pressure source. Distinct fluidic flow patterns are activated through the dynamic control of a single pressure input, akin to the analog responses of passive electrical circuits composed of resistors, capacitors and diodes.

This research constitutes a thorough study of the relationship between the chemical stability, aging state and global molecular motion on the one hand, and microscopic local mobility in multi‐component systems on the other hand. The objective of the present work was to determine whether annealing a glass below T g affects its chemical stability and determine if the rate of chemical degradation couples with global relaxation times determined using calorimetery, and/or with T 1 and T 1rho relaxation times measured using ssNMR. Model compounds chosen for this research were lyophilized aspartame/sucrose and aspartame/trehalose (1:10 w/w) formulations. The chemical degradation was assessed at various temperatures using high‐performance liquid chromatography (HPLC) to determine the impact of annealing on chemical stability. The rate constant for chemical degradation was estimated using stretched time kinetics. The results support the hypothesis that thermal history affects the molecular mobility required for structural relaxation and such effect is critical for chemical stability, that is, a stabilization effect upon annealing is observed. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5240–5251, 2008