Nanoporous Copper-Silver Alloys by Additive-Controlled Electrodeposition for the Selective Electroreduction of CO2 to Ethylene and Ethanol.

Electrodeposition of CuAg alloy films from plating baths containing 3,5-diamino-1,2,4-triazole (DAT) as an inhibitor yields high surface area catalysts for the active and selective electroreduction of CO2 to multicarbon hydrocarbons and oxygenates. EXAFS shows the co-deposited alloy film to be homogeneously mixed. The alloy film containing 6% Ag exhibits the best CO2 electroreduction performance, with the Faradaic efficiency for C2H4 and C2H5OH production reaching nearly 60 and 25%, respectively, at a cathode potential of just -0.7 V vs RHE and a total current density of ∼ - 300 mA/cm2. Such high levels of selectivity at high activity and low applied potential are the highest reported to date. In situ Raman and electroanalysis studies suggest the origin of the high selectivity toward C2 products to be a combined effect of the enhanced stabilization of the Cu2O overlayer and the optimal availability of the CO intermediate due to the Ag incorporated in the alloy.

Electroreduction of Carbon Dioxide to Hydrocarbons Using Bimetallic Cu-Pd Catalysts with Different Mixing Patterns.

Electrochemical conversion of CO2 holds promise for utilization of CO2 as a carbon feedstock and for storage of intermittent renewable energy. Presently Cu is the only metallic electrocatalyst known to reduce CO2 to appreciable amounts of hydrocarbons, but often a wide range of products such as CO, HCOO-, and H2 are formed as well. Better catalysts that exhibit high activity and especially high selectivity for specific products are needed. Here a range of bimetallic Cu-Pd catalysts with ordered, disordered, and phase-separated atomic arrangements (Cuat:Pdat = 1:1), as well as two additional disordered arrangements (Cu3Pd and CuPd3 with Cuat:Pdat = 3:1 and 1:3), are studied to determine key factors needed to achieve high selectivity for C1 or C2 chemicals in CO2 reduction. When compared with the disordered and phase-separated CuPd catalysts, the ordered CuPd catalyst exhibits the highest selectivity for C1 products (>80%). The phase-separated CuPd and Cu3Pd achieve higher selectivity (>60%) for C2 chemicals than CuPd3 and ordered CuPd, which suggests that the probability of dimerization of C1 intermediates is higher on surfaces with neighboring Cu atoms. Based on surface valence band spectra, geometric effects rather than electronic effects seem to be key in determining the selectivity of bimetallic Cu-Pd catalysts. These results imply that selectivities to different products can be tuned by geometric arrangements. This insight may benefit the design of catalytic surfaces that further improve activity and selectivity for CO2 reduction.

"Click Chip" Conjugation of Bifunctional Chelators to Biomolecules.

There is a growing demand for diagnostic procedures including in vivo tumor imaging. Radiometal-based imaging agents are advantageous for tumor imaging because radiometals (i) have a wide range of half-lives and (ii) are easily incorporated into imaging probes via a mild, rapid chelation event with a bifunctional chelator (BFC). Microfluidic platforms hold promise for synthesis of radiotracers because they can easily handle minute volumes, reduce consumption of expensive reagents, and minimize personnel exposure to radioactive compounds. Here we demonstrate the use of a "click chip" with an immobilized Cu(I) catalyst to facilitate the "click chemistry" conjugation of BFCs to biomolecules (BMs); a key step in the synthesis of radiometal-based imaging probes. The "click chip" was used to synthesize three different BM-BFC conjugates with minimal amounts of copper present in reaction solutions (∼20 ppm), which reduces or obviates the need for a copper removal step. These initial results are promising for future endeavors of synthesizing radiometal-based imaging agents completely on chip.

Gold Nanoparticles on Polymer-Wrapped Carbon Nanotubes: An Efficient and Selective Catalyst for the Electroreduction of CO2.

Multiple approaches will be needed to reduce the atmospheric CO2 levels, which have been linked to the undesirable effects of global climate change. The electroreduction of CO2 driven by renewable energy is one approach to reduce CO2 emissions while producing chemical building blocks, but current electrocatalysts exhibit low activity and selectivity. Here, we report the structural and electrochemical characterization of a promising catalyst for the electroreduction of CO2 to CO: Au nanoparticles supported on polymer-wrapped multiwall carbon nanotubes. This catalyst exhibits high selectivity for CO over H2 : 80-92 % CO, as well as high activity: partial current density for CO as high as 160 mA cm-2 . The observed high activity, originating from a high electrochemically active surface area (23 m2 g-1 Au), in combination with the low loading (0.17 mg cm-2 ) of the highly dispersed Au nanoparticles underscores the promise of this catalyst for efficient electroreduction of CO2 .

Elasticity in Macrophage-Synthesized Biocrystals.

Supramolecular crystalline assembly constitutes a rational approach to bioengineer intracellular structures. Here, biocrystals of clofazimine (CFZ) that form in vivo within macrophages were measured to have marked curvature. Isolated crystals, however, showed reduced curvature suggesting that intracellular forces bend these drug crystals. Consistent with the ability of biocrystals to elastically deform, the inherent crystal structure of the principal molecular component of the biocrystals-the hydrochloride salt of CFZ (CFZ-HCl)-has a corrugated packing along the (001) face and weak dispersive bonding in multiple directions. These characteristics were previously found to be linked to the elasticity of other organic crystals. Internal stress in bent CFZ-HCl led to photoelastic effects on the azimuthal orientation of polarized light transmittance. We propose that elastic, intracellular crystals can serve as templates to construct functional microdevices with different applications.

The effect of electrolyte composition on the electroreduction of CO2 to CO on Ag based gas diffusion electrodes.

The electroreduction of CO2 to C1-C2 chemicals can be a potential strategy for utilizing CO2 as a carbon feedstock. In this work, we investigate the effect of electrolytes on the electroreduction of CO2 to CO on Ag based gas diffusion electrodes. Electrolyte concentration was found to play a major role in the process for the electrolytes (KOH, KCl, and KHCO3) studied here. Several fold improvements in partial current densities of CO (jCO) were observed on moving from 0.5 M to 3.0 M electrolyte solution independent of the nature of the anion. jCO values as high as 440 mA cm(-2) with an energy efficiency (EE) of ≈ 42% and 230 mA cm(-2) with EE ≈ 54% were observed when using 3.0 M KOH. Electrochemical impedance spectroscopy showed that both the charge transfer resistance (Rct) and the cell resistance (Rcell) decreased on moving from a 0.5 M to a 3.0 M KOH electrolyte. Anions were found to play an important role with respect to reducing the onset potential of CO in the order OH(-) (-0.13 V vs. RHE) < HCO3(-) (-0.46 V vs. RHE) < Cl(-) (-0.60 V vs. RHE). A decrease in Rct upon increasing electrolyte concentration and the effect of anions on the cathode can be explained by an interplay of different interactions in the electrical double layer that can either stabilize or destabilize the rate limiting CO2˙(-) radical. EMIM based ionic liquids and 1 : 2 choline Cl urea based deep eutectic solvents (DESs) have been used for CO2 capture but exhibit low conductivity. Here, we investigate if the addition of KCl to such solutions can improve conductivity and hence jCO. Electrolytes containing KCl in combination with EMIM Cl, choline Cl, or DESs showed a two to three fold improvement in jCO in comparison to those without KCl. Using such mixtures can be a strategy for integrating the process of CO2 capture with CO2 conversion.

Thiol-based antioxidants elicit mitochondrial oxidation via respiratory complex III.

Excessive oxidation is widely accepted as a precursor to deleterious cellular function. On the other hand, an awareness of the role of reductive stress as a similar pathological insult is emerging. Here we report early dynamic changes in compartmentalized glutathione (GSH) redox potentials in living cells in response to exogenously supplied thiol-based antioxidants. Noninvasive monitoring of intracellular thiol-disulfide exchange via a genetically encoded biosensor targeted to cytosol and mitochondria revealed unexpectedly rapid oxidation of the mitochondrial matrix in response to GSH ethyl ester or N-acetyl-l-cysteine. Oxidation of the probe occurred within seconds in a concentration-dependent manner and was attenuated with the membrane-permeable ROS scavenger tiron. In contrast, the cytosolic sensor did not respond to similar treatments. Surprisingly, the immediate mitochondrial oxidation was not abrogated by depolarization of mitochondrial membrane potential or inhibition of mitochondrial GSH uptake. After detection of elevated levels of mitochondrial ROS, we systematically inhibited multisubunit protein complexes of the mitochondrial respiratory chain and determined that respiratory complex III is a downstream target of thiol-based compounds. Disabling complex III with myxothiazol completely blocked matrix oxidation induced with GSH ethyl ester or N-acetyl-l-cysteine. Our findings provide new evidence of a functional link between exogenous thiol-containing antioxidants and mitochondrial respiration.

A method of cryoprotection for protein crystallography by using a microfluidic chip and its application for in situ X-ray diffraction measurements.

We demonstrate a seamless and contactless method from protein crystallization to X-ray analysis using a microfluidic chip with the aim of obtaining a complete crystallographic data set of a protein crystal under cryogenic conditions. Our microfluidics-based approach did not require direct manipulation of the protein crystal. Therefore, the microfluidic chip approach is suitable for novices of X-ray analysis of protein crystals. We also investigated the effect of stepwise cryoprotection on the quality of protein crystals. Protein crystals with cryoprotection via on-chip manipulation did not show deterioration of crystallographic quality of the protein crystal. The complete diffraction data set of a protein crystal, which is required for determining the 3D structure of the target protein, is obtainable by a simple manipulation.

Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition.

In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition, and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen. The NMR, Raman, and powder X-ray diffraction (p-XRD) spectra of CLDIs isolated from the spleens of CFZ-treated mice matched the spectra of pure, CFZ hydrochloride crystals (CFZ-HCl). Elemental analysis revealed a 237-fold increase in chlorine content in CLDIs compared to untreated tissue samples and a 5-fold increase in chlorine content compared to CFZ-HCl, suggesting that the formation of CLDIs occurs through a chloride mediated crystallization mechanism. Single crystal analysis revealed that CFZ-HCl crystals had a densely packed orthorhombic lattice configuration. In vitro, CFZ-HCl formed at a pH of 4-5 only if chloride ions were present at sufficiently high concentrations (>50:1 Cl(-)/CFZ), indicating that intracellular chloride transport mechanisms play a key role in the formation of CLDIs. While microscopy and pharmacokinetic analyses clearly revealed crystallization and intracellular accumulation of the drug in vivo, the chemical and structural characterization of CLDIs implicates a concentrative, chloride transport mechanism, paralleling and thermodynamically stabilizing the massive bioaccumulation of a weakly basic drug.

Region specific enhancement of quantum dot emission using interleaved two-dimensional photonic crystals.

The power efficiency, spectral characteristics, and output directionality of light emitting diodes (LEDs) used for lighting and video display may be tailored by integrating nanostructures that interact with photon emitters. In this work, we demonstrate an approach in which visible-wavelength-emitting quantum dots (QDs) are integrated within a polymer-based photonic crystal (PC) and excited by an ultraviolet-emitting LED. The PC design incorporates two interleaved regions, each with distinct periods in orthogonal directions. The structure enables simultaneous resonant coupling of ultraviolet excitation photons to the QDs and visible QD emission at two different wavelengths to efficiently extract photons normal to the PC surface. The combined excitation and extraction enhancements result in a 5.8X increase in the QD output intensity. Further, we demonstrate multiple QD-doped PCs combined on a single surface to optimally couple with distinct populations of QDs, offering a means for blending color output and directionality of multiple wavelengths. Devices are fabricated upon flexible plastic surfaces by a manufacturable replica molding approach.

Triazine-based tool box for developing peptidic PET imaging probes: syntheses, microfluidic radiolabeling, and structure-activity evaluation.

This study was aimed at developing a triazine-based modular platform for targeted PET imaging. We synthesized mono- or bis-cyclo(RGDfK) linked triazine-based conjugates specifically targeting integrin αvß3 receptors. The core molecules could be easily linked to targeting peptide and radiolabeled bifunctional chelator. The spacer core molecule was synthesized in 2 or 3 steps in 64-80% yield, and the following conjugation reactions with cyclo(RGDfK) peptide or bifunctional chelator were accomplished using "click" chemistry or amidation reactions. The DOTA-TZ-Bis-cyclo(RGDfK) 13 conjugate was radiolabeled successfully with (64)Cu(OAc)2 using a microfluidic method, resulting in higher specific activity with above 95% labeling yields compared to conventional radiolabeling (SA ca. 850 vs 600 Ci/mmol). The dimeric cyclo(RGDfK) peptide was found to display significant bivalency effect using I(125)-Echistatin binding assay with IC50 value as 178.5 ± 57.1 nM, which displayed a 3.6-fold enhancement of binding affinity compared to DOTA-TZ-cyclo(RGDfK) 14 conjugate on U87MG human glioblastoma cell. Biodistribution of all four conjugates in female athymic nude mice were evaluated. DOTA-"Click"-cyclo(RGDfK) 15 had the highest tumor uptake among these four at 4 h p.i. with 1.90 ± 0.65%ID/g, while there was no clear bivalency effect for DOTA-TZ-BisRGD in vivo, which needs further experiments to address the unexpected questions.

Thiolene and SIFEL-based Microfluidic Platforms for Liquid-Liquid Extraction.

Microfluidic platforms provide several advantages for liquid-liquid extraction (LLE) processes over conventional methods, for example with respect to lower consumption of solvents and enhanced extraction efficiencies due to the inherent shorter diffusional distances. Here, we report the development of polymer-based parallel-flow microfluidic platforms for LLE. To date, parallel-flow microfluidic platforms have predominantly been made out of silicon or glass due to their compatibility with most organic solvents used for LLE. Fabrication of silicon and glass-based LLE platforms typically requires extensive use of photolithography, plasma or laser-based etching, high temperature (anodic) bonding, and/or wet etching with KOH or HF solutions. In contrast, polymeric microfluidic platforms can be fabricated using less involved processes, typically photolithography in combination with replica molding, hot embossing, and/or bonding at much lower temperatures. Here we report the fabrication and testing of microfluidic LLE platforms comprised of thiolene or a perfluoropolyether-based material, SIFEL, where the choice of materials was mainly guided by the need for solvent compatibility and fabrication amenability. Suitable designs for polymer-based LLE platforms that maximize extraction efficiencies within the constraints of the fabrication methods and feasible operational conditions were obtained using analytical modeling. To optimize the performance of the polymer-based LLE platforms, we systematically studied the effect of surface functionalization and of microstructures on the stability of the liquid-liquid interface and on the ability to separate the phases. As demonstrative examples, we report (i) a thiolene-based platform to determine the lipophilicity of caffeine, and (ii) a SIFEL-based platform to extract radioactive copper from an acidic aqueous solution.

Effects of detergent ß-octylglucoside and phosphate salt solutions on phase behavior of monoolein mesophases.

Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent ß-octylglucoside (ßOG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures of MO with salt solutions above the hydration boundary; and 2), quaternary mixtures of MO with ßOG and salt solutions over a wide range of hydration conditions. All quaternary mixtures showed highly regular lyotropic phase behavior with the same sequence of phases (Lα, Ia3d, and Pn3m) as MO/water mixtures at similar temperatures. The effects of additives in quaternary systems agreed qualitatively with those found in ternary mixtures in which only one additive is present. However, quantitative differences in the effects of additives on the lattice parameters of fully hydrated mesophases were found between ternary and quaternary mixtures. We discuss the implications of these findings for mechanistic investigations of membrane protein crystallization in lipidic mesophases and for studies of the suitability of precipitants for mesophase-based crystallization methods.

The non-receptor tyrosine kinase Lyn controls neutrophil adhesion by recruiting the CrkL-C3G complex and activating Rap1 at the leading edge.

Establishing new adhesions at the extended leading edges of motile cells is essential for stable polarity and persistent motility. Despite recent identification of signaling pathways that mediate polarity and chemotaxis in neutrophils, little is known about molecular mechanisms governing cell-extracellular-matrix (ECM) adhesion in these highly polarized and rapidly migrating cells. Here, we describe a signaling pathway in neutrophils that is essential for localized integrin activation, leading edge attachment and persistent migration during chemotaxis. This pathway depends upon G(i)-protein-mediated activation and leading edge recruitment of Lyn, a non-receptor tyrosine kinase belonging to the Src kinase family. We identified the small GTPase Rap1 as a major downstream effector of Lyn to regulate neutrophil adhesion during chemotaxis. Depletion of Lyn in neutrophil-like HL-60 cells prevented chemoattractant-induced Rap1 activation at the leading edge of the cell, whereas ectopic expression of Rap1 largely rescued the defects induced by Lyn depletion. Furthermore, Lyn controls spatial activation of Rap1 by recruiting the CrkL-C3G protein complex to the leading edge. Together, these results provide novel mechanistic insights into the poorly understood signaling network that controls leading edge adhesion during chemotaxis of neutrophils, and possibly other amoeboid cells.

Transient light-induced intracellular oxidation revealed by redox biosensor.

We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

Cell-laden hydrogels in integrated microfluidic devices for long-term cell culture and tubulogenesis assays.

A hydrogel biochip combining microfluidic mixing and orthogonal supplementation strategies is developed and validated to allow facile generation of libraries of optically transparent 3D culture microenvironments. Live, on-chip tracing of embryonic stem cell differentiation and endothelial cell tubulogenesis confirms that the platform can be used to both create communities of discrete 3D microenvironments as well as to locally monitor subsequent divergent responses at both single cell and multi-cell scales.

An X-ray transparent microfluidic platform for screening of the phase behavior of lipidic mesophases.

Lipidic mesophases are a class of highly ordered soft materials that form when certain lipids are mixed with water. Understanding the relationship between the composition and the microstructure of mesophases is necessary for fundamental studies of self-assembly in amphiphilic systems and for applications, such as the crystallization of membrane proteins. However, the laborious formulation protocol for highly viscous mesophases and the large amounts of material required for sample formulation are significant obstacles in such studies. Here we report a microfluidic platform that facilitates investigations of the phase behavior of mesophases by reducing sample consumption 300-fold, and automating and parallelizing sample formulation. The mesophases were formulated on-chip using less than 80 nL of material per sample and their microstructure was analyzed in situ using small-angle X-ray scattering (SAXS). The 220 µm-thick X-ray compatible platform was comprised of thin polydimethylsiloxane (PDMS) layers sandwiched between cyclic olefin copolymer (COC) sheets. Uniform mesophases were prepared using an active on-chip mixing strategy coupled with periodic cooling of the sample to reduce viscosity. We validated the platform by preparing and analyzing mesophases of the lipid monoolein (MO) mixed with aqueous solutions of different concentrations of ß-octylglucoside (ßOG), a detergent frequently used in membrane protein crystallization. Four samples were prepared in parallel on chip, by first metering and automatically diluting ßOG to obtain detergent solutions of different concentration, then metering MO, and finally mixing by actuation of pneumatic valves. Integration of detergent dilution and subsequent mixing significantly reduced the number of manual steps needed for sample preparation. Three different types of mesophases typical for MO were successfully identified in SAXS data from on-chip samples. Microstructural parameters of identical samples formulated in different chips showed excellent agreement. Phase behavior of samples on-chip (~80 nL per sample) corresponded well with that of samples prepared via the traditional coupled-syringe method using at least two orders of magnitude more material ("off-chip", 35-40 µL per sample), further validating the applicability of the microfluidic platform for on-chip characterization of mesophase microstructure.

Nitrogen-based catalysts for the electrochemical reduction of CO2 to CO.

The synthesis and application of carbon-supported, nitrogen-based organometallic silver catalysts for the reduction of CO(2) is studied using an electrochemical flow reactor. Their performance toward the selective formation of CO is similar to the performance achieved when using Ag as the catalyst, but comparatively at much lower silver loading. Faradaic efficiencies of the organometallic catalyst are higher than 90%, which are comparable to those of Ag. Furthermore, with the addition of an amine ligand to Ag/C, the partial current density for CO increases significantly, suggesting a possible co-catalyst mechanism. Additional improvements in activity and selectivity may be achieved as greater insight is obtained on the mechanism of CO(2) reduction and on how these complexes assemble on the carbon support.

Fabrication of X-ray compatible microfluidic platforms for protein crystallization.

This paper reports a method for fabricating multilayer microfluidic protein crystallization platforms using different materials to achieve X-ray transparency and compatibility with crystallization reagents. To validate this approach, three soluble proteins, lysozyme, thaumatin, and ribonuclease A were crystallized on-chip, followed by on-chip diffraction data collection. We also report a chip with an array of wells for screening different conditions that consume a minimal amount of protein solution as compared to traditional screening methods. A large number of high quality isomorphous protein crystals can be grown in the wells, after which slices of X-ray data can be collected from many crystals still residing within the wells. Complete protein structures can be obtained by merging these slices of data followed by further processing with crystallography software. This approach of using an x-ray transparent chip for screening, crystal growth, and X-ray data collection enables room temperature data collection from many crystals mounted in parallel, which thus eliminates crystal handling and minimizes radiation damage to the crystals.