Rapid and highly efficient growth of graphene on copper by chemical vapor deposition of ethanol

The growth of graphene by chemical vapor deposition on metal foils is a promising technique to deliver large-area films with high electron mobility. Nowadays, the chemical vapor deposition of hydrocarbons on copper is the most investigated synthesis method, although many other carbon precursors and metal substrates are used too. Among these, ethanol is a safe and inexpensive precursor that seems to offer favorable synthesis kinetics. We explored the growth of graphene on copper from ethanol, focusing on processes of short duration (up to one min). We investigated the produced films by electron microscopy, Raman and X-ray photoemission spectroscopy. A graphene film with high crystalline quality was found to cover the entire copper catalyst substrate in just 20 s, making ethanol appear as a more efficient carbon feedstock than methane and other commonly used precursors.

The effects of metabolic work rate and ambient environment on physiological tolerance times while wearing explosive and chemical personal protective equipment

This study evaluated the physiological tolerance times when wearing explosive and chemical (>35kg) personal protective equipment (PPE) in simulated environmental extremes across a range of differing work intensities. Twelve healthy males undertook nine trials which involved walking on a treadmill at 2.5, 4 and 5.5 km.h-1 in the following environmental conditions, 21, 30 and 37 °C wet bulb globe temperature (WBGT). Participants exercised for 60 min or until volitional fatigue, core temperature reached 39 °C, or heart rate exceeded 90% of maximum. Tolerance time, core temperature, skin temperature, mean body temperature, heart rate and body mass loss were measured. Exercise time was reduced in the higher WBGT environments (WBGT37

Physical, chemical, biological and ecotoxicological properties of wastewater discharged from Davis Station, Antarctica

The properties and toxicity of untreatedwastewater at Davis Station, East Antarctica,were investigated to inform decisions regarding the appropriate level of treatment for local discharge purposes and more generally, to better understand the risk associated with dispersal and impact of wastewaters in Antarctica. Suspended solids, nutrients (nitrogen, phosphorus), biological oxygen demand (BOD), metals, organic contaminants, surfactants and microbiological load were measured at various locations throughout the wastewater discharge system. Wastewater quality and properties varied greatly between buildings on station, each ofwhich has separate holding tanks. Nutrients, BOD and settleable solid levelswere higher than standard municipal wastewaters. Microbiological loads were typical of untreated wastewater. Contaminants detected in the wastewater included metals and persistent organic compounds, mainly polybrominated diphenyl ethers (PBDEs). The toxicity of wastewater was also investigated in laboratory bioassays using two local Antarctic marine invertebrates, the amphipod Paramoera walkeri and the microgastropod Skenella paludionoides. Animals were exposed to a range of wastewater concentrations from3% to 68% (test 1) or 63% (test 2) over 21 days with survival monitored daily. Significant mortality occurred in all concentrations of wastewater after 14 to 21 days, and at higher concentrations (50–68% wastewater) mortality occurred after only one day. Results indicate that the local receiving marine environment at Davis Station is at risk from existing wastewater discharges, and that advanced treatment is required both to remove contaminants shown to cause toxicity to biota, as well as to reduce the environmental risks associated with non-native micro-organisms in wastewater.

The harnessing of peptide-monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.

Using DNA as a drug - bioprocessing and delivery strategies

DNA may take a leading role in a future generation of blockbuster therapeutics. DNA has inherent advantages over other biomolecules such as protein, RNA and virus-like particles including safety, production simplicity and higher stability at ambient temperatures. Vaccination is the principal measure for preventing influenza and reducing the impact of pandemics; however, vaccines take up to 8-9 months to produce, and the global production capacity is woefully low. With production times as short as 2 weeks, improved safety and stability, bioprocess engineering developments, and the ability to perform numerous therapeutic roles, DNA has the potential to meet the demands of emerging and existing diseases. DNA is experiencing sharp growths in demand as indicated by its use in gene therapy trials and DNA vaccine related patents. Of particular interest for therapeutic use is plasmid DNA (pDNA), a form of non-genomic DNA that makes use of cellular machinery to express proteins or antigens. The production stages of fermentation and downstream purification are considered in this article. Forward looking approaches to purifying and delivering DNA are reported, including affinity chromatography and nasal inhalation. The place that pDNA may take in the preparation for and protection against pandemics is considered. If DNA therapeutics and vaccines prove to be effective, the ultimate scale of production will be huge which shall require associated bioprocess engineering research and development for purification of this large, unique biomolecule.

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

Rapid production of a plasmid DNA encoding a malaria vaccine candidate via amino-functionalized poly(GMA-co-EDMA) monolith

Malaria is a global health problem; an effective vaccine is urgently needed. Due to the relative poverty and lack of infrastructure in malaria endemic areas, DNA-based vaccines that are stable at ambient temperatures and easy to formulate have great potential. While attention has been focused mainly on antigen selection, vector design and efficacy assessment, the development of a rapid and commercially viable process to manufacture DNA is generally overlooked. We report here a continuous purification technique employing an optimized stationary adsorbent to allow high-vaccine recovery, low-processing time, and, hence, high-productivity. A 40.0 mL monolithic stationary phase was synthesized and functionalized with amino groups from 2-Chloro-N,N- diethylethylamine hydrochloride for anion-exchange isolation of a plasmid DNA (pDNA) that encodes a malaria vaccine candidate, VR1020-PyMSP4/5. Physical characterization of the monolithic polymer showed a macroporous material with a modal pore diameter of 750 nm. The final vaccine product isolated after 3 min elution was homogeneous supercoiled plasmid with gDNA, RNA and protein levels in keeping with clinical regulatory standards. Toxicological studies of the pVR1020-PyMSP4/5 showed a minimum endotoxin level of 0.28 EU/m.g pDNA. This cost-effective technique is cGMP compatible and highly scalable for the production of DNA-based vaccines in commercial quantities, when such vaccines prove to be effective against malaria. © 2008 American Institute of Chemical Engineers.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

Performance of R-N(R′)-R″ functionalised poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolithic sorbent for plasmid DNA adsorption

High-throughput plasmid DNA (pDNA) manufacture is obstructed predominantly by the performance of conventional stationary phases. For this reason, the search for new materials for fast chromatographic separation of pDNA is ongoing. A poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (GMA-EGDMA) monolithic material was synthesised via a thermal-free radical reaction, functionalised with different amino groups from urea, 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) and ammonia in order to investigate their plasmid adsorption capacities. Physical characterisation of the monolithic polymer showed a macroporous polymer having a unimodal pore size distribution pivoted at 600 nm. Chromatographic characterisation of the functionalised polymers using pUC19 plasmid isolated from E. coli DH5α-pUC19 showed a maximum plasmid adsorption capacity of 18.73 mg pDNA/mL with a dissociation constant (KD) of 0.11 mg/mL for GMA-EGDMA/DEAE-Cl polymer. Studies on ligand leaching and degradation demonstrated the stability of GMA-EGDMA/DEAE-Cl after the functionalised polymers were contacted with 1.0 M NaOH, which is a model reagent for most 'cleaning in place' (CIP) systems. However, it is the economic advantage of an adsorbent material that makes it so attractive for commercial purification purposes. Economic evaluation of the performance of the functionalised polymers on the grounds of polymer cost (PC)/mg pDNA retained endorsed the suitability of GMA-EGDMA/DEAE-Cl polymer.

Plasmid DNA purification and formulation for vaccine applications

Plasmid DMA offers the promise of a new generation of pharmaceuticals that will address the often overlooked issue of vaccine production by offering a simple and reproducible method for producing a vaccine. Through reverse engineering, production could be reduced from up to 9 months to as little as 1 month. Simplified development and faster turn-around times means that DMA offers a solution to the vaccine crisis and will help to contain future viral outbreaks by enabling the production of a vaccine against new viral strains in the shortest possible time. Work currently being completed in the area of plasmid DMA production, purification and encapsulation will be presented.

Preparation, Analysis and Use of an Affinity Adsorbent for the Purification of GST Fusion Protein

Methods are presented for the preparation, ligand density analysis and use of an affinity adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed and expanded bed chromatographic processes. The protein is composed of GST fused to a zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification, is covalently immobilized to a solid-phase adsorbent (Streamline™). The GST–ZnF fusion protein displays a dissociation constant of 0.6 x10-6 M to glutathione immobilized to Streamline™. Ligand density optimization, fusion protein elution conditions (pH and glutathione concentration) and ligand orientation are briefly discussed.