Naval Research Laboratory Center for Bio/Molecular Science and Engineering

Cooperative phenomena, described by one-dimensional statistical physical methods, are observed between the enantiomeric characteristics of monomeric materials and the polymers they produce. The effect of minute energies associated with this amplified chirality, although currently not interpretable, can be easily measured. Nonlinear relationships between enantiomeric excess or enantiomeric content and polymer properties may offer the possibility of developing chiral catalysts and chiral chromatographic materials in which the burden of large enantiomeric excess or content may be considerably alleviated. New approaches to information and sensor technology may become possible.

The use of molecular self-assembly to fabricate microstructures suitable for advanced material development is described. Templating techniques that transform biomolecular self-assemblies into rugged and stable nano- and microstructures are described. By using a lipid-based microcylinder (tubule) as a paradigm, the path followed from research and development to emerging technological applications is detailed. This process includes modification of the lipid molecular structure, the formation and subsequent characterization of cylindrical microstructures, the use of these structures as templates for metallization, and the characterization and assessment of these hollow metal microcylinders for several potential applications.

Free-standing anisotropic side chain liquid crystalline elastomer films have been prepared using mesogens with laterally affixed polymerizable side chains. We present data on two networks: one containing the monomer of 4‘-acryloyloxybutyl 2,5-(4‘-butyloxybenzoyloxy)benzoate and another from a 50/50 mol % mixture of the above with 4‘-acryloyloxybutyl 2,5-di(4‘-pentylcyclohexyloyloxy)benzoate. The cross-linking was achieved using 10 mol % of 1,6-hexanediol diacrylate. The calculated cross-linking density, as determined from the Young's modulus, was in the 10 -5 mol/cm3 range. Thermoelastic responses show strain changes through the nematic−isotropic phase transition to be 30−45%. The order parameters of the oriented films were determined from the dichroic ratio of IR absorption at 3343 cm-1 to the in-plane aromatic stretching overtone of the LC mesogen core. The variation of the order parameter with temperature scales similar to the strain changes at constant stress. Isostrain studies, conducted through the nematic to isotropic phase transition, show that the two networks behave as true elastomers with significant differences in the force developed. Dynamic shear measurements near the nematic to isotropic phase transition region show that the mechanical relaxation peak appears above 100 Hz, and that viscoelastic relaxations are minimal in the nematic to isotropic phase transition region below 5−10 Hz.

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, we prepared bioinorganic conjugates made with highly luminescent semiconductor nanocrystals (CdSe-ZnS core-shell QDs) and antibodies to perform multiplexed fluoroimmunoassays. Sandwich immunoassays for the detection of cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B were performed simultaneously in single wells of a microtiter plate. Initially the assay performance for the detection of each toxin was examined. We then demonstrated the simultaneous detection of the four toxins from a single sample probed with a mixture of all four QD-antibody reagents. Using a simple linear equation-based algorithm, it was possible to deconvolute the signal from mixed toxin samples, which allowed quantitation of all four toxins simultaneously.

Förster resonance energy transfer (FRET), which involves the nonradiative transfer of excitation energy from an excited donor fluorophore to a proximal ground-state acceptor fluorophore, is a well-characterized photophysical tool. It is very sensitive to nanometer-scale changes in donor-acceptor separation distance and their relative dipole orientations. It has found a wide range of applications in analytical chemistry, protein conformation studies, and biological assays. Luminescent semiconductor nanocrystals (quantum dots, QDs) are inorganic fluorophores with unique optical and spectroscopic properties that could enhance FRET as an analytical tool, due to broad excitation spectra and tunable narrow and symmetric photoemission. Recently, there have been several FRET investigations using luminescent QDs that focused on addressing basic fundamental questions, as well as developing targeted applications with potential use in biology, including sensor design and protein conformation studies. Herein, we provide a critical review of those developments. We discuss some of the basic aspects of FRET applied to QDs as both donors and acceptors, and highlight some of the advantages offered (and limitations encountered) by QDs as energy donors and acceptors compared to conventional dyes. We also review the recent developments made in using QD bioreceptor conjugates to design FRET-based assays.

We provide an overview of the progress made in the past few years in investigating fluorescence resonance energy transfer (FRET) using semiconductor quantum dots (QDs) and the application of QD-based FRET to probe specific biological processes. We start by providing some of the pertinent conceptual elements involved in resonance energy transfer, and then discuss why the Förster dipole-dipole mechanism applies to QD fluorophores. We then describe the unique QD photophysical properties of direct relevance to FRET and summarize the main advantages offered, along with some of the limitations encountered by QDs as exciton donors and/or acceptors. Next we describe the overall progress made and discuss a few representative examples where QD-based FRET sensing of specific biological processes has been demonstrated. We also detail some of the advances of single molecule FRET using QD-conjugates and highlight the unique information that can be extracted. We conclude by providing an assessment of where QD-based FRET investigations may be evolving in the near future.

Geobacteracea are distinct for their ability to reduce insoluble oxidants including minerals and electrodes without apparent reliance on soluble extracellular electron transfer (ET) mediators. This property makes them important anode catalysts in new generation microbial fuel cells (MFCs) because it obviates the need to replenish ET mediators otherwise necessary to sustain power. Here we report cyclic voltammetry (CV) of biofilms of wild type (WT) and mutant G. sulfurreducens strains grown on graphite cloth anodes acting as electron acceptors with acetate as the electron donor. Our analysis indicates that WT biofilms contain a conductive network of bound ET mediators in which OmcZ (outer membranec-type cytochrome Z) participates in homogeneous ET (through the biofilm bulk) while OmcB mediates heterogeneous ET (across the biofilm/electrode interface); that type IV pili are important in both reactions; that OmcS plays a secondary role in homogenous ET; that OmcE, important in Fe(III) oxide reduction, is not involved in either reaction; that catalytic current is limited overall by the rate of microbial uptake of acetate; that protons generated from acetate oxidation act as charge compensating ions in homogenous ET; and that homogenous ET, when accelerated by fast voltammetric scan rates, is limited by diffusion of protons within the biofilm. These results provide the first direct electrochemical evidence substantiating utilization of bound ET mediators by Geobacter biofilms and the distinct roles of OmcB and OmcZ in the extracellular ET properties of anode-reducing G. sulfurreducens.

Multilayer superlattices incorporating colloidal metal nanoparticles have been synthesized by an approach based on self‐assembly. Teh method of synthesis is described and the results are presented from X‐ray reflectivity measurements, optical spectroscopy, and various microscopies, showing that they provide a consistent picture of the structure of the resulting multilayer superlattices (see Figure). fig. magnified image

We describe the preparation and characterization of bioinorganic conjugates in which luminescent semiconductor CdSe-ZnS core-shell nanocrystal quantum dots (QDs) were coupled to antibodies through the use of an avidin bridge adsorbed to the nanocrystal surface via electrostatic self-assembly. Avidin, a highly positively charged protein, was found to adsorb tightly to QDs modified with dihydrolipoic acid, which gives their surface a homogeneous negative charge. QD conjugation to biotinylated antibodies subsequently is readily achieved. Fluoroimmunoassays utilizing these antibody conjugated QDs were successful in the detection of protein toxins (staphylococcal enterotoxin B, cholera toxin). QD-antibody conjugates formed in such a facile manner permit their use as a common immuno reagent, and in the development of multianalyte detection.

We have designed and synthesized a series of modular ligands based on poly(ethylene glycol) (PEG) coupled with functional terminal groups to promote water-solubility and biocompatibility of quantum dots (QDs). Each ligand is comprised of three modules: a PEG single chain to promote hydrophilicity, a dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring onto the QD surface, and a potential biological functional group (biotin, carboxyl, and amine) at the other end of the PEG. Water-soluble QDs capped with these functional ligands were prepared via cap exchange with the native hydrophobic caps. Homogeneous QD solutions that are stable over extended periods of time and over a broad pH range were prepared. Surface binding assays and cellular internalization and imaging showed that QDs capped with DHLA-PEG-biotin strongly interacted with either NeutrAvidin immobilized on surfaces or streptavidin coupled to proteins which were subsequently taken up by live cells. EDC coupling in aqueous buffer solutions was also demonstrated using resonance energy transfer between DHLA-PEG-COOH-functionalized QDs and an amine-terminated dye. The new functional surface ligands described here provide not only stable and highly water-soluble QDs but also simple and easy access to various biological entities.

Deep ultraviolet (UV) irradiation is shown to modify organosilane self-assembled monolayer (SAM) films by a photocleavage mechanism, which renders the surface amenable to further SAM modification. Patterned UV exposure creates alternating regions of intact SAM film and hydrophilic, reactive sites. The exposed regions can undergo a second chemisorption reaction to produce an assembly of SAMs in the same molecular plane with similar substrate attachment chemistry. The UV-patterned films are used as a template for selective buildup of fluorophores, metals, and biological cells.

We describe the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe-ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays. The conjugation strategy employs an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies. In this method, the number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed-composition conjugates of the molecular adaptor and a second two-domain protein that allows the use of affinity chromatography. QD-antibody conjugates were successfully used in fluoroimmunoassays for detection of both a protein toxin (staphylococcal enterotoxin B) and a small molecule (2,4,6-trinitrotoluene).

Introns interrupt almost every eukaryotic protein-coding gene, yet how the splicing apparatus interprets the genome during messenger RNA (mRNA) synthesis is poorly understood. We designed microarrays to distinguish spliced from unspliced RNA for each intron-containing yeast gene and measured genomewide effects on splicing caused by loss of 18 different mRNA processing factors. After accommodating changes in transcription and decay by using gene-specific indexes, functional relationships between mRNA processing factors can be identified through their common effects on spliced and unspliced RNA. Groups of genes with different dependencies on mRNA processing factors are also apparent. Quantitative polymerase chain reactions confirm the array-based finding that Prp17p and Prp18p are not dispensable for removal of introns with short branchpoint-to-3' splice site distances.

Charge transport studies across molecular length scales under symmetric and asymmetric metal-molecule contact conditions using a simple crossed-wire tunnel junction technique are presented. It is demonstrated that oligo(phenylene ethynylene), a conjugated organic molecule, acts like a molecular wire under symmetric contact conditions, but exhibits characteristics of a molecular diode when the connections are asymmetric. To understand this behavior, we have calculated current-voltage (I-V) characteristics using extended Huckel theory coupled with a Green's function approach. The experimentally observed I-V characteristics are in excellent qualitative agreement with the theory.

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).

Metal ion complexes are playing an increasing role in the development of antimicrobials. We review here the antimicrobial properties of cobalt coordination complexes in oxidation state 3+. In addition to reviewing the cobalt complexes containing polydentate donor ligands, we also focus on the antimicrobial activity of the homoleptic [Co(NH3)6]3+ ion.

Dissimilatory metal-reducing bacteria (DMRB), such as Geobacter and Shewanella spp., occupy a distinct metabolic niche in which they acquire energy by coupling oxidation of organic fuels with reduction of insoluble extracellular electron acceptors (i.e., minerals). Their unique extracellular electron transfer (EET) capabilities extend to reduction of anodes (electrodes maintained at sufficiently positive potentials) on which they form persistent, electric current generating biofilms. One hypothesis describing the mechanism of EET by Geobacter and Shewanella spp. involves superexchange in which electrons are conducted by a succession of electron transfer reactions among redox proteins associated with the outer cell membranes, aligned along pilus-like filaments (e.g.pili), and/or throughout the extracellular matrix. Here we present theory, previously developed to describe superexchange within abiotic redox polymers, to describe superexchange within DMRB biofilms grown on anodes. We show that this theory appears to apply to recent ex situ measurements of electrical conductivity by individual pilus-like filaments of S. oneidensis MR-1 and G. sulfurreducensDL1, referred to as microbial nanowires. Microbial nanowires have received much recent attention because they are thought by some to impart electrical conductivity to DMRB biofilms and because of the prospect of microbe-produced conductive nanomaterials. We also show that this theory appears to apply to preliminary in situ demonstration of electrical conductivity of an anode-grown G. sulfurreducensDL1 biofilm. Based on these results we suggest a role for nanowires of S. oneidensis and G. sulfurreducens in biofilm conductivity.

ABSTRACT Benthic microbial fuel cells are devices that generate modest levels of electrical power in seafloor environments by a mechanism analogous to the coupled biogeochemical reactions that transfer electrons from organic carbon through redox intermediates to oxygen. Two benthic microbial fuel cells were deployed at a deep‐ocean cold seep within Monterey Canyon, California, and were monitored for 125 days. Their anodes consisted of single graphite rods that were placed within microbial mat patches of the seep, while the cathodes consisted of carbon‐fibre/titanium wire brushes attached to graphite plates suspended ∼0.5 m above the sediment. Power records demonstrated a maximal sustained power density of 34 mW·m −2 of anode surface area, equating to 1100 mW m −2 of seafloor. Molecular phylogenetic analyses of microbial biofilms that formed on the electrode surfaces revealed changes in microbial community composition along the anode as a function of sediment depth and surrounding geochemistry. Near the sediment surface (20–29 cm depth), the anodic biofilm was dominated by micro‐organisms closely related to Desulfuromonas acetoxidans . At horizons 46–55 and 70–76 cm below the sediment–water interface, clone libraries showed more diverse populations, with increasing representation of δ‐proteobacteria such as Desulfocapsa and Syntrophus , as well as ɛ‐proteobacteria. Genes from phylotypes related to Pseudomonas dominated the cathode clone library. These results confound ascribing a single electron transport role performed by only a few members of the microbial community to explain energy harvesting from marine sediments. In addition, the microbial fuel cells exhibited slowly decreasing current attributable to a combination of anode passivation and sulfide mass transport limitation. Electron micrographs of fuel cell anodes and laboratory experiments confirmed that sulfide oxidation products can build up on anode surfaces and impede electron transfer. Thus, while cold seeps have the potential to provide more power than neighbouring ocean sediments, the limits of mass transport as well as the proclivity for passivation must be considered when developing new benthic microbial fuel cell designs to meet specific power requirements.

The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed. It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials. As a model system for study, we use Rhodamine red-labeled engineered variants of Escherichia coli maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core-shell QDs. Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD. In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface. Results indicate that MBP has a preferred orientation on the QD surface. The refined model is in agreement with other evidence, which indicates coordination of the protein to the QD occurs by means of its C-terminal pentahistidine tail, and the size of the QD estimated from the model is in good agreement with physical measurements of QD size. The approach detailed here may be useful in determining the orientation of proteins in other hybrid protein-nanoparticle materials. To our knowledge, this is the first structural model of a hybrid luminescent QD-protein receptor assembly elucidated by using spectroscopic measurements in conjunction with crystallographic and other data.

We present a molecular characterization of metal-affinity driven self-assembly between CdSe−ZnS core−shell quantum dots (QDs) and a series of proteins and peptides appended with various length polyhistidine tags. In particular, we investigated the kinetics of self-assembly between surface-immobilized QDs and proteins/peptides under flow conditions, as well as between freely diffusing QDs and proteins/peptides (solution phase). In the first configuration, QDs were immobilized onto functionalized substrates and then exposed to dye-labeled peptides/proteins. Using evanescent wave excitation, we assessed self-assembly by monitoring the time-dependent changes in the dye fluorescence. In solution, the kinetics of self-assembly was monitored via energy transfer between QDs and dye-labeled proteins/peptides. These measurements allowed determination of the kinetic parameters, including the association and dissociation rates (kon and koff) and the apparent binding constant (Kd). We find that self-assembly is rapid with an equilibrium constant Kd-1 ≈ 1 nM for solution self-assembly, confirming that metal-affinity interactions provide QD bioconjugates that are functional and stable.