Synthetic Porous Melanin.

Commonly known as a skin pigment, melanin has a vital role in UV radiation protection, primarily acting as a radical scavenger. However, a lesser known natural property of melanin, observed in some melanized organisms, is its capacity to adsorb toxins, including metals and organic molecules. Inspired by this, we set out to generate a synthetic porous melanin that would pave the way to enhancing the natural adsorbent properties of melanin and melanin-like materials. Here, we developed a method for the synthesis of porous polydopamine-based melanin utilizing a mesoporous silica (MS) nanoparticle template and characterized its physical properties. Through the oxidative polymerization of dopamine, followed by the etching of silica, we generated synthetic porous melanin (SPM) with the highest measured surface area of any known polydopamine-based material. The prepared SPM was effective for the uptake of various gases and organophosphate toxins, with the material exhibiting high selectivity for CO2 over CH4 and high potential for ammonia capture. Given the demonstrated advantages provided by synthetic porous melanin and melanin's role as an adsorbent in nature, we anticipate the discovery of porous analogues in biological systems.

Allomelanin: A Biopolymer of Intrinsic Microporosity.

Melanin is a ubiquitous natural pigment found in a diverse array of organisms. Allomelanin is a class of nitrogen-free melanin often found in fungi. Herein, we find artificial allomelanin analogues exhibit high intrinsic microporosity and describe an approach for further increasing and tuning that porosity. Notably, the synthetic method involves an oxidative polymerization of 1,8-DHN in water, negating the need for multiple complex templating steps and avoiding expensive or complex chemical precursors. The well-defined morphologies of these nanomaterials were elucidated by a combination of electron microscopy and scattering methods, yielding to high-resolution 3D reconstruction based on small-angle X-ray scattering (SAXS) results. Synthetic allomelanin nanoparticles exhibit high BET areas, up to 860 m2/g, and are capable of ammonia capture up to 17.0 mmol/g at 1 bar. In addition, these nanomaterials can adsorb nerve agent simulants in solution and as a coating on fabrics with high breathability where they prevent breakthrough. We also confirmed that naturally derived fungal melanin can adsorb nerve gas simulants in solution efficiently despite lower porosity than synthetic analogues. Our approach inspires further analysis of yet to be discovered biological materials of this class where melanins with intrinsic microporosity may be linked to evolutionary advantages in relevant organisms and may in turn inspire the design of new high surface area materials.

Development of a Colorimetric Sensor for Autonomous, Networked, Real-Time Application.

This review describes an ongoing effort intended to develop wireless sensor networks for real-time monitoring of airborne targets across a broad area. The goal is to apply the spectrophotometric characteristics of porphyrins and metalloporphyrins in a colorimetric array for detection and discrimination of changes in the chemical composition of environmental air samples. The work includes hardware, software, and firmware design as well as development of algorithms for identification of event occurrence and discrimination of targets. Here, we describe the prototype devices and algorithms related to this effort as well as work directed at selection of indicator arrays for use with the system. Finally, we review the field trials completed with the prototype devices and discuss the outlook for further development.

Field Demonstration of a Distributed Microsensor Network for Chemical Detection.

We have developed the ABEAM-15, a custom-built multiplexed reflectance device for the detection of vapor phase and aerosolized chemical plumes. The instrument incorporates fifteen individual sensing elements, has wireless communications, offers support for a battery pack, and is capable of both live and fully autonomous operation. Two housing options have been fabricated: a compact open housing for indoor use and a larger weather-sealed housing for outdoor use. Previously developed six-plex analysis algorithms are extended to 15-plex format and implemented on a laptop computer. We report the results of recent outdoor field trials with this instrument in Denver, CO in a stadium security scenario. Through software, the wireless modules on each instrument were configured to form a six-instrument, star-point topology, distributed microsensor network with live reporting and real-time data analysis. The network was tested with aerosols of methyl salicylate.

Development of a Detection Algorithm for Use with Reflectance-Based, Real-Time Chemical Sensing.

Here, we describe our efforts focused on development of an algorithm for identification of detection events in a real-time sensing application relying on reporting of color values using commercially available color sensing chips. The effort focuses on the identification of event occurrence, rather than target identification, and utilizes approaches suitable to onboard device incorporation to facilitate portable and autonomous use. The described algorithm first excludes electronic noise generated by the sensor system and determines response thresholds. This automatic adjustment provides the potential for use with device variations as well as accommodating differing indicator behaviors. Multiple signal channels (RGB) as well as multiple indicator array elements are combined for reporting of an event with a minimum of false responses. While the method reported was developed for use with paper-supported porphyrin and metalloporphyrin indicators, it should be equally applicable to other colorimetric indicators. Depending on device configurations, receiver operating characteristic (ROC) sensitivities of 1 could be obtained with specificities of 0.87 (threshold 160 ppb, ethanol).

Proteolytic activity at quantum dot-conjugates: kinetic analysis reveals enhanced enzyme activity and localized interfacial "hopping".

Recent studies show that polyvalent, ligand-modified nanoparticles provide significantly enhanced binding characteristics compared to isolated ligands. Here, we assess the ability of substrate-modified nanoparticles to provide enhanced enzymatic activity. Energy transfer assays allowed quantitative, real-time measurement of proteolytic digestion at polyvalent quantum dot-peptide conjugates. Enzymatic progress curves were analyzed using an integrated Michaelis-Menten (MM) formalism, revealing mechanistic details, including deviations from classic MM-behavior. A "hopping" mode of proteolysis at the nanoparticle was identified, confirming enhanced activity.

Toward in situ monitoring of water contamination by nitroenergetic compounds.

We have previously described the application of novel porous organosilicate materials to the preconcentration of nitroenergetic targets from aqueous solution prior to HPLC analysis. The performance of the sorbents and the advantages of these types of materials over commercially available solid phase extraction sorbents have been demonstrated. Here, the development of systems for application of those sorbents to in situ monitoring is described. Considerations such as column pressure, particulate filtration, and component durability are discussed. The diameter of selected column housings, the sorbent bed depth, and the frits utilized significantly impact the utility of the sorbent columns in the prototype system. The impact of and necessity for improvements in the morphological characteristics of the sorbents as they relate to reduction in column pressure are detailed. The results of experiments utilizing a prototype system are presented. Data demonstrating feasibility for use of the sorbents in preconcentration prior to ion mobility spectrometry is also presented.

Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.

Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid Loligo vulgaris and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 °C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.

Multifunctional compact zwitterionic ligands for preparing robust biocompatible semiconductor quantum dots and gold nanoparticles.

We describe the synthesis of a series of four different ligands which are used to prepare hydrophilic, biocompatible luminescent quantum dots (QDs) and gold nanoparticles (AuNPs). Overall, the ligands are designed to be compact while still imparting a zwitterionic character to the NPs. Ligands are synthesized appended to a bidentate dihydrolipoic acid- (DHLA) anchor group, allowing for high-affinity NP attachment, and simultaneously incorporate tertiary amines along with carboxyl and/or hydroxyl groups. These are placed in close proximity within the ligand structure and their capacity for joint ionization imparts the requisite zwitterionic nature to the nanocrystal. QDs functionalized with the four different compact ligands were subjected to extensive physical characterization including surface charge, wettability, hydrodynamic size, and tolerance to a wide pH range or high salt concentration over time. The utility of the compact ligand coated QDs was further examined by testing of direct conjugation to polyhistidine-appended protein and peptides, aqueous covalent-coupling chemistry, and the ability to engage in Förster resonance energy transfer (FRET). Conjugating cell penetrating peptides to the compact ligand coated QD series facilitated their rapid and efficient cellular uptake, while subsequent cytotoxicity tests showed no apparent decreases in cell viability. In vivo biocompatibility was also demonstrated by microinjecting the compact ligand coated QDs into cells and monitoring their stability over time. Inherent benefits of the ligand design could be extended beyond QDs as AuNPs functionalized with the same compact ligand series showed similar colloidal properties. The strong potential of these ligands to expand NP capabilities in many biological applications is highlighted.

Solid-phase extraction using hierarchical organosilicates for enhanced detection of nitroenergetic targets.

A novel porous organosilicate material was evaluated for application as a solid phase extraction sorbent for preconcentration of nitroenergetic targets from aqueous solution prior to HPLC analysis. The performance of the sorbent in spiked deionized water, groundwater, and surface water was evaluated. Targets considered included 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, RDX, HMX, and nitroglycerin. The sorbent was shown to provide improved performance over Sep-Pak RDX. The impact of complex matrices on target preconcentration by the sorbent was also found to be less dramatic than that observed for LiChrolut EN. The impact of changes in pH on target preconcentration was considered. Aqueous soil extracts generated from samples collected at sites of ordnance testing were also used to evaluate the materials. The results presented here demonstrate the potential of this novel sorbent for application as a solid phase extraction material for the preconcentration of nitroenergetic targets from aqueous solutions.

Porphyrin-embedded silicate materials for detection of hydrocarbon solvents.

The development of porphyrin-embedded mesoporous organosilicate materials for application to the detection of volatile hydrocarbon solvents is described. Design of the receptor and optical indicator construct begins with parallel selection of the porphyrin indicator and design of the mesoporous sorbent. For the porphyrin indicator, high binding affinity and strong changes in spectrophotometric character upon target interaction are desired. The sorbent should provide high target binding capacity and rapid binding kinetics. A number of porphyrin/metalloporphyrin variants and organosilicate sorbents were evaluated to determine the characteristics of their interaction with the targets, benzene, toluene, and hexane. The selected porphyrin candidates were covalently immobilized within a benzene-bridged sorbent. This construct was applied to the detection of targets using both fluorescence- and reflectance-based protocols. The use of red, green, and blue (RGB) color values from the constructs in a highly simplified detection scheme is described.

Bile acids stimulate ATP hydrolysis in the purified cholesterol transporter ABCG5/G8.

ABCG5 and ABCG8 are half-size ABC transporters that function as heterodimers (ABCG5/G8) to reduce sterol absorption in the intestines and increase sterol excretion from the liver. Previous studies demonstrated that bile acids increased ABCG5/G8 specific cholesterol efflux in cell models. In this study we tested the effects of bile acids on ATP hydrolysis in Pichia pastoris purified ABCG5/G8 and found that they stimulated hydrolysis approximately 20-fold in wild-type ABCG5/G8 but not in a hydrolysis-deficient mutant. Nonconjugated cholate supported the highest ATPase activity in ABCG5/G8 (256 +/- 9 nmol min(-1) mg(-1)). ATP hydrolysis was also stimulated by other conjugated bile acids and a mixture of bile acids resembling human bile with activities ranging from 129 +/- 4 to 147 +/- 14 nmol min(-1) mg(-1). The kinetic parameters, inhibitor profiles, and lipid requirements of bile acid stimulated ATP hydrolysis were characterized. Cholate-stimulated ATP hydrolysis was maximal at concentrations of >or=10 mM MgATP and had a relatively high K(M) (MgATP) of approximately 1 mM. Orthovanadate, BeFx, and AlFx effectively inhibited ABCG5/G8 at concentrations of 1 mM. Various lipid mixtures supported bile acid-stimulated ATP hydrolysis, which increased when cholesterol was present. The data demonstrate that bile acids together with lipids and cholesterol increase ATP hydrolysis in purified ABCG5/G8. Bile acids may promote an active conformation of purified ABCG5/G8 either by global stabilization of the transporter or by binding to a specific site on ABCG5/G8.

Mesoporous materials in sensing: morphology and functionality at the meso-interface.

Mesoporous materials are finding increasing utility in sensing applications. These applications can benefit from a surface area that may exceed 1,000 m(2) g(-1) and fast diffusion of analytes through a porous structure. This article reviews recent developments in mesoporous materials-based sensing and provides examples of the impact of different surface functionality, pore structure, and macro-morphology in an attempt to illustrate the contribution of these factors to the selectivity and sensitivity of a sensor response. The materials discussed include ordered mesoporous silicates synthesized with surfactants, hard templated ordered mesoporous carbons, and metal oxides with porous textures which have been applied to advantage in various detection schemes. Chemical functionalization of mesoporous materials through silane grafting, co-condensation, and adsorption are also addressed.

Fluorescent silicate materials for the detection of paraoxon.

Porphyrins are a family of highly conjugated molecules that strongly absorb visible light and fluoresce intensely. These molecules are sensitive to changes in their immediate environment and have been widely described for optical detection applications. Surfactant-templated organosilicate materials have been described for the semi-selective adsorption of small molecule contaminants. These structures offer high surface areas and large pore volumes within an organized framework. The organic bridging groups in the materials can be altered to provide varied binding characteristics. This effort seeks to utilize the tunable binding selectivity, high surface area, and low materials density of these highly ordered pore networks and to combine them with the unique spectrophotometric properties of porphyrins. In the porphyrin-embedded materials (PEMs), the organosilicate scaffold stabilizes the porphyrin and facilitates optimal orientation of porphyrin and target. The materials can be stored under ambient conditions and offer exceptional shelf-life. Here, we report on the design of PEMs with specificity for organophosphates and compounds of similar structure.

Impact of cranberry on Escherichia coli cellular surface characteristics.

The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

Mesoporous Silicate Materials in Sensing.

Mesoporous silicas, especially those exhibiting ordered pore systems and uniform pore diameters, have shown great potential for sensing applications in recent years. Morphological control grants them versatility in the method of deployment whether as bulk powders, monoliths, thin films, or embedded in coatings. High surface areas and pore sizes greater than 2 nm make them effective as adsorbent coatings for humidity sensors. The pore networks also provide the potential for immobilization of enzymes within the materials. Functionalization of materials by silane grafting or through cocondensation of silicate precursors can be used to provide mesoporous materials with a variety of fluorescent probes as well as surface properties that aid in selective detection of specific analytes. This review will illustrate how mesoporous silicas have been applied to sensing changes in relative humidity, changes in pH, metal cations, toxic industrial compounds, volatile organic compounds, small molecules and ions, nitroenergetic compounds, and biologically relevant molecules.

Deposition of Porous Sorbents on Fabric Supports.

A microwave deposition technique for silanes, previously described for production of oleophobic fabrics, is adapted to provide a fabric support material that can be subsequently treated by dip coating. Dip coating with a sol preparation provides a supported porous layer on the fabric. In this case, the porous layer is a porphyrin functionalized sorbent system based on a powdered material that has been demonstrated previously for the capture and conversion of phosgene. A representative coating is applied to cotton fabric at a loading level of 10 mg/g. This coating has minimal impact on water vapor transport through the fabric (93% of the support fabric rate) while significantly reducing transport of 2-chloroethyl ethyl sulfide (CEES) through the material (7% of support fabric rate). The described approaches are suitable for use with other fabrics providing amine and hydroxyl groups for modification and can be used in combination with other sol preparations to produce varying functionality.

Environmental Decontamination of a Chemical Warfare Simulant Utilizing a Membrane Vesicle-Encapsulated Phosphotriesterase.

While technologies for the remediation of chemical contaminants continue to emerge, growing interest in green technologies has led researchers to explore natural catalytic mechanisms derived from microbial species. One such method, enzymatic degradation, offers an alternative to harsh chemical catalysts and resins. Recombinant enzymes, however, are often too labile or show limited activity when challenged with nonideal environmental conditions that may vary in salinity, pH, or other physical properties. Here, we demonstrate how phosphotriesterase encapsulated in a bacterial outer membrane vesicle can be used to degrade the organophosphate chemical warfare agent (CWA) simulant paraoxon in environmental water samples. We also carried out remediation assays on solid surfaces, including glass, painted metal, and fabric, that were selected as representative materials, which could potentially be contaminated with a CWA.

Development of a Genetic System for Marinobacter atlanticus CP1 (sp. nov.), a Wax Ester Producing Strain Isolated From an Autotrophic Biocathode.

Here, we report on the development of a genetic system for Marinobacter sp. strain CP1, previously isolated from the Biocathode MCL community and shown to oxidize iron and grow as a cathodic biofilm. Sequence analysis of the small and large subunits of the 16S rRNA gene of CP1, as well as comparison of select conserved proteins, indicate that it is most closely related to Marinobacter adhaerens HP15 and Marinobacter sp. ES.042. In silico DNA-DNA hybridization using the genome-to-genome distance calculator (GGDC) predicts CP1 to be a new species of Marinobacter described here as Marinobacter atlanticus. CP1 is competent for transformation with plasmid DNA using conjugation with Escherichia coli donor strain WM3064 and constitutive expression of green fluorescent protein (GFP) is stable in the absence of antibiotic selection. Targeted double deletion mutagenesis of homologs for the M. aquaeoli fatty acyl-CoA reductase (acrB) and fatty aldehyde reductase (farA) genes resulted in a loss of production of wax esters; however, single deletion mutants for either gene resulted in an increase in total wax esters recovered. Genetic tools presented here for CP1 will enable further exploration of wax ester synthesis for biotechnological applications, as well as furthering our efforts to understand the role of CP1 within the Biocathode MCL community.

Improving Sorbents for Glycerol Capture in Biodiesel Refinement.

Biodiesel is produced by transesterification of animal fat, vegetable oil, or waste cooking oil with alcohol. After production costs, the economic viability of biodiesel is dependent on what steps are necessary to remove impurities following synthesis and the effectiveness of quality control analysis. Solid-phase extraction offers a potentially advantageous approach in biodiesel processing applications. Nanoporous scaffolds were investigated for adsorption of glycerol, a side product of biodiesel synthesis that is detrimental to engine combustion when present. Materials were synthesized with varying pore wall composition, including ethane and diethylbenzene bridging groups, and sulfonated to promote hydrogen bonding interactions with glycerol. Materials bearing sulfonate groups throughout the scaffold walls as well as those post-synthetically grafted onto the surfaces show notably superior performance for uptake of glycerol. The sorbents are effective when used in biodiesel mixtures, removing greater than 90% of glycerol from a biodiesel preparation.