Evaluation of apparent non-specific protein loss due to adsorption on sample tube surfaces and/or altered immunogenicity

The non-specific loss of protein analytes can have a major effect on assay results particularly where the concentrations of such analytes are extremely low and the matrix is complex. This report assesses how the protein incubated in sample tubes may be lost due to adsorption. Use of proteins, such as bovine serum albumin (BSA), may be used to pre-treat tubes to reduce such losses. However, such losses may also be associated with structural perturbations leading to changes in immunogenicity (as a result of alterations in specific epitope-related conformations). This can lead to erroneous results or lack of comparability with a range of methodologies such as the bicinchoninic protein assay and immunoassays or when surface plasmon resonance (SPR)-based approaches are used. A model system to evaluate these phenomena is proposed.     

Self-organization in an Open Reaction Network for Developing High-function Organized Molecular Systems**

Self-organized molecular systems such as liposomes and supramolecules have attracted considerable attention due to their characteristic properties. An open reaction network (ORN) is another interesting candidate for such systems; however, no stabilization mechanism has been clarified. This work reveals, by computer simulation and experiments, that a network of irreversible processes such as an ORN can be stabilized by self-organization through a full balance between all the involved irreversible processes, thus forming a steady state. The formation of a steady state indicates that a large spontaneous order is formed; specifically, self-organization occurs. Computer simulations also reveal that such a steady state characteristically evolves toward a high-efficiency state through the development of highly ordered structures. These findings indicate that ORN provides a new method for developing high-function organized molecular systems, such as an efficient catalytic system in a composite of ORN and equilibrium molecular structures such as supramolecules and polymers.     

A new photostable terrylene diimide dye for applications in single molecule studies and membrane labeling

A new terrylene diimide-based dye (WS-TDI) that is soluble in water has been synthesized, and its photophysical properties are characterized. WS-TDI forms nonfluorescing H-aggregates in water that show absorption bands being blue-shifted with respect to those of the fluorescing monomeric form. The ratio of monomeric WS-TDI to aggregated WS-TDI was determined to be 1 in 14 400 from fluorescence correlation spectroscopy (FCS) measurements, suggesting the presence of a large amount of soluble, nonfluorescent aggregates in water. The presence of a surfactant such as Pluronic P123 or CTAB leads to the disruption of the aggregates due to the formation of monomers in micelles. This is accompanied by a strong increase in fluorescence. A single molecule study of WS-TDI in polymeric films of PVA and PMMA reveals excellent photostability with respect to photobleaching, far above the photostability of other common water-soluble dyes, such as oxazine-1, sulforhodamine-B, and a water-soluble perylenediimide derivative. Furthermore, labeling of a single protein such as avidin is demonstrated by FCS and single molecule photostability measurements. The high tendency of WS-TDI to form nonfluorescent aggregates in water in connection with its high affinity to lipophilic environments is used for the fluorescence labeling of lipid membranes and membrane containing compartments such as artificial liposomes or endosomes in living HeLa cells. The superior fluorescence imaging quality of WS-TDI in such applications is demonstrated in comparison to other well-known membrane staining dyes such as Alexa647 conjugated with dextran and FM 4-64 lipophilic styryl dye.     

Redefining the terminology of stereoisomerism to produce a mathematics-based taxonomy paradigm

The traditional energy-based taxonomy of stereoisomers is replaced by a geometry-based one. A new paradigm using positive, rather than negative, definition of important terms such as diastereoisomers, and amenable to higher coordination and to incorporating newer taxonomy classes, such as topological isomers, is proposed. Using graph theoretical distance, as well as an additional layer of choice regarding metric distance in a standardized model, key terms, such as configuration vs. conformation, are redefined; also ‘antiquated’ terms, such as ‘geometrical’ isomerism, are reconstituted and given an important place in modern chemistry.     

Polymer dynamics with inertia

Some important recent advances in polymer science involve situations where molecular inertia plays an important role, for example, in the mass spectroscopy of large macromolecules. Only recently have we begun to understand how to analyze dynamics in such situations and what effects inertia has. In this review, we describe what is known about such effects and how nonlinearity plays a crucial role in generating damping, which is of a very different nature than is normally assumed. Not only is such analysis important in understanding polymers in low density environments but also in understanding the internal friction of a polymer chain in solution. The understanding of such situations is still in its infancy and we close by propounding a set of diverse questions for further study. © 2012 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2012     

Experimental measurement of the strength of a C alpha-H...O bond in a lipid bilayer

Due to the apolar nature of the lipid bilayer, the weak Calpha-H...O H-bond is thought to contribute significantly toward the stability of transmembrane helical bundles such as glycophorin A (GPA). Here for the first time we measured the strength of such a bond, using vibrational frequency shifts of a dimeric and nondimeric variants of GPA containing a Gly CD2 label. Although the resulting estimated bond strength of 0.88 kcal/mol is relatively weak, several such bonds could contribute significantly toward bundle stabilization.     

Bichromophoric perylene derivatives: energy transfer from non-fluorescent chromophores

The energy of excitation at non-fluorescent chromophores such as fluorenone and anthraquinone has been trapped by a fast energy transfer to the highly fluorescent perylene bisimides. To this end, anthraquinone, fluorenone and anthracene derivatives have been linked to the perylene bisimides by non-conjugating spacers and fluorescence quantum yield of such assemblies have been determined as a function of the wavelengths of excitation. Energy transfer in such assemblies is strongly influenced by the orientation of the two chromophores. This is of interest for the construction of fluorescence switches.     

Isoconfigurational molecular dynamics study of the kinetics of ice crystal growth

Spontaneous self-assembling, such as formation of molecular crystals, is a fascinating topic for investigation. Ability to initiate and control such transformations promises numerous benefits, but our knowledge of underlying mechanisms of such processes is rather limited. The process of freezing of water is an excellent testing ground for such studies. In this paper we report the results of a systematic molecular dynamics study of ice growth at three different temperatures below the melting point initiated from a number of initial interface structures within the isoconfigurational ensemble. It is shown that a specific structure at a growing ice-water interface is able to affect the growth process over a time scale of 1-2 ns. This structural effect can be characterized in terms of relative growth propensities. On the basis of the differences in the shape between isoconfigurational rate distributions and the rate distribution typical of the specific temperature several different kinds of relative growth propensities have been identified. The initial interfacial configurations employed in this work have been assigned using the proposed classification and possible mechanisms of propensity realization have been suggested for selected cases. Results reported in this paper clearly indicate that local structure effects can have significant impact on tendency for a particular ice surface to grow (or melt). The structural effect on ordering propensities is, most probably, a more universal behaviour and might be expected to be seen in other similar problems such as, for example, protein folding.     

Ring-shaped neuronal networks: a platform to study persistent activity

Persistent activity in the brain is involved in working memory and motor planning. The ability of the brain to hold information 'online' long after an initiating stimulus is a hallmark of brain areas such as the prefrontal cortex. Recurrent network loops such as the thalamocortical loop and reciprocal loops in the cortex are potential substrates that can support such activity. However, native brain circuitry makes it difficult to study mechanisms underlying such persistent activity. Here we propose a platform to study synaptic mechanisms of such persistent activity by constraining neuronal networks to a recurrent loop like geometry. Using a polymer stamping technique, adhesive proteins are transferred onto glass substrates in a precise ring shape. Primary rat hippocampal cultures were capable of forming ring-shaped networks containing 40-60 neurons. Calcium imaging of these networks show evoked persistent activity in an all-or-none manner. Blocking inhibition with bicuculline methaiodide (BMI) leads to an increase in the duration of persistent activity. These persistent phases were abolished by blockade of asynchronous neurotransmitter release by ethylene glycol tetraacetic acid (EGTA-AM).     

Micropollutant sorption to membrane polymers: a review of mechanisms for estrogens

Organic micropollutants such as estrogens occur in water in increasing quantities from predominantly anthropogenic sources. In water such micropollutants partition not only to surfaces such as membrane polymers but also to any other natural or treatment related surfaces. Such interactions are often observed as sorption in treatment processes and this phenomenon is exploited in activated carbon filtration, for example. Sorption is important for polymeric materials and this is used for the concentration of such micropollutants for analytical purposes in solid phase extraction. In membrane filtration the mechanism of micropollutant sorption is a relatively new discovery that was facilitated through new analytical techniques. This sorption plays an important role in micropollutant retention by membranes although mechanisms of interaction are to date not understood. This review is focused on sorption of estrogens on polymeric surfaces, specifically membrane polymers. Such sorption has been observed to a large extent with values of up to 1.2 ng/cm(2) measured. Sorption is dependent on the type of polymer, micropollutant characteristics, solution chemistry, membrane operating conditions as well as membrane morphology. Likely contributors to sorption are the surface roughness as well as the microporosity of such polymers. While retention-and/or reflection coefficient as well as solute to effective pore size ratio-controls the access of such micropollutants to the inner surface, pore size, porosity and thickness as well as morphology or shape of inner voids determines the available area for sorption. The interaction mechanisms are governed, most likely, by hydrophobic as well as solvation effects and interplay of molecular and supramolecular interactions such as hydrogen bonding, π-cation/anion interactions, π-π stacking, ion-dipole and dipole-dipole interactions, the extent of which is naturally dependent on micropollutant and polymer characteristics. Systematic investigations are required to identify and quantify both relative contributions and strength of such interactions and develop suitable surface characterisation tools. This is a difficult endeavour given the complexity of systems, the possibility of several interactions taking place simultaneously and the generally weaker forces involved.     

Sample preparation techniques for the determination of trace residues and contaminants in foods

The determination of trace residues and contaminants in complex matrices, such as food, often requires extensive sample extraction and preparation prior to instrumental analysis. Sample preparation is often the bottleneck in analysis and there is a need to minimise the number of steps to reduce both time and sources of error. There is also a move towards more environmentally friendly techniques, which use less solvent and smaller sample sizes. Smaller sample size becomes important when dealing with real life problems, such as consumer complaints and alleged chemical contamination. Optimal sample preparation can reduce analysis time, sources of error, enhance sensitivity and enable unequivocal identification, confirmation and quantification. This review considers all aspects of sample preparation, covering general extraction techniques, such as Soxhlet and pressurised liquid extraction, microextraction techniques such as liquid phase microextraction (LPME) and more selective techniques, such as solid phase extraction (SPE), solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE). The applicability of each technique in food analysis, particularly for the determination of trace organic contaminants in foods is discussed.     

Radiochemical neutron activation analysis of rare earth elements in peridotitic rocks

The understanding of the geochemistry of basalt petrogenesis and the nature of the upper mantle requires the examination of such rocks as peridotites which in many cases are thought to represent upper mantle material. The mineralogical composition of peridotitic rocks in such that they accommodate large proportions of such trace elements as the transition elements but very small amounts of the rare earths and hygromagmaphile elements. The last two groups are often able to provide a large amount of information leading to petrogenetic models. Owing to the extremely low concentrations of the REE in peridotitic rocks it is necessary to separate them from other elements which will interfere with their precise determination. We have used a radiochemical neutron activation analytical method which is adapted from various published methods. It involves a post-irradiation sample fusion, two separate ion-exchange chromatographic stages and finally a fluoride precipitation. The RNAA procedure is capable of providing very precise REE data for perioditic samples and we have used it for the analysis of such rocks from several geodynamic environments.     

Characterization of advanced morphologies in polymer dispersions by AUC and HDC

Polymer latex particles are used in a wide spectrum of applications that are directly influenced by the surface characteristics as well as particle size and size distribution of these polymer particles. Accurate analysis of such characteristics is required to efficiently control the behavior of such particles. Analytical ultracentrifugation (AUC) and hydrodynamic chromatography (HDC) are the particle characterization methods of high relevance owing to their statistical ability, and the combination of these two techniques also allows to generate information about the surface morphology of the particles. Such a comparison is facilitated owing to different principles of particle characterization in these two methods. AUC relies on the density difference between the particles and the suspending medium to correlate to the particle size, whereas HDC is based on the measurement of hydrodynamic diameter by UV absorption. When particles functionalized by a thin layer of hydrophilic polymer are analyzed, these two methods by the virtues of their characterization principles allow to detect the presence of such a functionalizing polymer layer on the surface. Subsequently, these methods also provide accurate estimation of the thickness of such a layer. The comparisons can also be carried out as a function of time or amount of surface functionalization to tune the properties according to the requirement. In the case where a thick polymer layer is present in the surface of the particles, the comparisons are more qualitative in nature owing to the bridging and aggregation of the particles especially noticed in HDC. However, even in such a case, the combination of these methods is the only way to characterize the special morphology of the particles.     

Quantum mechanical reactive scattering theory

In this article we briefly discuss a number of promising approaches to the formulation of a general, numerically exact, quantum mechanical theory of fully three-dimensional reactive atom-diatom collisions. It is noted that the ability to treat the three-dimensional H + H₂ exchange reactions is pre-requisite to any demonstration of the success of a general quantum mechanical formalism for reactivescattering. However, it is also noted that the time has come for any such theory to further demonstrate the ability to go beyond this basic, elementary atom-diatom exchange reaction. It is speculated that there now exist a number of approaches which can potentially provide such a general framework for quantum reactive scattering theory. Finally, a new generalizationof the Faddeev formalism for such systems, which is capable of inclusion ofthree body forces with a completely general form for the potential surface, is presented.     

Automated interpretation of mass spectra of complex mixtures by matching of isotope peak distributions

Mass spectrometry is now firmly established as a powerful technique for the identification and characterization of proteins when used in conjunction with sequence databases. Various approaches involving stable-isotope labeling have been developed for quantitative comparisons between paired samples in proteomic expression analysis by mass spectrometry. However, interpretation of such mass spectra is far from being fully automated, mainly due to the difficulty of analyzing complex patterns resulting from the overlap of multiple peaks arising from the assortment of natural isotopes. In order to facilitate the interpretation of a complex mass spectrum of such a mixture, such as an MS spectrum of a stable-isotope-enriched ion species, we report on the development of a software application, 'Matching' (web accessible), that enables the automatic matching of theoretical isotope envelopes to multiple ion peaks in a raw spectrum. It is particularly useful for resolving the relative abundances of narrow-split paired peaks caused by enrichment with a stable isotope, such as 18O, 13C, 2H, or 15N.     

Separation-Free Electrochemical Immunosensor Strategies

ABSTRACT

The simplification of the assay strategy is an essential development for the commercial success of immunosensors. The problems associated with assay simplification stem from the fact that immune recognition is not accompanied by an easily detectable event. Direct methods such as those based on optical systems have circumvented such problems by measuring mass changes. However, such devices are not so amenable to instrumental simplification. Electrochemical methods have so far offered the best prospects for commercial biosensors. Success has only occurred with enzyme-based biosensors measuring simple biochemical molecules such as glucose, lactate and creatinine. The use of antibody-based assay strategies requires the introduction of electrochemical labels. Introduction of such species results in greater complexity, accompanied by a series of assay steps. This extension in assay complexity is in direct opposition to the concept of the biosensor: simplicity. Several strategies have been taken to remove these assay steps and reduce the complexity of the immunoassay. Such strategies may contribute to the application of immunosensors as a serious commercial proposition.     

The diffusion coefficient of a swollen microgel particle

The drag on a permeable particle traversing through a Newtonian liquid is calculated. This is in terms of a single dimensionless group, Da, the Darcy number, which relates the particle permeability to the radius. For small values of the Darcy number the solution reverts to the well-known Stokes drag for smooth hard particles. For larger values of the Darcy number the drag is reduced. This drag allows calculation of the diffusion coefficient of such particles, again as a function of the Darcy number. The results are discussed in relation to microgel particles, which display swelling under certain conditions. The size of such particles is typically measured with dynamic light scattering, which measures the diffusion coefficient of particles and as such the analysis presented here shows the conditions under which dynamic light scattering is accurate. The Darcy number for microgel particles is estimated to be on the order of 10(-7).     

Analyzing censored data in the analysis of multiplicative repeatability and reproducibility studies

Measurements from instruments are sometimes censored at or below the instrument’s lower or upper limit of detection because the instruments have a calibrated response only over a certain data range. Near the ends of the range, the system reports a censored value such as “measurand value is less than threshold,” or “measurand value is more than threshold.” How should one analyze data that includes such “less than” and/or “more than” results and what is the impact of such censoring on estimation of variance components? To answer these two questions, this article makes three contributions: (1) It illustrates a straightforward numerical Bayesian analysis of such censored data based on the likelihood function, (2) it provides a simulation study to show the impact of increased amounts of censoring, and (3) it shows that if the true likelihood has an unknown form for measurand values near the threshold and another known form away from the threshold, then it is prudent for the measurement system to report censored values rather than actual values. Open source software to analyze censored data is provided as supplementary material.     

Regiospecific Intramolecular Aldol Condensation Induced by Conjugate Addition of lithium dimethylcuprate to ζ-Oxo-α, β-enones preliminary communication [1]

ζ-Oxo-α, β-enones react with lithium dimethylcuprate to give cyclic aldols such as hydroxydecalone 1 and hydroxyspiro[4.5]decanone 2 in a regio- and stereospecific manner. This new reaction, a combination of conjugate methyl addition to α, β-enones with directed intramolecular aldolisation, is suggested to proceed via either enoate anions, such as 8 and 11 , or radical anions, such as 9 → 10 and 12 → 13 , as intermediates.     

Identification of Four Process-Related Impurities in the Bulk Drug Butalbital Using HPLC-UV Photodiode Array Detection,Particle Beam MS,and NMR

Abstract

During the production of bulk active pharmaceutical ingredients, many opportunities for the generation of impurities may arise. In cases such as this, the impurities often result from “primary” impurities in raw materials, which are carried through the manufacturing process. Since these primary sources often are similar to the raw material in which they occur, the net effect is generation of impurities which may have a highly similar structure to the finished product. Due to such a comparable structure many characteristics which permit analytical resolution, such as the partitioning, chromatographic retention, and spectral characteristics (to name a few) of the impurities are also similar to the finished product. Using a combination of analytical techniques, however, it is possible to accurately describe the impurities. This is often necessary in closely regulated industries such as pharmaceutical manufacturing, where generation of accurate impurity profile methodologies is critical to GLP compliance¹.