Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening

The yolk protein precursor vitellogenin (Vtg) in plasma has proved to be a simple and sensitive biomarker for assessing exposure of fish to environmental estrogens. Within international bodies such as the Organization for Economic Cooperation and Development (OECD) work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISAs) for Vtg in common carp/fathead minnow, zebrafish and Japanese medaka. The assays were developed using a combination of monoclonal and polyclonal fish Vtg antibodies in a sandwich format, using stabilized Vtg from the test species as a standard. The carp Vtg ELISA has a working range of 1–63 ng/mL, a minimal detection limit of 0.6 ng/mL, and may also be used for quantification of Vtg in fathead minnow. In fathead minnow whole-body homogenate samples, the practical detection limit is 400 ng/mL due to the matrix effect. The zebrafish Vtg ELISA has a working range of 0.5–63 ng/mL, a minimal detection limit of 0.4 ng/mL, and a practical detection limit of 200 ng/mL in whole-body homogenate samples. The medaka Vtg ELISA has a working range of 0.25–16 ng/mL, a minimal detection limit of 0.1 ng/mL, and a practical detection limit of 125 ng/mL in whole-body homogenate samples. The intra- and inter-assay variations were below 20% for all assays. The assays were evaluated with sets of representative samples spanning the wide dynamic range of Vtg-levels found in fish exposed to environmental estrogens, and all three assays are currently undergoing international inter-laboratory validation.     

Analytical characterization of a facile porous polymer monolithic trypsin microreactor enabling peptide mass mapping using mass spectrometry

A simple and rapid single-step method is presented to fabricate an enzyme reactor using trypsin immobilized on a macroporous polymer monolith. A reactor produced in a capillary format is ready to use within 1 h of preparation. The monomers making up the monolith, including N-acryloxysuccinimide for covalent immobilization of the enzyme, are mixed with trypsin and introduced into the column by capillary force for polymerization/immobilization. The enzyme activity from column-to-column is reproducible below 5% relative standard deviation (RSD), while the reactor is durable for at least 20 weeks when stored at room temperature. The apparent kinetic constants V(max) and K(m) are of value similar to those obtained by free trypsin in solution. Enzymatic digestion of proteins was shown to be feasible on a time-scale of seconds and submicromolar concentrations enabling peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.     

Understanding the effect of cobalt particle size on Fischer-Tropsch synthesis: surface species and mechanistic studies by SSITKA and kinetic isotope effect

Co/γ-Al(2)O(3) catalysts with particle sizes in the range of 4-15 nm were investigated by isothermal hydrogenation (IH), temperature programmed hydrogenation (TPH), and steady-state isotopic transient kinetic analysis (SSITKA). Kinetic isotope effect experiments were used to probe possible mechanisms on Co/γ-Al(2)O(3) with different particle size. It was found that CO dissociated on Co/γ-Al(2)O(3) catalysts at 210 °C. The total amount of CO(2) formed following the dissociation depends on the cobalt crystal size. O-Co binding energy was found to be highly dependent on the Co metal particle size, whereas similar C-Co binding energy was found on catalysts with different Co particle size. Very strongly bonded carbon and oxygen surface species increased with decreasing particle size and acted as site blocking species in the methanation reaction. SSITKA experiments showed that the intrinsic activity (1/τ(CH(x))) remained constant as the particle size increased from 4 to 15 nm. The number of surface intermediates (N(CH(x))) increased with increasing particle size. The apparent activation energies were found similar for these catalysts, about 85 kJ/mol. D(2)-H(2) switches further confirmed that the particle size did not change the kinetically relevant steps in the reaction. The reactivity of the active sites on the 4 nm particles was the same as those on the 8, 11, and 15 nm particles, and only the number of total available surface active sites was less on the 4 nm particles than on the others.     

Molecular recognition: minimizing the acid–base interaction of a tunable host–guest system changes the selectivity of binding

Two new receptors incorporating a 4-n-butyl aniline moiety has been designed, synthesized and evaluated for their binding properties towards a series of ureido-glycine derivatives. The host design is based on an urea adamantyl host motif known from large generations of poly(propylene imine) dendrimers functionalized with urea adamantyl moieties on the periphery. The design of the host molecules was directed towards a study of the effects of basicity of an amine function versus the effect of molecular recognition on the binding strength as seen from comparing the results obtained in the present work with previously guest–host studies. The guest–host interaction features an electrostatic interaction and multiple hydrogen binding interactions, where the main difference between the hosts described here and previously described is a substitution from an amine to aniline. Anilines are weaker bases than aliphatic amines and they generally give lower binding constants when treated with acidic guest molecules. The association constants have been measured using NMR titrations and the nature of the guest–host system is discussed based on these results. A general decrease in binding affinities is observed upon changing from the trialkyl amine hosts to the dialkyl aniline based hosts. One exception was observed where the weaker base host had stronger affinity to one of the guests. Thus, when the basicity of the host is decreased other factors influence the binding such as a better geometric fit. A crystal structure of one of the receptors has been solved and it shows no intramolecular hydrogen bonding.     

Reconstruction of Solar Spectral Surface UV Irradiances Using Radiative Transfer Simulations

UV radiation exerts several effects concerning life on Earth, and spectral information on the prevailing UV radiation conditions is needed in order to study each of these effects. In this paper, we present a method for reconstruction of solar spectral UV irradiances at the Earth's surface. The method, which is a further development of an earlier published method for reconstruction of erythemally weighted UV, relies on radiative transfer simulations, and takes as input (1) the effective cloud optical depth as inferred from pyranometer measurements of global radiation (300–3000 nm); (2) the total ozone column; (3) the surface albedo as estimated from measurements of snow depth; (4) the total water vapor column; and (5) the altitude of the location. Reconstructed daily cumulative spectral irradiances at Jokioinen and Sodankylä in Finland are, in general, in good agreement with measurements. The mean percentage difference, for instance, is mostly within ±8%, and the root mean square of the percentage difference is around 10% or below for wavelengths over 310 nm and daily minimum solar zenith angles (SZA) less than 70°. In this study, we used pseudospherical radiative transfer simulations, which were shown to improve the performance of our method under large SZA (low Sun).     

A p‐Type NiO‐Based Dye‐Sensitized Solar Cell with an Open‐Circuit Voltage of 0.35 V

In tandem : Employing a molecular dyad and a cobalt‐based electrolyte gives a threefold‐increase in open‐circuit voltage (V_OC) for a p‐type NiO device (V_OC=0.35 V), and a fourfold better energy conversion efficiency. Incorporating these improvements in a TiO₂/NiO tandem dye‐sensitized solar cell (TDSC), results in a TDSC with a V_OC=0.91 V (see figure; CB=conductance band, VB= valence band).

     

A liquid chromatography/electrospray ionization-tandem mass spectrometry method for quantification of specific organophosphorus pesticide biomarkers in human urine

Organophosphorus pesticides are commonly used in both agricultural and residential settings. The widespread use of these chemicals makes it almost impossible for humans to avoid exposure. In order to determine background human exposure, there is a need for fast, reliable, and sensitive analytical methods. We have developed a sensitive method to quantify specific biomarkers of the organophosphorus pesticides acephate, azinphos, chlorpyrifos, coumaphos, diazinon, isazofos, malathion, methamidophos, parathion and pirimiphos or their O,O-dimethyl analogues in human urine, as their selective metabolites or as the intact pesticide. Isotopically labeled internal standards were used for eight of the analytes. The use of labeled internal standards in combination with high-performance liquid chromatography electrospray ionization–tandem mass spectrometry provided a high degree of specificity. Repeated analysis of urine samples fortified with high and low concentrations of the analytes gave relative standard deviations (RSD) of less than 10% for the analytes with an isotopically labeled standard. Analytes without isotopically labeled standards had higher RSD. For all compounds except methamidophos and acephate, the recoveries were greater than 70%. The limits of quantification for most of the analytes were in the range of 0.1 to 1 ng/mL. We detected concentrations of most of these pesticides and/or their metabolites in urine samples from non-occupationally exposed persons using our method. Our frequencies of detection for the analytes measured ranged from 1% to 98%.     

Methods for research on immune complement activation on modified sensor surfaces

We have developed a methodological system consisting of a new surface sensitive quartz crystal microbalance with dissipation monitoring (QCM-D) sensor surfaces together with different surface modification methods for the investigation of surface associated complement activation in human sera. The QCM-D surface, 10 mm in diameter, was modified by spin-coating of poly(urethane urea) (PUUR) and polystyrene (PS). Some sensor surfaces were also sputtered with titanium (Ti) or modified by hydrophobic self-assembled monolayer (SAM) of an 18-carbon alkane thiol with a ---CH₃ end group. The amount of surface deposited complement protein was investigated by incubation of the modified sensor surfaces in human sera, followed by incubation with antibodies directed against complement factor 3c (C3c). The amounts of bound anti-C3c were then used as an arbitrary measure of surface induced complement activation. The order of complement activation of the different surfaces, as judged by three separate measurements per surface modification, was PUUR>PS=SAM>Ti. The Ti surface had a similar low degree of anti-C3c binding as the negative controls (heat inactivated sera). The novel QCM-D methodology was found to be very simple, accurate, sensitive and well suited as a screening method for complement activation and protein adsorption on different materials. We also compared the sensitivity of QCM-D method with surface plasmon resonance (SPR) for the quantification of protein adsorption and complement activation on gold sensor surfaces. The QCM-D method was equally sensitive as the SPR for the detection of protein adsorption from a solution independently if low flow rate (5 μl/min) was used. A slight increase in sensitivity was found at higher flow rate (30 μl/min). However, we found it difficult to use the SPR method on the Ti, PS and PUUR surfaces due to decreased light penetration of the modified SPR sensor chip.     

Nitrogen isotope analysis of ammonium in aqueous solutions using a perfluorosulfonated ionomer membrane for solid-phase microextraction

The use of custom-made solid-phase microextraction (SPME) fibers coated with a perfluorosulfonated ionomer, Nafion, was investigated for nitrogen isotopic analysis of ammonium in aqueous solutions. Aqueous ammonium was converted to ammonia by addition of a base, followed by absorption from the headspace, desorption in the injection port of a gas chromatograph, and analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Fibers coated with a Nafion tubing were chosen due to a higher fiber-gas distribution constant and a higher Nafion thickness than fibers coated with Nafion solution, both leading to a higher amount of ammonia absorbed at equilibrium. The Nafion membrane-coated fiber absorbed approximately 20 times more than a commercial polydimethylsiloxane (PDMS) fiber. The isotopic fractionation between fiber and gas was 1.0117 +/- 0.0009 (standard deviation, SD, of all measurements) at an initial ammonia gas concentration of 21-210 microM. At 390 microM initial gas concentration it was slightly lower. When sampling from liquid samples, an ammonium concentration of 10 mM was needed to obtain a sufficient amount of ammonia absorbed. Modeling of the absorption at different temperatures showed that the absorption was approximately constant in the temperature range suitable for SPME experiments. Absorption at room temperature was therefore used for simplicity. A pilot study was conducted in which absorption was achieved from a single 9 microL droplet of sample. The preliminary results showed that delta(15)N analysis was possible for only 0.4-0.5 micromol of ammonium with a SD of 0.8 per thousand (n = 5).     

Accelerated 18O-labeling in urinary proteomics

Proteolytic (18)O-labeling of peptides has been studied and optimized in order to improve the labeling efficiency and to accelerate the process without increasing the degree of incomplete labeling. Using peptides generated from tryptic digested bovine serum albumin (BSA) and cytochrome c as model proteins, it was shown that complete labeling was achieved after 2 h at pH 6. To increase the sample throughput in a bottom-up proteomic setup, tryptic digestion of proteins in-solution was replaced with tryptic digestion using immobilized trypsin. As a result, an integrated approach was made possible, where both digestion (pH 8) and (18)O/(16)O-labeling of the resulting peptides (pH 6) were done using immobilized trypsin beads. This simplified the sample handling and reduced the overall reaction time significantly: the setup enabled tryptic digestion and (18)O/(16)O-labeling without sample transfer steps within 3.5 h with average (18)O/(16)O-ratios of 0.96±0.13 in aqueous buffer. The initial results were confirmed with a more complex matrix, by spiking urine with the model proteins, yielding results comparable with the ratios obtained in buffer. Satisfying ratios were also achieved regarding urinary proteins identified in a full scale bottom-up experiment. Average (18)O/(16)O-peptide ratios of 0.83±0.13 and 0.91±0.27 indicated good performance in a highly relevant matrix for biomarker discovery.