The development of a silica nanoparticle-based label-free DNA biosensor

A silica nanoparticle-based DNA biosensor capable of detecting Bacillus anthracis bacteria through the use of unlabelled ss-oligonucleotides has been developed. The biosensor makes use of the optical changes that accompany a nanoparticle-immobilized cationic conjugated polymer (polythiophene) interacting with single-stranded vs. hybridized oligonucleotides, where a fluorescence signal appears only when hybridized DNA is present (i.e. only when the ss-oligonucleotide interacting with the polymer has hybridized with its complement). In order to enhance the sensitivity of the biosensor, two different nanoparticle architectures were developed and used to elucidate how the presence of neighboring fluorophores on the nanoparticle surface affects F?rster-resonant energy transfer (FRET) between the polythiophene/oligonucleotide complex (FRET donor) and the fluorophores (FRET acceptors). We demonstrate that the silica nanoparticle-based FRET platform lowers the limit of detection at least 10-fold in comparison to the polythiophene itself, and allows the detection of ～2 × 10(-12) moles of ss-oligonucleotide in a 100 μL sample with a standard fluorimeter (i.e. has a limit of detection of ～2 nM ssDNA). Such nanoparticle-based biosensor platforms are beneficial because of the robustness and stability inherent to their covalent assembly and they provide a valuable new tool that may allow for the sensitive, label-free detection (the target DNA that produces the fluorescence signal is unlabelled) without the use of polymerase chain reaction.     

Effect of chain rigidity on block copolymer micelle formation and dissolution as observed by 1H-NMR spectroscopy

Amphiphilic copolymers poly(methyl methacrylate-b-acrylic acid), poly(methyl methacrylate-b-methacrylic acid), poly(methyl acrylate-b-acrylic acid) and poly(methyl acrylate-b-methacrylic acid) were prepared by reversible addition fragmentation chain-transfer (RAFT) polymerization. The hydrophilic polyacid blocks were either synthesized directly or formed by the hydrolysis of poly(tert-butyl acrylate) or poly(tert-butyl methacrylate) blocks. The hydrophobic blocks consisted of either the more rigid, high glass transition temperature (T _g ) poly(methyl methacrylate) or more flexible, low T _g poly(methyl acrylate) material. The hydrophilic blocks were either poly(methacrylic acid) (rigid, high T _g ) or poly(acrylic acid) (flexible, low T _g ). The micellization behavior of the polymers was studied by proton nuclear magnetic resonance (¹H-NMR) spectroscopy in mixtures of 1,4-dioxane-d₈ and D₂O. All four polymers were soluble in neat dioxane. In solutions of higher water content, the polymers with the more rigid hydrophobic blocks formed into micelles as was evidenced by broadening of the resonances resulting from the protons in those blocks. At moderate water concentration (25–50%), dissolution of the micelles was observed upon heating the solution. No micellization was observed in polymers containing the less rigid poly(methyl acrylate) hydrophobic block regardless of the identity of the hydrophilic block. As further evidence of micellization formation and dissolution, the spin-lattice (T ₁) and spin-spin (T ₂) relaxation times of protons in the hydrophobic and hydrophilic blocks were measured. Significant differences in the relaxation times as functions of temperature and solvent concentration were observed between the hydrophilic and hydrophobic blocks of the micelle-forming polymers.     

Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution

We have used directed evolution methods to express a fungalenzyme, galactose oxidase (GOase), in functional form in Escherichiacoli. The evolved enzymes retain the activity and substratespecificity of the native fungal oxidase, but are more thermostable,are expressed at a much higher level (up to 10.8 mg/l of purifiedGOase), and have reduced negative charge compared to wild type,all properties which are expected to facilitate applicationsand further evolution of the enzyme. Spectroscopic characterizationof the recombinant enzymes reveals a tyrosyl radical of comparablestability to the native GOase from Fusarium.     

An organically modified zirconate film as a corrosion-resistant treatment for aluminum 2024-T3

Current coating systems for aircraft corrosion protection are based on a traditional chromate surface treatment, primer, and topcoat. The Air Force is seeking environmentally benign corrosion-resistant surface treatments for aluminum-skinned aircraft as a replacement for environmentally hazardous surface treatments involving chromates. Performance of replacement treatments must be able to satisfy the durability needed for dramatically extended lifetimes, be compatible with present and future environmental requirements, and be easily integrated into current primer/topcoat paint systems.

Organically modified zirconate sol–gel films were investigated as an environmentally compliant replacement for chromated surface treatments, which included functionalized components to tailor the chemistry at both the aluminum oxide substrate/surface treatment interface and the surface treatment/topcoat interface. Sol–gel films were applied to aluminum 2024-T3 alloy coupons, and the resulting film chemistry was investigated. FTIR was used to identify organic components in the film and X-ray photoelectron spectroscopy was used to investigate the interface chemistry. The result of the chemically modified sol–gel synthesis was a coating in which a concentration gradient was formed at the surface, which is discussed. The corrosion protection attributes of these films was also studied and the electrochemical evaluation of sol–gel films will be discussed, both as stand-alone coatings and as part of a full coating system.

Organically modified sol–gels exhibited significantly better protection in terms of barrier properties in comparison to a typical chromate-based processes. The resulting data from evaluations of sol–gel produced coatings show promise towards the goal of producing a robust chemical interaction/bonding of such corrosion-resistant coatings on the surface of aluminum-skinned aircraft without the use of environmentally hazardous chromate agents.     

Synthesis of magnetic nanocomposite microparticles for binding of chlorinated organics in contaminated water sources

In this work, the development of novel magnetic nanocomposite microparticles (MNMs) via free radical polymerization for their application in the remediation of contaminated water is presented. Acrylated plant-based polyphenols, curcumin multiacrylate (CMA) and quercetin multiacrylate (QMA), were incorporated as functional monomers to create high affinity binding sites for the capture of polychlorinated biphenyls (PCBs), as a model pollutant. The MNMs were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, dynamic light scattering, and UV–visible spectroscopy. The affinity of these novel materials for PCB 126 was evaluated and fitted to the nonlinear Langmuir model to determine binding affinities (K_D). The results suggest the presence of the polyphenolic moieties enhances the binding affinity for PCB 126, with K_D values comparable to that of antibodies. This demonstrates that these nanocomposite materials have promising potential as environmental remediation adsorbents for harmful contaminants.     

CuI and H2O2 Inactivate and FeII Inhibits [Fe]‐Hydrogenase at Very Low Concentrations

[Fe]‐Hydrogenase (Hmd) catalyzes reversible hydride transfer from H₂. It harbors an iron‐guanylylpyridinol as a cofactor with an Fe^II that is ligated to one thiolate, two COs, one acyl‐C, one pyridinol‐N, and solvent. Here, we report that Cu^I and H₂O₂ inactivate Hmd (half‐maximal rates at 1 μM Cu^I and 20 μM H₂O₂) and that Fe^II inhibits the enzyme with very high affinity (K_i=40 nM ). Infrared and EPR studies together with competitive inhibition studies with isocyanide indicated that Cu^I exerts its inhibitory effect most probably by binding to the active site iron‐thiolate ligand. Using the same methods, it was found that H₂O₂ binds to the active‐site iron at the solvent‐binding site and oxidizes Fe^II to Fe^III. Also it was shown that Fe^II reversibly binds away from the active site iron, with binding being competitive to the organic hydride acceptor; this inhibition is specific for Fe^II and is reminiscent of that for the [FeFe]‐hydrogenase second iron, which specifically interacts with H₂.     

Cloning and heterologous expression of three type II PKS gene clusters from Streptomyces bottropensis

Mensacarcin is a potent cytotoxic agent isolated from Streptomyces bottropensis. It possesses a high content of oxygen atoms and two epoxide groups, and shows cytostatic and cytotoxic activity comparable to that of doxorubicin, a widely used drug for antitumor therapy. Another natural compound, rishirilide A, was also isolated from the fermentation broth of S. bottropensis. Screening a cosmid library of S. bottropensis with minimal PKS-gene-specific primers revealed the presence of three different type II polyketide synthase (PKS) gene clusters in this strain: the msn cluster (mensacarcin biosynthesis), the rsl cluster (rishirilide biosynthesis), and the mec cluster (putative spore pigment biosynthesis). Interestingly, luciferase-like oxygenases, which are very rare in Streptomyces species, are enriched in both the msn cluster and the rsl cluster. Three cosmids, cos2 (containing the major part of the msn cluster), cos3 (harboring the mec cluster), and cos4 (spanning probably the whole rsl cluster) were introduced into the general heterologous host Streptomyces albus by intergeneric conjugation. Expression of cos2 and cos4 in S. albus led to the production of didesmethylmensacarcin (DDMM, a precursor of mensacarcin) and the production of rishirilide A and B (a precursor of rishirilide A), respectively. However, no product was detected from the expression of cos3. In addition, based on the results of isotope-feeding experiments in S. bottropensis, a putative biosynthesis pathway for mensacarcin is proposed.     

Application of β-Sitosterol + γ-Oryzanol-Structured Organogel as Migration Barrier in Filled Chocolate Products

Oil migration in filled pralines is a phenomenon that is highly correlated with the occurrence of chocolate bloom. In this study the potential to suppress or prevent oil migration by incorporation of sterol/sterolster-structured organogels was evaluated. Different quantities, 2.5 or 14% (w/w), of gel with structurant levels of either 10 or 25% (w/w) were studied in a layered model system. The gel was either a part of the nougat or of the chocolate phase, or as a separate layer. Samples were monitored regularly for a period of 24 weeks at storage temperatures of 10, 18 and 28 °C. The amount of migrated oil was determined via DSC analysis of a surface sample. The results indicate that, despite the additional oil brought into the system via the oleogel, the level of oil found in the chocolate layer is reduced through the presence of the gel. In particular, the three-layer system and gelled chocolate appear to be promising routes to either suppress oil migration or improve nutritional profiles by incorporation of liquid oils.     

Development of Biarylalkyl Carboxylic Acid Amides with Improved Anti-schistosomal Activity

The parasitic disease schistosomiasis is the cause of more than 200 000 human deaths per year. Although the disease is treatable, there is one major shortcoming: praziquantel has been the only drug used to combat these parasites since 1977. The risk of the emergence of resistant schistosomes is known to be increasing, as a reduced sensitivity of these parasites toward praziquantel has been observed. We developed a new class of substances, which are derived from inhibitors of human aldose reductase, and which showed promising activity against Schistosoma mansoni couples in vitro. Further optimisation of the compounds led to an increase in anti-schistosomal activity with observed phenotypes such as reduced egg production, vitality, and motility as well as tegumental damage and gut dilatation. Here, we performed structure–activity relationship studies on the carboxylic acid moiety of biarylalkyl carboxylic acids. Out of 82 carboxylic acid amides, we identified 10 compounds that are active against S. mansoni at 25 μm . The best five compounds showed an anti-schistosomal activity up to 10 μm and induced severe phenotypes. Cytotoxicity tests in human cell lines showed that two derivatives had no cytotoxicity at 50 or 100 μm . These compounds are promising candidates for further optimisation toward the new anti-schistosomal agents.