Dual-Functionalized Virus-Gold Nanoparticle Clusters for Biosensing.

Metallic nanoscale 3D architectures concentrate electromagnetic energy at precise spatial locations to enable sensing and photocatalysis applications. We have developed solution-based methods to reproducibly fabricate 3D gold nanostructures useful as efficient surface-enhanced Raman spectroscopy (SERS) biosensors. Virus capsids were recruited as templates to assemble gold nanoparticles on their surfaces at well-defined locations to prepare the nanoscale 3D structures. Cowpea mosaic virus (CPMV) and its variants were selected as specific templates due to their high symmetry, scalability, and stability, which have proven useful in materials science applications. While the methods described herein were optimized for the CPMV capsids, they also provide a useful starting point for researchers who are working toward the nanoassembly of metal nanoparticles on other protein scaffolds.

A virus-based nanoplasmonic structure as a surface-enhanced Raman biosensor.

Fabrication of nanoscale structures with localized surface plasmons allows for substantial increase in sensitivity of chem/bio sensors. The main challenge for realizing complex nanoplasmonic structures in solution is the high level of precision required at the nanoscale to position metal nanoparticles in 3D. In this study, we report a virus-like particle (VLP) for building a 3D plasmonic nanostructure in solution in which gold nanoparticles are precisely positioned on the VLP by directed self-assembly techniques. These structures allow for concentration of electromagnetic fields in the desired locations between the gold nanoparticles or "hot spots". We measure the efficiency of the optical field spatial concentration for the first time, which results in a ten-fold enhancement of the capsid Raman peaks. Our experimental results agree with our 3D finite element simulations. Furthermore, we demonstrate as a proof-of-principle that the plasmonic nanostructures can be utilized in DNA detection down to 0.25 ng/µl (lowest concentration tested), while the protein peaks from the interior of the nanoplasmonic structures, potentially, can serve as an internal tracer for the biosensors.

Kinetic analysis of the hydrolysis of methyl parathion using citrate-stabilized 10 nm gold nanoparticles.

"Ligand-free" citrate-stabilized 10 nm gold nanoparticles (AuNPs) promote the hydrolysis of the thiophosphate ester methyl parathion (MeP) on the surface of gold as a function of pH and two temperature values. At 50 °C, the active surface gold atoms show catalytic turnover ∼4 times after 8 h and little turnover of gold surface atoms at 25 °C with only 40% of the total atoms being active. From Michaelis-Menten analysis, k(cat) increases between pH 8 and 9 and decreases above pH 9. A global analysis of the spectral changes confirmed the stoichiometric reaction at 25 °C and the catalytic reaction at 50 °C and mass spectrometry confirmed the identity of p-nitrophenolate (PNP) product. Additional decomposition pathways involving oxidation and hydrolysis independent of the formation of PNP were also seen at 50 °C for both catalyzed and un-catalyzed reactions. This work represents the first kinetic analysis of ligand-free AuNP catalyzed hydrolysis of a thiophosphate ester.

Differences in Physical and Biochemical Properties of Thermus scotoductus SA-01 Cultured with Dielectric or Convection Heating.

A thermophile, Thermus scotoductus SA-01, was cultured within a constant-temperature (65°C) microwave (MW) digester to determine if MW-specific effects influenced the growth and physiology of the organism. As a control, T. scotoductus cells were also cultured using convection heating at the same temperature as the MW studies. Cell growth was analyzed by optical density (OD) measurements, and cell morphologies were characterized using electron microscopy imaging (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]), dynamic light scattering (DLS), and atomic force microscopy (AFM). Biophysical properties (i.e., turgor pressure) were also calculated with AFM, and biochemical compositions (i.e., proteins, nucleic acids, fatty acids) were analyzed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the fatty acid methyl esters extracted from cell membranes. Here we report successful cultivation of a thermophile with only dielectric heating. Under the MW conditions for growth, cell walls remained intact and there were no indications of membrane damage or cell leakage. Results from these studies also demonstrated that T. scotoductus cells grown with MW heating exhibited accelerated growth rates in addition to altered cell morphologies and biochemical compositions compared with oven-grown cells.

Suppressing the Neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors.

Neurospora crassa has been utilized as a model organism for studying biological, regulatory, and circadian rhythms for over 50 years. These circadian cycles are driven at the molecular level by gene transcription events to prepare for environmental changes. N. crassa is typically found on woody biomass and is commonly studied on agar-containing medium which mimics its natural environment. We report a novel method for disrupting circadian gene transcription while maintaining light responsiveness in N. crassa when held in a steady metabolic state using bioreactors. The arrhythmic transcription of core circadian genes and downstream clock-controlled genes was observed in constant darkness (DD) as determined by reverse transcription-quantitative PCR (RT-qPCR). Nearly all core circadian clock genes were up-regulated upon exposure to light during 11hr light/dark cycle experiments under identical conditions. Our results demonstrate that the natural timing of the robust circadian clock in N. crassa can be disrupted in the dark when maintained in a consistent metabolic state. Thus, these data lead to a path for the production of industrial scale enzymes in the model system, N. crassa, by removing the endogenous negative feedback regulation by the circadian oscillator.

Preparation and layer-by-layer solution deposition of Cu(In,Ga)O2 nanoparticles with conversion to Cu(In,Ga)S2 films.

We present a method of Cu(In,Ga)S2 (CIGS) thin film formation via conversion of layer-by-layer (LbL) assembled Cu-In-Ga oxide (CIGO) nanoparticles and polyelectrolytes. CIGO nanoparticles were created via a novel flame-spray pyrolysis method using metal nitrate precursors, subsequently coated with polyallylamine (PAH), and dispersed in aqueous solution. Multilayer films were assembled by alternately dipping quartz, Si, and/or Mo substrates into a solution of either polydopamine (PDA) or polystyrenesulfonate (PSS) and then in the CIGO-PAH dispersion to fabricate films as thick as 1-2 microns. PSS/CIGO-PAH films were found to be inadequate due to weak adhesion to the Si and Mo substrates, excessive particle diffusion during sulfurization, and mechanical softness ill-suited to further processing. PDA/CIGO-PAH films, in contrast, were more mechanically robust and more tolerant of high temperature processing. After LbL deposition, films were oxidized to remove polymer and sulfurized at high temperature under flowing hydrogen sulfide to convert CIGO to CIGS. Complete film conversion from the oxide to the sulfide is confirmed by X-ray diffraction characterization.

Virus-templated plasmonic nanoclusters with icosahedral symmetry via directed self-assembly.

The assembly of plasmonic nanoparticles with precise spatial and orientational order may lead to structures with new electromagnetic properties at optical frequencies. The directed self-assembly method presented controls the interparticle-spacing and symmetry of the resulting nanometer-sized elements in solution. The self-assembly of three-dimensional (3D), icosahedral plasmonic nanosclusters (NCs) with resonances at visible wavelengths is demonstrated experimentally. The ideal NCs consist of twelve gold (Au) nanospheres (NSs) attached to thiol groups at predefined locations on the surface of a genetically engineered cowpea mosaic virus with icosahedral symmetry. In situ dynamic light scattering (DLS) measurements confirm the NSs assembly on the virus. Transmission electron micrographs (TEM) demonstrate the ability of the self-assembly method to control the nanoscopic symmetry of the bound NSs, which reflects the icosahedral symmetry of the virus. Both, TEM and DLS show that the NCs comprise of a distribution of capsids mostly covered (i.e., 6-12 NS/capsid) with NSs. 3D finite-element simulations of aqueous suspensions of NCs reproduce the experimental bulk absorbance measurements and major features of the spectra. Simulations results show that the fully assembled NCs give rise to a 10-fold surface-averaged enhancement of the local electromagnetic field.

A programmable fluorescent viral nanoblock: sensing made easy in a single step.

Viral nanoblock provides specific positioning of recognition moieties and dye molecules which can be used as a signal-generating element and enhance detection sensitivity. The methods described herein use a 30 nm viral nanoblock to couple a variety of proteins and peptides for the incorporation of recognition elements along with a large number of dye molecules (200). The bioconjugation techniques were adapted and optimized over the years to fabricate nanoparticles that exhibit high fluorescence output while maintaining the selectivity of the target receptors. These complexes can be used for the detection of pathogens and toxins in a single step since both receptor and reporter are in the same viral nanoblock. Its stability and nanometer size allows for its utilization in well-established sensing platforms like microarrays.

Shewanella frigidimarina microbial fuel cells and the influence of divalent cations on current output.

The genes involved in the proposed pathway for Shewanella extracellular electron transfer (EET) are highly conserved. While extensive studies involving EET from a fresh water Shewanella microbe (S. oneidensis MR-1) to soluble and insoluble electron acceptors have been published, only a few reports have examined EET from marine strains of Shewanella. Thus, Shewanella frigidimarina (an isolate from Antarctic Sea ice) was used within miniature microbial fuel cells (mini-MFC) to evaluate potential power output. During the course of this study several distinct differences were observed between S. oneidensis MR-1 and S. frigidimarina under comparable conditions. The maximum power density with S. frigidimarina was observed when the anolyte was half-strength marine broth (1/2 MB) (0.28 µW/cm(2)) compared to Luria-Bertani (LB) (0.07 µW/cm(2)) or a defined growth minimal medium (MM) (0.02 µW/cm(2)). The systematic modification of S. frigidimarina cultured in 1/2 MB and LB with divalent cations shows that a maximum current output can be generated independent of internal ionic ohmic losses and the presence of external mediators.

Adaptation of the black yeast Wangiella dermatitidis to ionizing radiation: molecular and cellular mechanisms.

Observations of enhanced growth of melanized fungi under low-dose ionizing radiation in the laboratory and in the damaged Chernobyl nuclear reactor suggest they have adapted the ability to survive or even benefit from exposure to ionizing radiation. However, the cellular and molecular mechanism of fungal responses to such radiation remains poorly understood. Using the black yeast Wangiella dermatitidis as a model, we confirmed that ionizing radiation enhanced cell growth by increasing cell division and cell size. Using RNA-seq technology, we compared the transcriptomic profiles of the wild type and the melanin-deficient wdpks1 mutant under irradiation and non-irradiation conditions. It was found that more than 3000 genes were differentially expressed when these two strains were constantly exposed to a low dose of ionizing radiation and that half were regulated at least two fold in either direction. Functional analysis indicated that many genes for amino acid and carbohydrate metabolism and cell cycle progression were down-regulated and that a number of antioxidant genes and genes affecting membrane fluidity were up-regulated in both irradiated strains. However, the expression of ribosomal biogenesis genes was significantly up-regulated in the irradiated wild-type strain but not in the irradiated wdpks1 mutant, implying that melanin might help to contribute radiation energy for protein translation. Furthermore, we demonstrated that long-term exposure to low doses of radiation significantly increased survivability of both the wild-type and the wdpks1 mutant, which was correlated with reduced levels of reactive oxygen species (ROS), increased production of carotenoid and induced expression of genes encoding translesion DNA synthesis. Our results represent the first functional genomic study of how melanized fungal cells respond to low dose ionizing radiation and provide clues for the identification of biological processes, molecular pathways and individual genes regulated by radiation.

Function and regulation of Vibrio campbellii proteorhodopsin: acquired phototrophy in a classical organoheterotroph.

Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii. Pigmentation analysis, absorption spectroscopy and photoinduction assays using a heterologous over-expression system established the V. campbellii PR as a functional green light absorbing proton pump. In situ analyses comparing PR expression and function in wild type (WT) V. campbellii with an isogenic ΔpR deletion mutant revealed a marked absence of PR membrane localization, pigmentation and light-induced proton pumping in the ΔpR mutant. Comparative photoinduction assays demonstrated the distinct upregulation of pR expression in the presence of light and PR-mediated photophosphorylation in WT cells that resulted in the enhancement of cellular survival during respiratory stress. In addition, we demonstrate that the master regulator of adaptive stress response and stationary phase, RpoS1, positively regulates pR expression and PR holoprotein pigmentation. Taken together, the results demonstrate facultative phototrophy in a classical marine organoheterotrophic Vibrio species and provide a salient example of how this organism has exploited lateral gene transfer to further its adaptation to the photic zone.

Biotemplating rod-like viruses for the synthesis of copper nanorods and nanowires.

BACKGROUND: In the past decade spherical and rod-like viruses have been used for the design and synthesis of new kind of nanomaterials with unique chemical positioning, shape, and dimensions in the nanosize regime. Wild type and genetic engineered viruses have served as excellent templates and scaffolds for the synthesis of hybrid materials with unique properties imparted by the incorporation of biological and organic moieties and inorganic nanoparticles. Although great advances have been accomplished, still there is a broad interest in developing reaction conditions suitable for biological templates while not limiting the material property of the product. RESULTS: We demonstrate the controlled synthesis of copper nanorods and nanowires by electroless deposition of Cu on three types of Pd-activated rod-like viruses. Our aqueous solution-based method is scalable and versatile for biotemplating, resulting in Cu-nanorods 24-46 nm in diameter as measured by transmission electron microscopy. Cu2+ was chemically reduced onto Pd activated tobacco mosaic virus, fd and M13 bacteriophages to produce a complete and uniform Cu coverage. The Cu coating was a combination of Cu0 and Cu2O as determined by X- ray photoelectron spectroscopy analysis. A capping agent, synthesized in house, was used to disperse Cu-nanorods in aqueous and organic solvents. Likewise, reactions were developed to produce Cu-nanowires by metallization of polyaniline-coated tobacco mosaic virus. CONCLUSIONS: Synthesis conditions described in the current work are scalable and amenable for biological templates. The synthesized structures preserve the dimensions and shape of the rod-like viruses utilized during the study. The current work opens the possibility of generating a variety of nanorods and nanowires of different lengths ranging from 300 nm to micron sizes. Such biological-based materials may find ample use in nanoelectronics, sensing, and cancer therapy.

Aggrandizing power output from Shewanella oneidensis MR-1 microbial fuel cells using calcium chloride.

There are several interconnected metabolic pathways in bacteria essential for the conversion of carbon electron sources directly into electrical currents using microbial fuel cells (MFCs). This study establishes a direct exogenous method to increase power output from a Shewanella oneidensis MR-1 containing MFC by adding calcium chloride to the culture medium. The current output from each CaCl(2) concentration tested revealed that the addition of CaCl(2) to 1400 µM increased the current density by >80% (0.95-1.76 µA/cm(2)) using sodium lactate as the sole carbon source. Furthermore, polarization curves showed that the maximum power output could be increased from 157 to 330 µW with the addition of 2080 µM CaCl(2). Since the conductivity of the culture medium did not change after the addition of CaCl(2) (confirmed by EIS and bulk conductivity measurements), this increase in power was primarily biological and not based on ionic effects. Thus, controlling the concentration of CaCl(2) is a pathway to increase the efficiency and performance of S. oneidensis MR-1 MFCs.

Role of hexahistidine in directed nanoassemblies of tobacco mosaic virus coat protein.

A common challenge in nanotechnology is the fabrication of materials with well-defined nanoscale structure and properties. Here we report that a genetically engineered tobacco mosaic virus (TMV) coat protein (CP), to which a hexahistidine (His) tag was incorporated, can self-assemble into disks, hexagonally packed arrays of disks, stacked disks, helical rods, fibers, and elongated rafts. The insertion of a His tag to the C-terminus of TMV-CP was shown to significantly affect the self-assembly in comparison to the wild type, WT-TMV-CP. Furthermore, the His tag interactions attributed to the alternative self-assembly of His-TMV-CP can be controlled through ethanol and nickel-nitrilotriacetic acid (Ni-NTA) additions as monitored with atomic force microscopy.

Engineered T4 viral nanoparticles for cellular imaging and flow cytometry.

Viruses are of particular interest as scaffolds for biotechnology applications given their wide range of shapes and sizes and the possibility to modify them with a variety of functional moieties to produce useful virus-based nanoparticles (VNPs). In order to develop functional VNPs for cell imaging and flow cytometry applications, we used the head of the T4 bacteriophage as a scaffold for bioconjugation of fluorescent dyes. Bacteriophage T4 is a double-stranded DNA virus with an elongated icosahedron head and a contractile tail. The head is ∼100 nm in length and ∼90 nm in width. The large surface area of the T4 head is an important advantage for the development of functional materials since it can accommodate significantly larger numbers of functional groups, such as fluorescent dyes, in comparison with other VNPs. In this study, Cy3 and Alexa Fluor 546 were chemically incorporated into tail-less T4 heads (T4 nanoparticles) for the first time, and the fluorescent properties of the dye-conjugated nanoparticles were characterized. The T4 nanoparticles were labeled with up to 19 000 dyes, and in particular, the use of Cy3 led to fluorescent enhancements of up to 90% compared to free Cy3. We also demonstrate that the dye-conjugated T4 nanoparticles are structurally stable and that they can be used as molecular probes for cell imaging and flow cytometry applications.

Molecular electronics based nanosensors on a viral scaffold.

Assembling and interconnecting the building blocks of nanoscale devices and being able to electronically address or measure responses at the molecular level remains an important challenge for nanotechnology. Here we show the usefulness of bottom-up self-assembly for building electronic nanosensors from multiple components that have been designed to interact in a controlled manner. Cowpea mosaic virus was used as a scaffold to control the positions of gold nanoparticles. The nanoparticles were then interconnected using thiol-terminated conjugated organic molecules, resulting in a three-dimensional conductive network. Biotin molecules were attached to the virus scaffold using linkers to act as molecular receptors. We demonstrated that binding avidin to the biotin receptors on the self-assembled nanosensors causes a significant change in the network conductance that is dependent on the charge of the avidin protein.

Virus hybrids as nanomaterials for biotechnology.

The current review describes advances in the field of bionanotechnology in which viruses are used to fabricate nanomaterials. Viruses are introduced as protein cages, scaffolds, and templates for the production of biohybrid nanostructured materials where organic and inorganic molecules are incorporated in a precise and a controlled fashion. Genetic engineering enables the insertion or replacement of selected amino acids on virus capsids for uses from bioconjugation to crystal growth. The variety of nanomaterials generated in rod-like and spherical viruses is highlighted for tobacco mosaic virus (TMV), M13 bacteriophage, cowpea chlorotic mottle virus (CCMV), and cowpea mosaic virus (CPMV). Functional biohybrid nanomaterials find applications in biosensing, memory devices, nanocircuits, light-harvesting systems, and nanobatteries.

Cowpea mosaic virus nanoscaffold as signal enhancement for DNA microarrays.

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.

Reduction of Non-Specific Protein Adsorption Using Poly(ethylene) Glycol (PEG) Modified Polyacrylate Hydrogels In Immunoassays for Staphylococcal Enterotoxin B Detection.

Three PEG molecules (PEG-methacrylate, -diacrylate and -dimethacrylate) were incorporated into galactose-based polyacrylate hydrogels and their relative abilities to reduce non-specific protein adsorption in immunoassays were determined. Highly crosslinked hydrogels containing amine-terminated functionalities were formed and used to covalently attach antibodies specific for staphylococcal enterotoxin B (SEB). Patterned arrays of immobilized antibodies in the PEG-modified hydrogels were created with a PDMS template containing micro-channels for use in sandwich immunoassays to detect SEB. Different concentrations of the toxin were applied to the hydrogel arrays, followed with a Cy3-labeled tracer antibody specific for the two toxins. Fluorescence laser scanning confocal microscopy of the tracer molecules provided both qualitative and quantitative measurements on the detection sensitivity and the reduction in non-specific binding as a result of PEG incorporation. Results showed the PEG-modified hydrogel significantly reduced non-specific protein binding with a detection limit for SEB of 1 ng/mL. Fluorescence signals showed a 10-fold decrease in the non-specific binding and a 6-fold increase in specific binding of SEB.