Effect of sample preparation on the thermal degradation of metal-added biomass

The present study investigates the effect of different sample preparation methods on the pyrolysis behaviour of metal-added biomass; Willow samples were compared in the presence of two salts of zinc and lead containing sulphate and nitrate anions which were added to the wood samples with three different techniques as dry-mixing, impregnation and ion-exchange. The effect of acid and water wash as common demineralisation pre-treatments were also analysed to evaluate their roles in the thermal degradation of the biomass. Results from thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR) and pyrolysis-mass spectrometry (Py-MS) measurements indicated that these pre-treatments change the matrix and the physical-chemical properties of wood. Results suggested that these structural changes increase the thermal stability of cellulose during pyrolysis. Sample preparation was also found to be a crucial factor during pyrolysis; different anions of metal salts changed the weight loss rate curves of wood material, which indicates changes in the primary degradation process of the biomass. Results also showed that dry-mixing, impregnation or ion-exchange influence the thermal behaviour of wood in different ways when a chosen metal salt was and added to the wood material. © 2011 Elsevier B.V. All rights reserved.

Microscopy imaging of liposomes:from coverslips to environmental SEM

A plethora of techniques for the imaging of liposomes and other bilayer vesicles are available. However, sample preparation and the technique chosen should be carefully considered in conjunction with the information required. For example, larger vesicles such as multilamellar and giant unilamellar vesicles can be viewed using light microscopy and whilst vesicle confirmation and size prior to additional physical characterisations or more detailed microscopy can be undertaken, the technique is limited in terms of resolution. To consider the options available for visualising liposome-based systems, a wide range of microscopy techniques are described and discussed here: these include light, fluorescence and confocal microscopy and various electron microscopy techniques such as transmission, cryo, freeze fracture and environmental scanning electron microscopy. Their application, advantages and disadvantages are reviewed with regard to their use in analysis of lipid vesicles.

Environmental scanning electron microscope imaging of vesicle systems

The structural characteristics of liposomes have been widely investigated and there is certainly a strong understanding of their morphological characteristics. Imaging of these systems, using techniques such as freeze-fracturing methods, transmission electron microscopy, and cryo-electron imaging, has allowed us to appreciate their bilayer structures and factors that influence this. However, there are a few methods that study these systems in their natural hydrated state; commonly, the liposomes are visualized after drying, staining and/or fixation of the vesicles. Environmental scanning electron microscopy (ESEM) offers the ability to image a liposome in its hydrated state without the need for prior sample preparation. We were the first to use ESEM to study the liposomes and niosomes, and have been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses onto, or evaporates from, the sample in real-time. This provides an insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay for liposome formulation and stability.

Mass spectrometry imaging of pharmacological compounds in tissue sections

The use of MS imaging (MSI) to resolve the spatial and pharmacodynamic distributions of compounds in tissues is emerging as a powerful tool for pharmacological research. Unlike established imaging techniques, only limited a priori knowledge is required and no extensive manipulation (e.g., radiolabeling) of drugs is necessary prior to dosing. MS provides highly multiplexed detection, making it possible to identify compounds, their metabolites and other changes in biomolecular abundances directly off tissue sections in a single pass. This can be employed to obtain near cellular, or potentially subcellular, resolution images. Consideration of technical limitations that affect the process is required, from sample preparation through to analyte ionization and detection. The techniques have only recently been adapted for imaging and novel variations to the established MSI methodologies will further enhance the application of MSI for pharmacological research.

A solvent-free matrix application method for matrix-assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI-MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules.

Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.

Environmental scanning electron microscopy offers real-time morphological analysis of liposomes and niosomes

Liposomes have been imaged using a plethora of techniques. However, few of these methods offer the ability to study these systems in their natural hydrated state without the requirement of drying, staining, and fixation of the vesicles. However, the ability to image a liposome in its hydrated state is the ideal scenario for visualization of these dynamic lipid structures and environmental scanning electron microscopy (ESEM), with its ability to image wet systems without prior sample preparation, offers potential advantages to the above methods. In our studies, we have used ESEM to not only investigate the morphology of liposomes and niosomes but also to dynamically follow the changes in structure of lipid films and liposome suspensions as water condenses on to or evaporates from the sample. In particular, changes in liposome morphology were studied using ESEM in real time to investigate the resistance of liposomes to coalescence during dehydration thereby providing an alternative assay of liposome formulation and stability. Based on this protocol, we have also studied niosome-based systems and cationic liposome/DNA complexes. Copyright © Informa Healthcare.

Stabilisation of isocyanate cross linked polybutadiene binder

Isocyanate cross-linked hydroxy terminated polybutadiene is used as a binder for solid rocket propellant. Rocket motors containing this propellant require a storage life of at least 20 years. During storage it has been found that the important rubbery properties of the binder can be lost due to oxidative cross-linking of the polybutadiene chains. This could cause catastrophic failure when the rocket motor is required. At present the bis-hindered phenol Calco 2246 is used as a thermal oxidative stabiliser, but it's performance is only adequate. This has led to the search for a more efficient stabiliser system. To hasten the evaluation of new antioxidant systems the use of dynamic thermal analysis was investigated. Results showed that a tentative relationship existed between predictions by thermal analysis and the long term oven ageing for simple single antioxidant systems. But for more complex systems containing either autosynergistic or mixed antioxidants no relationship was observed suggesting that results for such an "accelerated" technique cannot be used for the purpose of extrapolation for long term performance. This was attributed to the short time and more aggressive condition used (hjgher temperature and oxygen rich atmosphere in thermal analysis) altering the mechanism of action of the antioxidants and not allowing time for co-operative effect of the combined antioxidant system to form. One potential problem for the binder system is the use of an diisocyanate as a cross-linking agent. This reacts with the hydroxyl hydrogen on the polymer as well as other active hydrogens such as those contained in a number of antioxidants, affecting both cross-linking and antioxidant effectiveness. Studies in this work showed that only antioxidants containing amine moieties have a significant affect on binder preparation, with the phenolic antioxidants not reacting. This is due to the greater nucleophilicity of the amines. Investigation of a range of antioxidant systems, including potentially homo, hetero and autosynergistic systems, has highlighted a number of systems which show considerably greater effectiveness than the currently used antioxidant Calco 2246. The only single antioxidant which showed improvement was the partially unhindered phenol y-Tocopherol. Of the mixed systems combinations of the sulphur containing antioxidants e.g. DLTP with higher levels of chain-breaking antioxidants, especially Calco 2246, were the most promising. Also the homosynergistic mix of an aromatic amine and a phenol was seen to be very effective but the results were inconsistent. This inconsistency could be explained by the method of sample preparation used. It was shown that the efficiency of a number of antioxidant.s could be dramatically improved by the use of ultrasound during the mixing stage of preparation. The reason for this increase in performance is unclear but in the case of the homosynergistic amine/phenol mix both more efficient mixing and/or the production of a novel mechanism of action are suggested

Chemical synthesis of isotopically labelled (M+4) purine nucleosides and their incorporation into DNA oligomers

Development of accurate and sensitive analytical methods to measure the level of biomarkers, such as 8-oxo-guanine or its corresponding nucleoside, 8-oxo-2’-deoxyguanosine, has become imperative in the study of DNA oxidative damage in vivo. Of the most promising techniques, HPLC-MS/MS, has many attractive advantages. Like any method that employs the MS technique, its accuracy depends on the use of multiply, isotopically-labelled internal standards. This project is aimed at making available such internal standards. The first task was to synthesise the multiply, isotopically-labelled bases (M+4) guanine and (M+4) 8-oxo-guanine. Synthetic routes for both (M+4) guanine and (M+4) 8-oxo-guanine were designed and validated using the unlabelled compounds. The reaction conditions were also optimized during the “dry runs”. The amination of the 4-hydroxy-2,6-dichloropyrimidine, appeared to be very sensitive to the purity of the commercial [15]N benzylamine reagent. Having failed, after several attempts, to obtain the pure reagent from commercial suppliers, [15]N benzylamine was successfully synthesised in our laboratory and used in the first synthesis of (M+4) guanine. Although (M+4) bases can be, and indeed have been used as internal standards in the quantitative analysis of oxidative damage, they can not account for the errors that may occur during the early sample preparation stages. Therefore, internal standards in the form of nucleosides and DNA oligomers are more desirable. After evaluating a number of methods, an enzymatic transglycolization technique was adopted for the transfer of the labelled bases to give their corresponding nucleosides. Both (M+4) 2-deoxyguanosine and (M+4) 8-oxo-2’-deoxyguanosine can be purified on micro scale by HPLC. The challenge came from the purification of larger scale (>50 mg) synthesis of nucleosides. A gel filtration method was successfully developed, which resulted in excellent separation of (M+4) 2’-deoxyguanosine from the incubation mixture. The (M+4) 2’-deoxyguanosine was then fully protected in three steps and successfully incorporated, by solid supported synthesis, into a DNA oligomer containing 18 residues. Thus, synthesis of 8-oxo-deoxyguanosine on a bigger scale for its future incorporation into DNA oligomers is now a possibility resulting from this thesis work. We believe that these internal standards can be used to develop procedures that can make the measurement of oxidative DNA damage more accurate and sensitive.

Deformation of sand in plane strain and oxisymmetric compression

Small scale laboratory experiments, in which the specimen is considered to represent an element of soil in the soil mass, are essential to the evolution of fundamental theories of mechanical behaviour. In this thesis, plane strain and axisymmetric compression tests, performed on a fine sand, are reported and the results are compared with various theoretical predictions. A new apparatus is described in which cuboidal samples can be tested in either axisymmetric compression or plane strain. The plane strain condition is simulated either by rigid side platens, in the conventional manner, or by flexible side platens which also measure the intermediate principal stress. Close control of the initial porosity of the specimens is achieved by a vibratory method of sample preparation. The strength of sand is higher in plane strain than in axisymmetric compression, and the strains required to mobilize peak strength are much smaller. The difference between plane strain and axisymmetric compression behaviour is attributed to the restrictions on particle movement enforced by the plane strain condition; this results in an increase in the frictional component of shear strength. The stress conditions at failure in plane strain, including the intermediate principal stress, are accurately predicted by a theory based on the stress- dilatancy interpretation of Mohr's circles. Detailed observations of rupture modes are presented and measured rupture plane inclinations are predicted by the stress-dilatancy theory. Although good correlation with the stress-dilatancy theory is obtained during virgin loading, in both axisymmetric compression and plane strain, the stress-dilatancy rule is only obeyed during reloading if the specimen has been unloaded to approximate ambient stress conditions. The shape of the stress-strain curves during pre-peak deformation, in both plane strain and axisymmetric compression, is accurately described bv a combined parabolic-hyperbolic specification.

Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system.

Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe2+, lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.

Application and use of isothermal calorimetry in pharmaceutical development

There are many steps involved in developing a drug candidate into a formulated medicine and many involve analysis of chemical interaction or physical change. Calorimetry is particularly suited to such analyses as it offers the capacity to observe and quantify both chemical and physical changes in virtually any sample. Differential scanning calorimetry (DSC) is ubiquitous in pharmaceutical development, but the related technique of isothermal calorimetry (IC) is complementary and can be used to investigate a range of processes not amenable to analysis by DSC. Typically, IC is used for longer-term stability indicating or excipient compatibility assays because both the temperature and relative humidity (RH) in the sample ampoule can be controlled. However, instrument design and configuration, such as titration, gas perfusion or ampoule-breaking (solution) calorimetry, allow quantification of more specific values, such as binding enthalpies, heats of solution and quantification of amorphous content. As ever, instrument selection, experiment design and sample preparation are critical to ensuring the relevance of any data recorded. This article reviews the use of isothermal, titration, gas-perfusion and solution calorimetry in the context of pharmaceutical development, with a focus on instrument and experimental design factors, highlighted with examples from the recent literature. © 2011 Elsevier B.V.

Accurate estimation of isoelectric point of protein and peptide based on amino acid sequences

Motivation: In any macromolecular polyprotic system - for example protein, DNA or RNA - the isoelectric point - commonly referred to as the pI - can be defined as the point of singularity in a titration curve, corresponding to the solution pH value at which the net overall surface charge - and thus the electrophoretic mobility - of the ampholyte sums to zero. Different modern analytical biochemistry and proteomics methods depend on the isoelectric point as a principal feature for protein and peptide characterization. Protein separation by isoelectric point is a critical part of 2-D gel electrophoresis, a key precursor of proteomics, where discrete spots can be digested in-gel, and proteins subsequently identified by analytical mass spectrometry. Peptide fractionation according to their pI is also widely used in current proteomics sample preparation procedures previous to the LC-MS/MS analysis. Therefore accurate theoretical prediction of pI would expedite such analysis. While such pI calculation is widely used, it remains largely untested, motivating our efforts to benchmark pI prediction methods. Results: Using data from the database PIP-DB and one publically available dataset as our reference gold standard, we have undertaken the benchmarking of pI calculation methods. We find that methods vary in their accuracy and are highly sensitive to the choice of basis set. The machine-learning algorithms, especially the SVM-based algorithm, showed a superior performance when studying peptide mixtures. In general, learning-based pI prediction methods (such as Cofactor, SVM and Branca) require a large training dataset and their resulting performance will strongly depend of the quality of that data. In contrast with Iterative methods, machine-learning algorithms have the advantage of being able to add new features to improve the accuracy of prediction. Contact: yperez@ebi.ac.uk Availability and Implementation: The software and data are freely available at https://github.com/ypriverol/pIR. Supplementary information: Supplementary data are available at Bioinformatics online.

The automation of protein crystal presentation for x ray diffraction experiments using standing acoustic waves in a microfluidic chip environment

As the pressure continues to grow on Diamond and the world's synchrotrons for higher throughput of diffraction experiments, new and novel techniques are required for presenting micron dimension crystals to the X ray beam. Currently this task is both labour intensive and primarily a serial process. Diffraction measurements typically take milliseconds but sample preparation and presentation can reduce throughput down to 4 measurements an hour. With beamline waiting times as long as two years it is of key importance for researchers to capitalize on available beam time, generating as much data as possible. Other approaches detailed in the literature [1] [2] [3] are very much skewed towards automating, with robotics, the actions of a human protocols. The work detailed here is the development and discussion of a bottom up approach relying on SSAW self assembly, including material selection, microfluidic integration and tuning of the acoustic cavity to order the protein crystals.