Exploring the Kinetic and Thermodynamic Relationship of Charge Transfer Reactions Used in Localized Electrodeposition and Patterning in a Scanning Bipolar Cell.

Bipolar electrochemistry involves spatial separation of charge balanced reduction and oxidation reactions on an electrically floating electrode, a result of intricate coupling of the work piece with the ohmic drop in the electrochemical cell and to the thermodynamics and kinetics of the respective bipolar reactions. When paired with a rastering microjet electrode, in a scanning bipolar cell (SBC), local electrodeposition and patterning of metals beneath the microjet can be realized without direct electrical connections to the workpiece. Here, we expand on prior research detailing electrolyte design guidelines for electrodeposition and patterning with the SBC, focusing on the relationship between kinetics and thermodynamics of the respective bipolar reactions. The kinetic reversibility or irreversibility of the desired deposition reaction influences the range of possible effective bipolar counter reactions. For kinetically irreversible deposition systems (i.e., nickel), a wider thermodynamic window is available for selection of the counter reaction. For kinetically reversible systems (i.e., copper or silver) that can be easily etched, tight thermodynamic windows with a small downhill driving force for spontaneous reduction are required to prevent metal patterns from electrochemical dissolution. Furthermore, additives used for the bipolar counter reaction can influence not only the kinetics of deposition, but also the morphology and microstructure of the deposit. Cyclic voltammetry measurements help elucidate secondary parasitic reduction reactions occurring during bipolar nickel deposition and describe the thermodynamic relationship of both irreversible and reversible bipolar couples. Finally, finite element method simulations explore the influence of bipolar electrode area on current efficiency and connect experimental observations of pattern etching to thermodynamic and kinetic relationships.

Inhibition of the Exoglucanase Cel7A by a Douglas-Fir-Condensed Tannin.

Douglas-fir forestry residues are a potential feedstock for saccharification-based biofuels, and condensed tannins are expected to make up ∼3% of the dry mass of this feedstock. Condensed tannins are well-known for their ability to interact with proteins and can bind and inhibit cellulase enzymes used in saccharification. In this study, we use molecular docking and classical molecular dynamics simulations to investigate how a characterized condensed tannin from Douglas-fir bark binds to the exoglucanase Cel7A from Trichoderma reesei. Through looking at the "occupancy" and "residency" of specific amino acid residue-tannin interactions, we find that the binding sites are characterized by many simultaneous tannin-enzyme interactions with the strongest occurring on the catalytic module as opposed to the carbohydrate-binding module. The simulations indicate that tannin inhibition can result from binding at or near the catalytic tunnel's entrance and exit. The analyzed tannin further prefers to bind to loops around the catalytic region and has affinity for aromatic and charged amino acid residues. These insights provide direction for the rational design of tannin-resistant cellulases.

Rational design of polymer-based absorbents: application to the fermentation inhibitor furfural.

BACKGROUND: Reducing the amount of water-soluble fermentation inhibitors like furfural is critical for downstream bio-processing steps to biofuels. A theoretical approach for tailoring absorption polymers to reduce these pretreatment contaminants would be useful for optimal bioprocess design. RESULTS: Experiments were performed to measure aqueous furfural partitioning into polymer resins of 5 bisphenol A diglycidyl ether (epoxy) and polydimethylsiloxane (PDMS). Experimentally measured partitioning of furfural between water and PDMS, the more hydrophobic polymer, showed poor performance, with the logarithm of PDMS-to-water partition coefficient falling between -0.62 and -0.24 (95% confidence). In contrast, the fast setting epoxy was found to effectively partition furfural with the logarithm of the epoxy-to-water partition coefficient falling between 0.41 and 0.81 (95% confidence). Flory-Huggins theory is used to predict the partitioning of furfural into diverse polymer absorbents and is useful for predicting these results. CONCLUSION: We show that Flory-Huggins theory can be adapted to guide the selection of polymer adsorbents for the separation of low molecular weight organic species from aqueous solutions. This work lays the groundwork for the general design of polymers for the separation of a wide range of inhibitory compounds in biomass pretreatment streams.

Separation and enhanced detection of anesthetic compounds using solid phase micro-extraction (SPME)-Raman spectroscopy.

Polydimethylsiloxane (PDMS)-based solid-phase micro-extraction (SPME) was used along with Raman spectroscopy (RS) to separate and enhance the detection of five anesthetic compounds (halothane, propofol, isoflurane, enflurane, and etomidate) from aqueous and serum phases. Raman signals in the spectral ranges 250-450 cm(-1) and 950-1050 cm(-1) allowed the unique characterization of all five compounds when extracted into the PDMS phase. The SPME-RS detection of clinically relevant concentrations of aqueous propofol (6.5 µM) and halothane (200 µM) is shown. We quantify the partition coefficient for aqueous halothane in PDMS as log K = 1.9 ± 0.2. Solid-phase micro-extraction of the anesthetics makes their detection possible without the strong autofluorescent interference of serum proteins. Because of low solubility and/or weak Raman scattering, we found it challenging to detect enflurane, isoflurane, and etomidate directly from the aqueous phase, but could we do so with SPME enhancement. These studies show the potential of SPME-RS as a method for the direct detection of anesthetics in blood.

Fast Pyrolysis of Wood for Biofuels: Spatiotemporally Resolved Diffuse Reflectance In†situ Spectroscopy of Particles.

Fast pyrolysis of woody biomass is a promising process capable of producing renewable transportation fuels to replace gasoline, diesel, and chemicals currently derived from nonrenewable sources. However, biomass pyrolysis is not yet economically viable and requires significant optimization before it can contribute to the existing oil-based transportation system. One method of optimization uses detailed kinetic models for predicting the products of biomass fast pyrolysis, which serve as the basis for the design of pyrolysis reactors capable of producing the highest value products. The goal of this work is to improve upon current pyrolysis models, usually derived from experiments with low heating rates and temperatures, by developing models that account for both transport and pyrolysis decomposition kinetics at high heating rates and high temperatures (>400 °C). A new experimental technique is proposed herein: spatiotemporally resolved diffuse reflectance in situ spectroscopy of particles (STR-DRiSP), which is capable of measuring biomass composition during fast pyrolysis with high spatial (10 µm) and temporal (1 ms) resolution. Compositional data were compared with a comprehensive 2D single-particle model, which incorporated a multistep, semiglobal reaction mechanism, prescribed particle shrinkage, and thermophysical properties that varied with temperature, composition, and orientation. The STR-DRiSP technique can be used to determine the transport-limited kinetic parameters of biomass decomposition for a wide variety of biomass feedstocks.

Quantitative solid-phase microextraction (SPME)-Raman spectroscopy for the detection of trace organics in water.

Solid-phase microextraction (SPME) was used along with Raman spectroscopy to quantify the partitioning of trace organics into polydimethylsiloxane (PDMS) matrices. PDMS has previously been utilized with SPME-Raman to pre-concentrate trace benzene, toluene, ethyl-benzene, and xylene fuel components from contaminated water, thereby enhancing detected Raman signals. Here, we show that SPME can increase Raman signals more than two orders of magnitude for the compounds investigated. We also demonstrate the quantitative features of SPME-Raman by estimating PDMS-organic partition coefficients for benzene [log(K) = 1.90 ± 10] and toluene [log(K) = 2.35 ± 20] by using linear regression fits in the dilute limit of concentrations. The K values obtained are within the range of values obtained with other quantitative SPME techniques. The method was also used to characterize quinoline, a pyridine-based organic, which yielded reasonable K values [log(K) = 1.20 ± 20]. Combining PDMS-based SPME with a technique such as Raman spectroscopy potentially enhances optical detection methods used in microfluidic systems, wherein PDMS is a common material of construction.

Electrodeposition modeling and optimization to improve thin film patterning with orchestrated structure evolution.

Orchestrated structure evolution is an alternative nanomanufacturing approach that combines the advantages of top-down patterning and bottom-up self-organizing growth. It relies upon tool-directed patterning to create 'seed' locations on a surface from which a subsequent deposition process produces the final, merged film. Despite its demonstrated ability to reduce patterning time by orders of magnitude, our prior reliance on mass transfer limited deposition and square seed arrays resulted in extraneous film growth along pattern edges, thereby limiting the pattern quality of the final film. Here, quality improvements are demonstrated by modeling and tuning the growth mechanism of the deposition step to include charge transfer effects. In addition, a seed positioning optimization technique derived from simulated annealing is introduced as a method for relocating the seeds to minimize film overgrowth at the pattern edges. These improvements enable OSE to maintain geometric quality while substantially reducing the time and cost compared to traditional direct-write manufacturing methods.

Laying out ground rules for protein-aided nanofabrication: ZnO synthesis at 70°C as a case study.

Designer proteins that incorporate solid-binding peptides hold promise to control the nucleation, growth, morphology, and assembly of inorganic phases under mild conditions of temperature and pressure. However, protein-aided nanofabrication remains more art than science and some materials can only be synthesized at temperatures that cause most mesophilic proteins to unfold. Using zinc oxide (ZnO) synthesis at 70°C as case study, we show here that seemingly unimportant variables, such as the carry-over concentration of Tris buffer and the "empty" host protein scaffold can exert a significant influence on materials morphology. We also show that, once well-controlled conditions are established, thermodynamic predictions and adsorption isotherms are powerful tools to understand how various ZnO-binding sequence inserted within the thermostable framework of Escherichia coli thioredoxin A (TrxA) affect inorganic morphogenesis.

Biomineralization and size control of stable calcium phosphate core-protein shell nanoparticles: potential for vaccine applications.

Calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of Escherichia coli thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50-70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high-affinity ACP binding as elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers 3 weeks postinjection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.

Hydrodynamic tweezers: impact of design geometry on flow and microparticle trapping.

Here we explore the role of microfabricated device geometry on frequency-dependent low Reynolds number steady streaming flow and particle trapping behavior. In our system, flow and particle trapping is induced near an obstruction or cavity located in an otherwise rectilinear oscillating flow of frequency ω and amplitude s in a fluid of kinematic viscosity ν. This work expands prior studies to characterize nine distinct obstruction/cavity geometries. The imaged microeddy flows show that the device geometry affects the eddy number, shape, structure, and strength. Comparison of measured particle trap locations with the computed eddy flow structure shows that particles trap closer to the wall than the eddy core. Trapping strength and location are controlled by the geometry and the oscillation frequency. In most cases, the trapping behavior is linearly proportional to the Stokes layer thickness, δ(AC) ~ O((ν/ω)(1/2)). We show that steady streaming in microfluidic eddies can be a flexible and versatile method for noncontact microparticle trapping, and hence we call this class of devices "hydrodynamic tweezers".

Orchestrated structure evolution: modeling growth-regulated nanomanufacturing.

Orchestrated structure evolution (OSE) is a scalable manufacturing method that combines the advantages of top-down (tool-directed) and bottom-up (self-propagating) approaches. The method consists of a seed patterning step that defines where material nucleates, followed by a growth step that merges seeded islands into the final patterned thin film. We develop a model to predict the completed pattern based on a computationally efficient approximate Green's function solution of the diffusion equation plus a Voronoi diagram based approach that defines the final grain boundary structure. Experimental results rely on electron beam lithography to pattern the seeds, followed by the mass transfer limited growth of copper via electrodeposition. The seed growth model is compared with experimental results to quantify nearest neighbor seed-to-seed interactions as well as how seeds interact with the pattern boundary to impact the local growth rate. Seed-to-seed and seed-to-pattern interactions are shown to result in overgrowth of seeds on edges and corners of the shape, where seeds have fewer neighbors. We explore how local changes to the seed location can be used to improve the patterning quality without increasing the manufacturing cost. OSE is shown to enable a unique set of trade-offs between the cost, time, and quality of thin film patterning.

Orchestrated structure evolution: accelerating direct-write nanomanufacturing by combining top-down patterning with bottom-up growth.

Direct-write nanomanufacturing with scanning beams and probes is flexible and can produce high quality products, but it is normally slow and expensive to raster point-by-point over a pattern. We demonstrate the use of an accelerated direct-write nanomanufacturing method called 'orchestrated structure evolution' (OSE), where a direct-write tool patterns a small number of growth 'seeds' that subsequently grow into the final thin film pattern. Through control of seed size and spacing, it is possible to vary the ratio of 'top-down' to 'bottom-up' character of the patterning processes, ranging from conventional top-down raster patterning to nearly pure bottom-up space-filling via seed growth. Electron beam lithography (EBL) and copper electrodeposition were used to demonstrate trade-offs between process time and product quality over nano- to microlength scales. OSE can reduce process times for high-cost EBL patterning by orders of magnitude, at the expense of longer (but inexpensive) copper electrodeposition processing times. We quantify the degradation of pattern quality that accompanies fast OSE patterning by measuring deviations from the desired patterned area and perimeter. We also show that the density of OSE-induced grain boundaries depends upon the seed separation and size. As the seed size is reduced, the uniformity of an OSE film becomes more dependent on details of seed nucleation processes than normally seen for conventionally patterned films.

Single-pot biofabrication of zinc sulfide immuno-quantum dots.

Quantum dots (QDs) are a powerful alternative to organic dyes and fluorescent proteins for biological and biomedical applications. These semiconductor nanocrystals are traditionally synthesized above 200 degrees C in organic solvents using toxic and costly precursors, and further steps are required to conjugate them to a biological ligand. Here, we describe a simple, aqueous route for the one-pot synthesis of antibody-derivatized zinc sulfide (ZnS) immuno-QDs. In this strategy, easily expressed and purified fusion proteins perform the dual function of nanocrystal mineralizers through ZnS binding sequences identified by cell surface display and adaptors for immunoglobin G (IgG) conjugation through a tandem repeat of the B domain of Staphylococcus aureus protein A. Although approximately 4.3 nm ZnS wurtzite cores could be biomineralized from either zinc chloride or zinc acetate precursors, only the latter salt gives rise to protein-coated QDs with long shelf life and narrow hydrodynamic diameters (8.8 +/- 1.4 nm). The biofabricated QDs have a quantum yield of 2.5% and blue-green ensemble emission with contributions from the band-edge at 340 nm and from trap states at 460 and 665 nm that are influenced by the identity of the protein shell. Murine IgG(1) antibodies exhibit high affinity (K(d) = 60 nM) for the protein shell, and stable immuno-QDs with a hydrodynamic diameter of 14.1 +/- 1.3 nm are readily obtained by mixing biofabricated nanocrystals with human IgG.

Simulation study on the use of strippable coatings for radiocesium decontamination of concrete.

The contamination and decontamination of concrete by a soluble contaminant (radiocesium) was modeled using the finite element method. The decontamination process relied on the application of a hyper-accumulating strippable polymer (HASP™) [corrected] to sequester contaminant that was transported to the concrete surface. The model accounted for the transport of cesium by diffusion, capillary pressure-driven convection, and equilibrium adsorption of cesium within the concrete substrate and HASP™ [corrected] coating. The influence of HASP™ [corrected] properties (porosity, thickness, and cesium distribution coefficient), and a wide range of operation variables (HASP™ [corrected] contact time, delay time until HASP™ [corrected] decontamination begins, influence of evaporative boundary conditions) on final decontamination efficacy were explored. Transient saturation (moisture content) and cesium concentration profiles were used to understand key factors in the decontamination process, and water wicking experiments were performed to validate the capillary convection model. The results showed that prompt HASP™ [corrected] application after the initial contamination event is critical for high decontamination efficacies. A 30-day HASP™ [corrected] treatment removed about 90% of the contaminant when HASP™ [corrected] was applied within 30min of the initial contamination event, whereas the decontamination process removed less than half as much cesium if months were allowed to elapse months prior to decontamination. Multiple applications of fresh HASP™ [corrected] were also shown to improve decontamination performance under certain circumstances.

Three-dimensional architecture of inorganic nanoarrays electrodeposited through a surface-layer protein mask.

Transmission electron microscopy was used to analyze the three-dimensional (3D) architecture of cuprous oxide electrochemically deposited through the pores of the hexagonally packed intermediate surface-layer protein from Deinococcus radiodurans SARK. Imaging at multiple tilt angles and averaging from five different samples allowed approximately 3 nm computed 3D reconstructions of the inorganic deposit and protein template. We show that the electrodeposition process used here was able to fully access the pore structure that penetrates the protein layer, allowing the fabrication of a polycrystalline nanoarray with 18 nm periodicity and lateral interconnectivity among the pores with 3-fold symmetry. At the resolution of the reconstruction, the 6-fold symmetry pores also appear filled but are not connected laterally to the rest of the deposit. These results show that electrochemical deposition can produce interconnected 3D structures at dimensions an order of magnitude smaller than the most advanced integrated circuits (IC), boding well for continued down-scaling of electrodeposition to meet the needs for future generations of IC device interconnects.

A genetic approach for controlling the binding and orientation of proteins on nanoparticles.

Although silver nanoparticles are excellent surface enhancers for Raman spectroscopy, their use to probe the conformation of large proteins at interfaces has been complicated by the fact that many polypeptides adsorb weakly or with a random orientation to colloidal silver. To address these limitations, we sought to increase binding affinity and control protein orientation by fusing a silver-binding dodecapeptide termed Ag4 to the C-terminus of maltose-binding protein (MBP), a well-characterized model protein with little intrinsic silver binding affinity. Quartz crystal microbalance measurements conducted with the MBP-Ag4 fusion protein revealed that its affinity for silver (Kd approximately 180 nM) was at least 1 order of magnitude higher than a control protein, MBP2, containing a non-silver-specific C-terminal extension. Under our experimental conditions, MBP-Ag4 SERS spectra exhibited 2-4 fold higher signal-to-background relative to MPB2 and contained a number of amino acid-assigned vibrational modes that were either weak or absent in control experiments performed with MBP2. Changes in amino acid-assigned peaks before and after MBP-Ag4 bound maltose were used to assess protein orientation on the surface of silver nanoparticles. The genetic route described here may prove useful to study the orientation of other proteins on a variety of SERS-active surfaces, to improve biosensors performance, and to control functional nanobiomaterials assembly.

Conformational control of inorganic adhesion in a designer protein engineered for cuprous oxide binding.

Combinatorial selection of peptides that bind technological materials has emerged as a valuable tool for room-temperature nucleation and assembly of complex nanostructured materials. At present, the parameters that control peptide-solid binding are poorly understood, but such knowledge is needed to build the next generation of hybrid materials. Here, we use a derivative of the DNA binding protein TraI engineered with a disulfide-bonded cuprous oxide binding sequence called CN225 to probe the influence of sequence composition and conformation on Cu2O binding affinity. We previously reported a statistically significant enrichment in paired arginines (RR) among a family of cuprous oxide binding peptides and hypothesized that this is a key motif for binding. However, systematic alanine (A) substitutions in the CN225 RR motif (creating RA, AR, and AA pairs) do not support the hypothesis that RR is critical for Cu2O binding by CN225. Instead, we find that the presentation of the peptide in a disulfide-constrained loop (i.e., the conformation present during combinatorial selection) is crucial for binding to the metal oxide. Our results suggest that caution should be exerted when extrapolating from statistical data and that, in some cases, conformation is more important than composition in determining peptide-inorganic adhesion.

Electrochemical factors controlling the patterning of metals on SAM-coated substrates.

Alkanethiol self-assembled monolayers (SAMs) have been used in electrochemical microfabrication processes. The reductive desorption potential of alkanethiol SAMs, Edes, can be comparable to, greater than, or less than the metal reduction potential during electrodeposition, Emet. As a result, the SAM layer can passivate the surface or desorb simultaneously with metal deposition. We show that these electrochemical traits can be combined with a rastering microjet electrode to pattern SAMs directly and create patterned metal films without lithography steps. For the case of copper deposition on 1-octanethiol (OT)- and 1-dodecanethiol (DT)-coated substrates, Edes is significantly negative of Emet, resulting in high-resolution metal patterns with poor nucleation and poor adhesion to the substrate. However, nickel patterns deposited on 1-butanethiol (BT), OT, and DT have traits similar to bare gold (excellent nucleation and adhesion) because Edes is positive of Emet. Substrates with SAMs also suppress adventitious chemistries that occur distant from the rastering microjet electrode, such as oxygen reduction, making samples more corrosion resistant and improving the overall patterning process that we call electrochemical printing.

Selection and analysis of solid-binding peptides.

Nature has long used peptide- and protein-based manufacturing to create structures whose remarkable mechanical, transport, optical, and even magnetic properties are determined by a fine control of composition and architecture extending from the nanoscale to the macroscale. Although there is much to learn from the tools and strategies that have been evolutionary selected for building biomaterials, accessing compositions and architectures of engineering interest is crucial to the development of the next generation of hybrid functional materials. In recent years, portable amino acid sequences selected from combinatorial libraries and supporting the assembly, nucleation, and geometrical organization of solid phases have emerged as attractive tools for bionanofabrication. Here, we review how these polypeptides are selected and progress in the understanding of their interaction with inorganic and synthetic materials.

Stability of S-layer proteins for electrochemical nanofabrication.

Crystalline cell surface layer proteins (S-layers) can be used in electrochemical fabrication to create nanoscale arrays of metals and oxides on surfaces so long as the proteins maintain their long-range order during processing. We have explored the stability of the HPI layer protein (the S-layer protein from the microorganism Deinococcus radiodurans) adsorbed onto platinum surfaces after immersion in sulfuric acid or sodium hydroxide electrolytes ranging in pH from 0 to 14 over time periods ranging from 1 to 1000s. Topographic data obtained by atomic force microscopy (AFM) was used to characterize the protein stability, judged by its retention of long-range order after immersion. The compiled data revealed that, under these solution conditions and in this environment, the HPI layer protein has a dose-dependent structural stability "envelope" in the acidic range from 1