Strategies for the development of haemocompatible dialysis membranes

Haemocompatibility is the number one target of membrane development for haemodialysis, because chronic renal failure and the subsequent dialysis-treatment is affecting more and more patients. Two strategies are currently in use to achieve the goal of new blood compatible polymers: Firstly, existing cellulosic membranes are modified by introducing functional groups through ether and ester bonds and secondly, synthetic polymers are mixed with bulk additives or copolymerized with other polymers. Biological responses as a consequence of blood/material interaction are common observations. However, blood compatibility in general cannot be improved by a single membrane surface feature. The assessment of a series of surface properties shows that physiological defense systems, like complement and cell activation, or coagulation are differently stimulated by means of the same surface structures of biomaterials. Thus, a compromise has to be found, already during membrane development. We synthetized a series of modifications of cellulosic membranes with the aim to investigate whether the type of substituent and its degree of substitution has an influence on the blood compatibility of these dialysis membranes. Data indicate that alterations in the lipophilic/ hydrophilic balance (HLB) on the polymer surface may explain substituent-related changes in haemocompatibility. Optimal degrees of substitution are characteristic for a given type of substituent and enables the membrane manufacturer to tailor materials for improved blood compatibility.     

Performance of the reproducible polyvinyl alcohol fracturing fluid system

Russian Journal of Applied Chemistry - A novel organic boron cross-linker for the polyvinyl alcohol fracturing fluid system was synthesized. The formulas of the fracturing fluid system operating at...     

A microfluidic system for high-speed reproducible DNA sizing and quantitation

Microfabrication technology was used to develop a system consisting of disposable glass chips containing etched channels, reagents including polymer matrix and size standards, computer-controlled instrumentation for performing electrophoretic separations and fluorescence detection of double-stranded DNA, and software for automated data analysis. System performance was validated for separation and quantitation reproducibility using samples varying in amount and size of DNA fragments, buffer composition, and salt concentrations. Several applications of the microfluidic system for DNA analysis have been demonstrated, such as of polymerase chain reaction (PCR) products, sizing of plasmid digests, and detection of point mutations by restriction fragment length polymorphism (RFLP) mapping.     

A simple highly reproducible gradient system for micro column HPLC

Summary A gradient delivery system for microbore and open tubular liquid chromatography is described. A gradient mixing vessel of 5–100l, or 25–1000l allows the generation of binary gradients with high reproducibility. The suitability of the system for short packed micro bore columns and open tubular columns is demonstrated.     

REPROVIS-DB: a benchmark system for ligand-based virtual screening derived from reproducible prospective applications

Benchmark calculations are essential for the evaluation of virtual screening (VS) methods. Typically, classes of known active compounds taken from the medicinal chemistry literature are divided into reference molecules (search templates) and potential hits that are added to background databases assumed to consist of compounds not sharing this activity. Then VS calculations are carried out, and the recall of known active compounds is determined. However, conventional benchmarking is affected by a number of problems that reduce its value for method evaluation. In addition to often insufficient statistical validation and the lack of generally accepted evaluation standards, the artificial nature of typical benchmark settings is often criticized. Retrospective benchmark calculations generally overestimate the potential of VS methods and do not scale with their performance in prospective applications. In order to provide additional opportunities for benchmarking that more closely resemble practical VS conditions, we have designed a publicly available compound database (DB) of reproducible virtual screens (REPROVIS-DB) that organizes information from successful ligand-based VS applications including reference compounds, screening databases, compound selection criteria, and experimentally confirmed hits. Using the currently available 25 hand-selected compound data sets, one can attempt to reproduce successful virtual screens with other than the originally applied methods and assess their potential for practical applications.     

Spreader-CDR system for efficient,reproducible, and frugal western blot

Efficient antibody incubation is a vital step for successful western blot. During the incubation, a thin antibody-depleted layer is created around the blotting membrane, which limits antibody binding. Although the conventional batch shaking method is ineffective against it, this layer can be easily disrupted by cyclic draining and replenishing (CDR) of the antibody solution during membrane incubation. Previously, we introduced a closed and rotating cylindrical chamber as a tool to implement CDR for western blots (rCDR). A new open bucket-style chamber was devised for easier operation and the possibility of process automation. Instead of rotation as in rCDR, rocking it back and forth achieved the CDR antibody incubation (R-CDR). The chamber was then equipped with a spreader-rod to facilitate the uniform movement of the antibody solution across the membrane surface. Hence, it was named spreader CDR (S-CDR). Compared to the batch incubation method, both the S-CDR and R-CDR devices produced significantly enhanced signals and developed faster results. There were several additional benefits of using the spreader-rod, which included uniform antibody binding across the membrane, reduced usage of antibodies, and the ability to recover results even from mishandled, creased membranes. The S-CDR device ensures better blots and can be easily implemented in existing western blot protocols.     

The spectrophysics of warfarin: implications for protein binding

The photophysical behavior of the isomers of the anticoagulant drug warfarin in various solvents and solvent mixtures was investigated using absorption, 1H NMR, and steady-state and time-resolved fluorescence spectroscopies in conjunction with B3LYP-based theoretical treatments. Complex absorption patterns were observed, indicative of the presence of different isomers of warfarin in the various solvents studied. In alkaline aqueous solution, the deprotonated open side form of warfarin is highly dominant and only one S0-->S1 singlet transition could be observed in the absorption spectrum centered at 320 nm. These observations were supported by theoretical density functional calculations (B3LYP) in which the geometries of nine isomers of warfarin were optimized and their respective eight lowest singlet and three lowest triplet excitation energy levels were predicted. Examination of the fluorescence excitation and emission spectra of the isomers in nonpolar and polar organic solvents showed the presence of the deprotonated open side chain form of warfarin in 2-propanol, ethanol, and acetonitrile. Time-resolved fluorescence experiments revealed a short decay time constant, tau1, in all solvents studied while in more polar environments a second longer one, tau2, was evident varying between 0.5 and 1.6 ns depending on solvent polarity. The variation of number and length of fluorescence lifetimes as a function of solvent environment has provided a tool for examining warfarin protein binding. Studies on the binding of warfarin to human serum albumin (HSA) have been undertaken, and different modes of binding were observed which are indicative of binding to the anion-selective Sudlow I and, second, a lower affinity mode of interaction.     

Fluorescence turn-on detection of a protein through the displaced single-stranded DNA binding protein binding to a molecular beacon

A new approach has been developed for the highly sensitive and selective sensing of a protein. Lysozyme binding to its aptamer prevents SSB protein binding, and the subsequent binding of the free SSB protein to a molecular beacon results in a turn-on fluorescence signal, which can be used for lysozyme quantification.     

High-throughput screening of protein binding by equilibrium dialysis combined with liquid chromatography and mass spectrometry

A new approach for screening plasma protein binding is presented. The method is based on equilibrium dialysis combined with rapid generic LC-MS bioanalysis by using a sample pooling approach enabling high-throughput screening of protein binding in the drug discovery phase. The method is evaluated by a comparison of measured unbound free fractions f(u) (%) between single and pooled compounds for a test set of structurally diverse compounds with a wide range of unbound fractions. Test compounds include 1 acidic and 10 basic drug standards along with 36 new chemical entities. A good correlation (R2>0.95) of f(u) (%) between the single and pooled compounds is found, suggesting that at least 10 compounds can be simultaneously measured with acceptable accuracy. A simplified drug-protein binding model is applied to calculate the f(u) (%) of drugs at various drug and protein concentrations and this is applied to elucidate the applicability of the sample pooling approach from a theoretical standpoint. Moreover, pH shifts in the plasma were observed after dialysis when using different types of buffers and the impact of that on the f(u) is illustrated in association with their physicochemical properties, in particular the ionization state of compounds by the profile of effective mobility as a function of pH. A new buffer is proposed being able to minimize the pH shift of plasma during the dialysis. In addition, the application of the proposed buffer does not necessarily require adjusting plasma pH before the dialysis and utilizing a CO2 incubator during the dialysis. The effect of the ionic strengths of different buffers on MS signals is investigated with regard to ion suppression. The sample pooling method not only significantly reduces the plasma volume required but also the number of bioanalysis samples as compared to the single compound measurements by a conventional approach. The new proposed approach is especially beneficial for measuring in vitro protein binding in matrices such as mouse plasma where plasma is available only in limited amounts. The current new development will facilitate the drug discovery process by more rapidly assessing the protein binding potential of drug candidates.     

Immobilisation of a repressor protein for binding of plasmid DNA

The use of plasmid DNA in gene therapy and genetic vaccination has increased the need for scalable and sustainable production processes. One key challenge for bioprocess engineering is the separation of plasmid DNA from structurally related impurities. Affinity purification procedures allow a highly selective capturing of the target molecule. In this paper, we present the isolation of a his-tagged lac repressor, its non-covalent immobilisation to different matrices and binding of DNA, thus enabling us to screen for combinations of ligands and stationary phases by using a building block principle.     

The investigation of an equilibrium dependent reaction for the formation of enamines in a microchemical system

The paper describes the equilibrium dependant reaction for the formation of enamines in a microchemical system utilising electroosmotic flow (EOF) for fluid mobilisation. The authors have shown that the reaction can be carried out without the presence of a Lewis acid catalyst, in addition the enamine intermediate was synthesised at room temperature using mild solvent conditions. A 42% conversion of cyclohexanone into the enamine has been achieved to date.     

The equilibrium phase diagram of the copper-indium system: a new investigation

The entire phase diagram of the Cu-In system was investigated using differential scanning calorimetry (DSC), powder X-ray diffraction (XRD), metallography and electron probe microanalysis (EPMA) studies. The results indicate the existence of a solubility region of In in Cu with the limit of the solid solution at 5.20 at.% In at 400 °C and of six intermediate phases, the three low-temperature phases δ, η and Cu₁₁In₉, and the three high-temperature phases γ, η′ and β. The boundaries of each phase were defined with respect to temperature and composition.     

Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines

A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman’s test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3–5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described.     

The development of a sensitive near-IR Raman system

This paper describes the development of a highly sensitive near-IR laser Raman system which has been designed and built in our laboratory. The system comprises both a micro and macro sample handling facility. The approach that we have adopted uses a conventional spectrograph with a charge coupled device detector and optics optimized for the 700–1000 nm spectral region. The laser source is a tunable titanium sapphire laser which provides intense radiation in the 700–900 nm spectral region. We show that the spatial resolution and spectral sensitivity that can be obtained with the microscope attachment are excellent. A comparison between data obtained from our system and that from an FT-Raman system is given.     

An automated flow-injection serial dynamic dialysis technique for drug-protein binding studies

An automated flow-injection analyzer is interfaced with a dialysis unit to study drug-protein protein binding interactions. The binding of some sulfonamides to bovine serum albumin is studied by means of the automated system and gives results similar to those obtained by other procedures. A usual time for a complete run is 100 min, including calibration. The dialysable sulfonamides are quantified spectrophotometrically by using a modified Bratton-Marshall method. The system is also used for the calculation of dialysis rate constants.     

A thermodynamic investigation on the binding of mercury ion with myelin basic protein at different temperatures

A thermodynamic study on the interaction of myelin basic protein with mercury ion was studied by using isothermal titration calonmetry,ITC,at 300.15,310.15 and 320.15 K in Tris buffer solution at pH 7.The enthalpies of MBP + Hg²⁺ interaction are reported and analysed in terms of the extended solvation model.It was found that MBP has two identical and non-cooperative binding sites for Hg²⁺ ions.The intrinsic dissociation equilibrium constants are 99.904,112.968 and 126.724μmol/L,and the molar enthalpy of binding are -11.634,-10.768 and -10.117kJ mol^-1 at 300.15,310.15 and 320.15 K,respectively.     

A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli

A sensing system for nickel based on the nickel binding protein (NBP) from Escherichia coli is shown to be feasible. The versatility of NBP was demonstrated by its use in three different assay formats. When the NBP binds nickel, it undergoes a conformational change that can be used as the basis for an optical sensing system for nickel. The NBP gene was overexpressed in E. coli and the protein purified in a single step using perfusion anion-exchange chromatography. A unique cysteine residue at position 15 in the NBP was labeled with the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). In a spectrofluorimetric assay, there was a maximum of 65% quenching of the fluorescence signal produced by NBP-MDCC in the presence of nickel. A response curve for nickel using NBP-MDCC revealed a detection limit of 8 x 10(-8) mol L(-1). NBP-MDCC was also used to develop assays in microtiter plate and fiber optic bundle formats. Detection limits for nickel using these formats were also in the submicromolar range. Selectivity studies conducted with other divalent metals, including copper, cobalt, iron, cadmium, and manganese, showed that fluorescence quenching for cobalt was similar in magnitude but with a detection limit more than 10-fold higher than for nickel. The quenching responses were lower for the other metals, with detection limits at least 10 to 100 times higher than for nickel. These results suggest that fluorescently labeled NBP is potentially useful in the development of a sensing system for nickel.