Supramolecular aggregation of block copolymers in the solid state as assisted by the selective formation of inclusion crystals

Supramolecular self-assembly of a host molecule with selected blocks of triblock copolymers enabled the formation of inclusion 2D nanocrystals that connect consecutive copolymer chains. Indeed, the selective inclusion of ethylene oxide (EO) blocks in inclusion crystals and the phase segregation of PO blocks of poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) (EO(n)PO(m)EO(n)) triblock copolymers provide an efficient route to create alternated crystalline lamellae and amorphous layers, forming a well-organized material. The spontaneous formation of the supramolecular architectures was realized by a solvent-free mechanochemical approach or by thermal treatment of the copolymer and host (tris-o-phenylenedioxycyclotriphosphazene), as demonstrated by in situ synchrotron X-ray diffraction. The driving force for the fabrication of crystalline inclusion compounds with selected EO segments is based on the establishment of cooperative noncovalent intermolecular interactions, while steric effects prevent the formation of the inclusion crystal with the remaining PO blocks. The 2D (1)H-(13)C solid state and fast-(1)H MAS NMR provide direct evidence of the intimate interactions between the host and EO block and the topology of the block copolymer in the material. The large magnetic susceptibility generated by the aromatic host nanochannels surrounding the included EO chains was interpreted by ab initio calculations (HF-GIAO/DGDZVP) that carefully reproduce the chemical shifts associated with the effects of guest-host interactions. The theoretical calculations enable the measurement of short intermolecular distances between the host and the target block, demonstrating the existence of a diffuse network of multiple CH···π host-guest interactions that improve the robustness of the supramolecular architecture. The overall evidence enforces the strategy of combining the use of block copolymers and clathrate-forming molecules to fabricate organized materials through noncovalent interactions.     

Nature-driven photochemistry for catalytic solar hydrogen production: a Photosystem I-transition metal catalyst hybrid

Solar energy conversion of water into the environmentally clean fuel hydrogen offers one of the best long-term solutions for meeting future energy demands. Nature provides highly evolved, finely tuned molecular machinery for solar energy conversion that exquisitely manages photon capture and conversion processes to drive oxygenic water-splitting and carbon fixation. Herein, we use one of Nature's specialized energy-converters, the Photosystem I (PSI) protein, to drive hydrogen production from a synthetic molecular catalyst comprised of inexpensive, earth-abundant materials. PSI and a cobaloxime catalyst self-assemble, and the resultant complex rapidly produces hydrogen in aqueous solution upon exposure to visible light. This work establishes a strategy for enhancing photosynthetic efficiency for solar fuel production by augmenting natural photosynthetic systems with synthetically tunable abiotic catalysts.     

Broad distribution of energetically important contacts across an extended protein interface

Infection of cells by HIV depends upon profound structural rearrangements within the trimeric viral protein gp41. Critical to this process is the formation of a six-helix bundle in which a set of three N-terminal heptad repeat (NHR) helices assemble to form a core displaying long grooves that provide docking sites for three C-terminal heptad repeat (CHR) helices. We report experiments designed to discriminate between two alternative hypotheses regarding the source of affinity between individual CHR helices and the complementary groove: (1) affinity is dominated by interactions of a small cluster of side chains at one end of the CHR helix; or (2) affinity depends upon interactions distributed across the long CHR helix. We have employed two complementary experimental designs, and results from both favor the latter hypothesis.     

Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BG) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BG, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BG which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes.     

The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC(50) of 0.01 ng mL(-1) and 0.16 ng mL(-1) respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.     

Formation of kinetically trapped nanoscopic unilamellar vesicles from metastable nanodiscs

Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., [DMPG]/[DMPC] = 0.01 and 0.001) and two solution salinities (i.e., [NaCl] = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 °C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 °C), but transform into uniform size, nanoscopic ULVs after incubation at 10 °C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 °C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 °C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical applications that depend on monodisperse lipid-based systems for therapeutic and diagnostic purposes.     

Spectroscopic and computational studies of matrix-isolated iso-CHBr3: structure, properties, and photochemistry of iso-bromoform

Iso-polyhalomethanes are known reactive intermediates that play a pivotal role in the photochemistry of halomethanes in condensed phases. In this work, iso-bromoform (iso-CHBr(3)) and its deuterated isotopomer were characterized by matrix isolation infrared and UV/visible spectroscopy, supported by ab initio and density functional theory calculations, to further probe the structure, spectroscopy, and photochemistry of this important intermediate. Selected wavelength laser irradiation of CHBr(3) isolated in Ar or Ne matrices at ~5 K yielded iso-CHBr(3); the observed infrared and UV/visible absorptions are in excellent agreement with computational predictions, and the energies of various stationary points on the CHBr(3) potential energy surface were characterized computationally using high-level methods in combination with correlation consistent basis sets. These calculations show that, while the corresponding minima lie ~200 kJ/mol above the global CHBr(3) minimum, the isomer is bound by some 60 kJ/mol in the gas phase with respect to the CHBr(2) + Br asymptote. The photochemistry of iso-CHBr(3) was investigated by selected wavelength laser irradiation into the intense S(0) → S(3) transition, which resulted in back photoisomerization to CHBr(3). Intrinsic reaction coordinate calculations confirmed the existence of a first-order saddle point connecting the two isomers, which lies energetically below the threshold of the radical channel. Subsequently, natural bond orbital analysis and natural resonance theory were used to characterize the important resonance structures of the isomer and related stationary points, which demonstrate that the isomerization transition state represents a crossover from dominantly covalent to dominantly ionic bonding. In condensed phases, the ion-pair dominated isomerization transition state structure is preferentially stabilized, so that the barrier to isomerization is lowered.     

Application of dispersive liquid-liquid microextraction for the determination of aflatoxins B1, B2, G1 and G2 in cereal products

The application of dispersive liquid-liquid microextraction (DLLME) technique for the rapid analysis of aflatoxins B(1), B(2), G(1) and G(2) in maize, rice and wheat products has been evaluated. After extraction of aflatoxins from cereal matrices with a mixture of methanol/water 8:2 (v/v), the analytes were rapidly transferred from the extract to another small volume of organic solvent, chloroform, by DLLME. Aflatoxins were determined using high performance liquid chromatography with florescence detection and photochemical post-column derivatization. Parameters affecting both extraction and DLLME procedures, such as extraction solvent, type and volume of DLLME extractant, volume of water and salt effect, were systematically investigated and optimized to achieve the best extraction efficiency. Under the optimal experimental conditions, the whole analytical method provides enrichment factors around 2.5 times and detection limits (0.01-0.17 μg kg(-1)) below the maximum levels imposed by current regulation for aflatoxins in cereals and cereal products intended for direct human consumption. Recoveries (67-92%) and repeatability (RSD<10, n=3), tested in three different cereal matrices, meet the performance criteria required by EC Regulation No. 401/2006 for the determination of the levels of mycotoxins in foodstuffs. The proposed method was successfully applied to the analysis of retail cereal products with quantitative results comparable to the immunoaffinity chromatography (IAC). The main advantages of developed method are the simplicity of operation, the rapidity to achieve a very high sample throughput and low cost.