Mesostructured Materials with Controllable Long-Range Orientational Ordering and Anisotropic Properties.

Inorganic-organic mesophase materials provide a wide range of tunable properties, which are often highly dependent on their nano-, micro-, or meso-scale compositions and structures. Among these are macroscopic orientational order and corresponding anisotropic material properties, the adjustability of which are difficult to achieve. This is due to the complicated transient and coupled transport, chemical reaction, and surface processes that occur during material syntheses. By understanding such processes, general criteria are established and used to prepare diverse mesostructured materials with highly aligned channels with uniform nanometer dimensions and controllable directionalities over macroscopic dimensions and thicknesses. This is achieved by using a micropatterned semipermeable poly(dimethylsiloxane) stamp to manage the rates, directions, and surfaces at which self-assembling phases nucleate and the directions that they grow. This enables mesostructured surfactant-directed silica and titania composites, including with functional guest species, and mesoporous carbons to be prepared with high degrees of hexagonal order, as well as controllable orthogonal macroscopic orientational order. The resulting materials exhibit novel anisotropic properties, as demonstrated by the example of direction-dependent photocurrent generation, and are promising for enhancing the functionality of inorganic-organic nanocomposite materials in separations, catalysis, and energy conversion applications.

Modeling control and transduction of electrochemical gradients in acid-stressed bacteria.

Transmembrane electrochemical gradients drive solute uptake and constitute a substantial fraction of the cellular energy pool in bacteria. These gradients act not only as "homeostatic contributors," but also play a dynamic and keystone role in several bacterial functions, including sensing, stress response, and metabolism. At the system level, multiple gradients interact with ion transporters and bacterial behavior in a complex, rapid, and emergent manner; consequently, experiments alone cannot untangle their interdependencies. Electrochemical gradient modeling provides a general framework to understand these interactions and their underlying mechanisms. We quantify the generation, maintenance, and interactions of electrical, proton, and potassium potential gradients under lactic acid-stress and lactic acid fermentation. Further, we elucidate a gradient-mediated mechanism for intracellular pH sensing and stress response. We demonstrate that this gradient model can yield insights on the energetic limitations of membrane transport, and can predict bacterial behavior across changing environments.

Identification of Microorganisms that Bind Specifically to Target Materials of Interest Using a Magnetophoretic Microfluidic Platform.

Discovery of microorganisms and their relevant surface peptides that specifically bind to target materials of interest can be achieved through iterative biopanning-based screening of cellular libraries having high diversity. Recently, microfluidics-based biopanning methods have been developed and exploited to overcome the limitations of conventional methods where controlling the shear stress applied to remove cells that do not bind or only weakly bind to target surfaces is difficult and the overall experimental procedure is labor-intensive. Despite the advantages of such microfluidic methods and successful demonstration of their utility, these methods still require several rounds of iterative biopanning. In this work, a magnetophoretic microfluidic biopanning platform was developed to isolate microorganisms that bind to target materials of interest, which is gold in this case. To achieve this, gold-coated magnetic nanobeads, which only attached to microorganisms that exhibit high affinity to gold, were used. The platform was first utilized to screen a bacterial peptide display library, where only the cells with surface peptides that specifically bind to gold could be isolated by the high-gradient magnetic field generated within the microchannel, resulting in enrichment and isolation of many isolates with high affinity and high specificity toward gold even after only a single round of separation. The amino acid profile of the resulting isolates was analyzed to provide a better understanding of the distinctive attributes of peptides that contribute to their specific material-binding capabilities. Next, the microfluidic system was utilized to screen soil microbes, a rich source of extremely diverse microorganisms, successfully isolating many naturally occurring microorganisms that show strong and specific binding to gold. The results show that the developed microfluidic platform is a powerful screening tool for identifying microorganisms that specifically bind to a target material surface of interest, which can greatly accelerate the development of new peptide-driven biological materials and hybrid organic-inorganic materials.

Bioelectronic control of a microbial community using surface-assembled electrogenetic cells to route signals.

We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.

Biofunctionalized Cellulose Nanofibrils Capable of Capture and Antiadhesion of Fimbriated Escherichia coli.

In this study, naturally derived cellulose nanofibrils (CNFs), a renewable and easily modified nanomaterial with low cytotoxicity, were rendered bioactive via one-step functionalization with mannopyranoside (CNFs-mannose) for use as a new glyconanomaterial platform for control of bacterial pathogenesis. The recognition affinity of the bioactive surfaces toward fimbriated Escherichia coli was assessed using genetically engineered strains as well as wild-type (WT) MG1655 bacteria. The results revealed high surface coverages of FimH+ (with overexpressed FimH) and WT bacteria on the films of CNFs-mannose due to specific interaction between prevalent mannose on nanofibrils and FimH receptors on E. coli fimbriae. The CNFs-mannose nanofibrils were capable of capturing E. coli from a liquid suspension, as demonstrated either by the nanofibril clusters or by the cellulose filter papers impregnated with CNFs-mannose. More importantly, CNFs-mannose efficiently inhibited adhesion of both FimH+ and WT E. coli to mannosylated surfaces even at a very low concentration, resulting in over 95% reduction of bacterial adhesion. Furthermore, the bioactive nanofibrils showed effective disruption of nonspecific binding of bacteria to abiotic surfaces in flow channel tests. These findings highlight the potential of cellulose nanofibrils as a biocompatible polyvalent nanoscale scaffold and exemplify sugar grafted nanofibrils as novel and effective tools in control of bacterial pathogenesis, bacterial removal, as well as in many other applications.

Functionally Active Membrane Proteins Incorporated in Mesostructured Silica Films.

A versatile synthetic protocol is reported that allows high concentrations of functionally active membrane proteins to be incorporated in mesostructured silica materials. Judicious selections of solvent, surfactant, silica precursor species, and synthesis conditions enable membrane proteins to be stabilized in solution and during subsequent coassembly into silica-surfactant composites with nano- and mesoscale order. This was demonstrated by using a combination of nonionic ( n-dodecyl-ß-d-maltoside or Pluronic P123), lipid-like (1,2-diheptanoyl- s n-glycero-3-phosphocholine), and perfluoro-octanoate surfactants under mild acidic conditions to coassemble the light-responsive transmembrane protein proteorhodopsin at concentrations up to 15 wt % into the hydrophobic regions of worm-like mesostructured silica materials in films. Small-angle X-ray scattering, electron paramagnetic resonance spectroscopy, and transient UV-visible spectroscopy analyses established that proteorhodopsin molecules in mesostructured silica films exhibited native-like function, as well as enhanced thermal stability compared to surfactant or lipid environments. The light absorbance properties and light-activated conformational changes of proteorhodopsin guests in mesostructured silica films are consistent with those associated with the native H+-pumping mechanism of these biomolecules. The synthetic protocol is expected to be general, as demonstrated also for the incorporation of functionally active cytochrome c, a peripheral membrane protein enzyme involved in electron transport, into mesostructured silica-cationic surfactant films.

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

"Living" dynamics of filamentous bacteria on an adherent surface under hydrodynamic exposure.

The potential advantages of cell-based biohybrid devices over conventional nonliving systems drive the interest to control the behavior of the underlying biological cells in microdevices. Here, the authors studied how shear influenced the geometry and elongation of fimbriated filaments on affinity substrates. The cells were engineered to express FimH, which binds to mannose with a high affinity. A microfluidic channel was functionalized with RNAse B, which is rich in mannose residues, and the device was used to control the hydrodynamic force on live Escherichia coli under filamentous growth. It was discovered that filamentous E. coli cells adopt buckled geometry when the shear rate is low, but assume an extended geometry at high shear and align with the flow direction. The extension moves from bidirectional to preferentially downstream as the shear rate increases. Furthermore, living filaments slide easily on the substrate, and detach from the substrates at a rate nearly ten times greater than unfilamented live E. coli at high shear conditions (1000-4000 s-1). The hydrodynamic force and binding force experienced by the cells are further analyzed by COMSOL simulation and atomic force microscopy measurements, respectively, to explore the mechanism behind the living cell dynamics. Knowledge from this work helps guide design of interfacial properties and shear environments to control the geometry of living filamentous bacteria.

Vapor-fed bio-hybrid fuel cell.

BACKGROUND: Concentration and purification of ethanol and other biofuels from fermentations are energy-intensive processes, with amplified costs at smaller scales. To circumvent the need for these processes, and to potentially reduce transportation costs as well, we have previously investigated bio-hybrid fuel cells (FCs), in which a fermentation and FC are closely coupled. However, long-term operation requires strictly preventing the fermentation and FC from harming each other. We introduce here the concept of the vapor-fed bio-hybrid FC as a means of continuously extracting power from ongoing fermentations at ambient conditions. By bubbling a carrier gas (N2) through a yeast fermentation and then through a direct ethanol FC, we protect the FC anode from the catalyst poisons in the fermentation (which are non-volatile), and also protect the yeast from harmful FC products (notably acetic acid) and from build-up of ethanol. RESULTS: Since vapor-fed direct ethanol FCs at ambient conditions have never been systematically characterized (in contrast to vapor-fed direct methanol FCs), we first assess the effects on output power and conversion efficiency of ethanol concentration, vapor flow rate, and FC voltage. The results fit a continuous stirred-tank reactor model. Over a wide range of ethanol partial pressures (2-8 mmHg), power densities are comparable to those for liquid-fed direct ethanol FCs at the same temperature, with power densities >2 mW/cm2 obtained. We then demonstrate the continuous operation of a vapor-fed bio-hybrid FC with fermentation for 5 months, with no indication of performance degradation due to poisoning (of either the FC or the fermentation). It is further shown that the system is stable, recovering quickly from disturbances or from interruptions in maintenance. CONCLUSIONS: The vapor-fed bio-hybrid FC enables extraction of power from dilute bio-ethanol streams without costly concentration and purification steps. The concept should be scalable to both large and small operations and should be generalizable to other biofuels and waste-to-energy systems.

Aspergillus oryzae-Saccharomyces cerevisiae Consortium Allows Bio-Hybrid Fuel Cell to Run on Complex Carbohydrates.

Consortia of Aspergillus oryzae and Saccharomyces cerevisiae are examined for their abilities to turn complex carbohydrates into ethanol. To understand the interactions between microorganisms in consortia, Fourier-transform infrared spectroscopy is used to follow the concentrations of various metabolites such as sugars (e.g., glucose, maltose), longer chain carbohydrates, and ethanol to optimize consortia conditions for the production of ethanol. It is shown that with proper design A. oryzae can digest food waste simulants into soluble sugars that S. cerevisiae can ferment into ethanol. Depending on the substrate and conditions used, concentrations of 13% ethanol were achieved in 10 days. It is further shown that a direct alcohol fuel cell (FC) can be coupled with these A. oryzae-enabled S. cerevisiae fermentations using a reverse osmosis membrane. This "bio-hybrid FC" continually extracted ethanol from an ongoing consortium, enhancing ethanol production and allowing the bio-hybrid FC to run for at least one week. Obtained bio-hybrid FC currents were comparable to those from pure ethanol-water mixtures, using the same FC. The A. oryzae-S. cerevisiae consortium, coupled to a bio-hybrid FC, converted food waste simulants into electricity without any pre- or post-processing.

Influences of Adhesion Variability on the "Living" Dynamics of Filamentous Bacteria in Microfluidic Channels.

Microfabricated devices have increasingly incorporated bacterial cells for microscale studies and exploiting cell-based functions in situ. However, the role of surface interactions in controlling the bacterial cell behavior is not well understood. In this study, microfluidic substrates of varied bacterial-binding affinity were used to probe the interaction-driven behavior of filamentous Escherichia coli. In particular, cell alignment under controlled shear flow as well as subsequent orientation and filamentation were compared between cells presenting distinct outer membrane phenotypes. We demonstrated that filaments retained position under flow, which allowed for dynamic single-cell monitoring with in situ elongation of over 100 µm for adherent cells. This maximum was not reached by planktonic cells and was, therefore, adhesion-dependent. The bound filaments initially aligned with flow under a range of flow rates and their continual elongation was traced in terms of length and growth path; analysis demonstrated that fimbriae-mediated adhesion increased growth rate, increased terminal length, as well as dramatically changed the adherent geometry, particularly buckling behavior. The effects to filament length and buckling were further exaggerated by the strongest, specificity-driven adhesion tested. Such surface-guided control of the elongation process may be valuable to yield interesting "living" filamentous structures in microdevices. In addition, this work may offer a biomedically relevant platform for further elucidation of filamentation as an immune-resistant morphology. Overall, this work should inspire broader exploration of microfabricated devices for the study and application of single bacterial cells.

Simple, fast, and accurate methodology for quantitative analysis using Fourier transform infrared spectroscopy, with bio-hybrid fuel cell examples.

The standard methodologies for quantitative analysis (QA) of mixtures using Fourier transform infrared (FTIR) instruments have evolved until they are now more complicated than necessary for many users' purposes. We present a simpler methodology, suitable for widespread adoption of FTIR QA as a standard laboratory technique across disciplines by occasional users.•Algorithm is straightforward and intuitive, yet it is also fast, accurate, and robust.•Relies on component spectra, minimization of errors, and local adaptive mesh refinement.•Tested successfully on real mixtures of up to nine components. We show that our methodology is robust to challenging experimental conditions such as similar substances, component percentages differing by three orders of magnitude, and imperfect (noisy) spectra. As examples, we analyze biological, chemical, and physical aspects of bio-hybrid fuel cells.

Conjugated gold nanoparticles as a tool for probing the bacterial cell envelope: The case of Shewanella oneidensis MR-1.

The bacterial cell envelope forms the interface between the interior of the cell and the outer world and is, thus, the means of communication with the environment. In particular, the outer cell surface mediates the adhesion of bacteria to the surface, the first step in biofilm formation. While a number of ligand-based interactions are known for the attachment process in commensal organisms and, as a result, opportunistic pathogens, the process of nonspecific attachment is thought to be mediated by colloidal, physiochemical, interactions. It is becoming clear, however, that colloidal models ignore the heterogeneity of the bacterial surface, and that the so-called nonspecific attachment may be mediated by specific regions of the cell surface, whether or not the relevant interaction is ligand-mediate. The authors introduce surface functionalized gold nanoparticles to probe the surface chemistry of Shewanella oneidensis MR-1 as it relates to surface attachment to ω-substituted alkanethiolates self-assembled monolayers (SAMs). A linear relationship between the attachment of S. oneidensis to SAM modified planar substrates and the number of similarly modified nanoparticles attached to the bacterial surfaces was demonstrated. In addition, the authors demonstrate that carboxylic acid-terminated nanoparticles attach preferentially to the subpolar region of the S. oneidensis and obliteration of that binding preference corresponds in loss of attachment to carboxylic acid terminated SAMs. Moreover, this region corresponds to suspected functional regions of the S. oneidensis surface. Because this method can be employed over large numbers of cells, this method is expected to be generally applicable for understanding cell surface organization across populations.

Effect of Modified Phospholipid Bilayers on the Electrochemical Activity of a Membrane-Spanning Conjugated Oligoelectrolyte.

The incorporation and electrochemical activity of a conjugated oligoelectrolyte (COE) in model phospholipid bilayers have been characterized using cyclic voltammetry and UV-vis absorption measurements. Several other modifiers were also incorporated into the phospholipid membranes to alter properties such as charge and alkyl chain disorder. Using potassium ferricyanide to measure charge transport, it was observed that bilayers that contained cholic acid, a negatively charged additive that also promotes alkyl chain disorder, had higher COE uptake and charge permeability than unmodified bilayers. In contrast, when the positively charged choline was incorporated, charge permeability decreased and COE uptake was similar to that of unmodified bilayers. The incorporation of cholesterol at low concentrations within the phospholipid membranes was shown to enhance the COE's effectiveness at increasing membrane charge permeability without increasing the COE concentration in the bilayer. Higher concentrations of cholesterol reduce membrane fluidity and membrane charge permeability. Collectively, these results demonstrate that changes in phospholipid membrane charge permeability upon COE incorporation depend not only on the concentration in the membrane but also on interactions with the phospholipid bilayer and other additives present in the membranes. This approach of manipulating the properties of phospholipid membranes to understand COE interactions is applicable to understanding the behavior of a wide range of molecules that impart useful properties to phospholipid membranes.

Importance of the donor:fullerene intermolecular arrangement for high-efficiency organic photovoltaics.

The performance of organic photovoltaic (OPV) material systems are hypothesized to depend strongly on the intermolecular arrangements at the donor:fullerene interfaces. A review of some of the most efficient polymers utilized in polymer:fullerene PV devices, combined with an analysis of reported polymer donor materials wherein the same conjugated backbone was used with varying alkyl substituents, supports this hypothesis. Specifically, the literature shows that higher-performing donor-acceptor type polymers generally have acceptor moieties that are sterically accessible for interactions with the fullerene derivative, whereas the corresponding donor moieties tend to have branched alkyl substituents that sterically hinder interactions with the fullerene. To further explore the idea that the most beneficial polymer:fullerene arrangement involves the fullerene docking with the acceptor moiety, a family of benzo[1,2-b:4,5-b']dithiophene-thieno[3,4-c]pyrrole-4,6-dione polymers (PBDTTPD derivatives) was synthesized and tested in a variety of PV device types with vastly different aggregation states of the polymer. In agreement with our hypothesis, the PBDTTPD derivative with a more sterically accessible acceptor moiety and a more sterically hindered donor moiety shows the highest performance in bulk-heterojunction, bilayer, and low-polymer concentration PV devices where fullerene derivatives serve as the electron-accepting materials. Furthermore, external quantum efficiency measurements of the charge-transfer state and solid-state two-dimensional (2D) (13)C{(1)H} heteronuclear correlation (HETCOR) NMR analyses support that a specific polymer:fullerene arrangement is present for the highest performing PBDTTPD derivative, in which the fullerene is in closer proximity to the acceptor moiety of the polymer. This work demonstrates that the polymer:fullerene arrangement and resulting intermolecular interactions may be key factors in determining the performance of OPV material systems.