Reassessing the rationale behind herbicide biosensors: The case of a photosystem II/redox polymer-based bioelectrode.

Interfacing photosynthetic protein complexes with electrodes is frequently used for the identification of electron transfer mechanisms and the fabrication of biosensors. Binding of herbicide compounds to the terminal plastoquinone QB at photosystem II (PSII) causes disruption of electron flow that is associated with a diminished performance of the associated biodevice. Thus, the principle of electron transport inhibition at PSII can be used for herbicide detection and has inspired the fabrication of several biosensors for this purpose. However, the biosensor performance may reveal a more complex behavior than generally expected. As we present here for a photobioelectrode constituted by PSII embedded in a redox polymer matrix, the effect caused by inhibitors does not only impact the electron transfer from PSII but also the properties of the polymer film used for immobilization and electrical wiring of the protein complexes. Incorporation of phenolic inhibitors into the polymer film surprisingly translates into enhanced photocurrents and, in particular cases, in a higher stability of the overall electrode architecture. The achieved results stress the importance to evaluate first the possible influence of analytes of interest on the biosensor architecture as a whole and provide important insights for consideration in future design of bioelectrochemical devices.

Electrochemical sensing of 2-methyl-4, 6-dinitrophenol by nanomagnetic core shell linked to carbon nanotube modified glassy carbon electrode.

Electrochemical behavior and sensing of 2-methyl-4, 6-dinitrophenol as an herbicide has been investigated by cyclic voltammetry (CV) and square wave voltammetry (SWV). We have synthesized a cefazolin (CEF) immobilized nanomagnetic core-shell (Fe3O4@SiO2-(CH2)3-CEF) and attached it on the modified glassy carbon electrode (GCE/MWCNT) through electrostatic adsorption (GCE/MWCNT/CEF-SiO2@Fe3O4). 2-Methyl-4, 6-dinitrophenol has an oxidation peak that is related to the formation of nitrosamine from hydroxylamine species. This peak was applied for quantifying determination of 2-methyl-4, 6-dinitrophenol. The electrochemical oxidation involves two electron transfers accompanied by two protons. The electrochemical process was controlled by adsorption. The good agreement between the obtained computational studies and the experimental results has demonstrated that outer sphere electron transfer occurred on modified electrode. The new electrochemical sensor was applied to determination of 2-methyl-4, 6-dinitrophenol by SWV in the range of 1.0 × 10-10 to 1.5 × 10-7 M with a detection limit of 0.1 nM. The proposed sensor was applied for determination of 2-methyl-4, 6-dinitrophenol in water samples.

Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.

Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.

[Use of the analytical method LC-MS/MS for finding dinoseb and dinoterb in agricultural products, livestock products and seafood].

A simple determination method of dinoseb and dinoterb in agricultural products, livestock products and seafood by LC-MS/MS was developed. Agricultural samples were extracted with acetone (in the case of rice, soybean and tea leaf, phosphoric acid was added). An aliquot of crude extract was partitioned with hexane and sat. sodium chloride solution. In the case of livestock products and seafood, samples were extracted with a mixture of acetone, hexane, water and sodium chloride, and the organic layer was collected. Clean-up was performed using a PSA mini column. The LC separation was performed on a C18 column with methanol-water (19 : 1) containing 0.005 v/v% acetic acid as a mobile phase, and MS with negative ion electrospray ionization was used for detection. The calibration curve was linear between 0.0005 to 0.04 µg/mL for each compound. Average recoveries (n=5) of dinoseb and dinoterb from 20 kinds of agricultural products, livestock products and seafood fortified at the MRLs were 77-111%, and the relative standard deviations were 2-15%. The limits of quantitation were 0.001 µg/g for both compounds.

Dynamic hollow-fibre liquid phase microextraction of dinitrophenols from human plasma: optimization of an extraction flow system using experimental design methodology.

The utility of a dynamic hollow-fibre liquid phase microextraction method (optimized using a four-variable experimental design and response surface modelling) for extracting dinitrophenolic compounds from human plasma samples was evaluated. The investigated variables were donor phase salt concentration (10-400 mM), donor phase pH (2-6), acceptor phase pH (7-12), and donor/acceptor phase flow rates (30/7.5 to 70/17.5 microL min(-1)). Four dinitrophenol pesticides were used as model substances at concentrations of 0.1 microg mL(-1) in spiked human plasma samples. Extraction efficiencies ranging from 42 to 77% with RSDs below 9 were achieved with the optimized method. The flow rate and acceptor pH were shown to strongly affect the extraction efficiency for all compounds, while the donor phase pH and salt concentration had minor effects. With a well-defined acceptor phase pH and flow rate the system exhibited high robustness. The limits of quantification for the investigated compounds, using the presented extraction method followed by liquid chromatography/electrospray ionization mass spectrometry in selected ion monitoring mode, ranged from 0.05 to 0.1 microg mL(-1) plasma.

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: competition kinetics as a tool for enzyme specificity investigation.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide-substrate's N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.

Evaluation of a TiO2 photocatalysis treatment on nitrophenols and nitramines contaminated plant wastewaters by solid-phase extraction coupled with ESI HPLC-MS.

Nitration reactions of aromatic compounds are commonly involved in different industrial processes for pharmaceutical, pesticide or military uses. For many years, most of the manufacturing sites used lagooning systems to treat their process effluents. In view of a photocatalytic degradation assay, the wastewater of a lagoon was investigated by using HPLC coupled with mass spectrometry. The wastewater was highly concentrated in RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and two herbicides Dinoterb (2-tert-butyl-4,6-dinitrophenol) and Dinoseb (2-sec-butyl-4,6-dinitrophenol). First of all, an analytical method using solid-phase extraction (SPE) combined with HPLC ESI MS/MS was put in work for identification and titration of RDX, HMX and the two dinitrophenols in a complex natural matrix. Then, the UV/TiO2 treatment was investigated for pollutants removal. Dinitrophenolic compounds were significantly degraded after a 8-h-exposition of the wastewater/TiO2 suspension, whereas RDX and HMX were poorly affected.

Effects of either glucose or fructose and metabolic regulators on bovine embryo development and lipid accumulation in vitro.

Our objective was to determine if replacing glucose with fructose would decrease cytoplasmic lipid accumulation during culture of embryos with or without regulators of metabolism. In vitro-produced bovine zygotes were cultured 60 hr in chemically defined medium-1 (CDM-1) plus 0.5% BSA and 0.5 mM fructose or glucose in Experiment 1, and glucose in Experiment 2. In both experiments, 8-cell embryos were next cultured 135 hr in CDM-2 plus 2 mM fructose or glucose in factorial combination with five treatments: (Experiment 1: control, 10% fetal calf serum (FCS), 0.3 microM phenazine ethosulfate (PES), 30 microM dinitrophenol (DNP), and PES + DNP), and (Experiment 2: control, PES, PES + DNP, and 1 and 3 microg/ml cerulenin (C1 and C3)). Day 7.5 blastocysts were stained with Sudan Black B to quantify cytoplasmic lipid droplets as small (SD, <2 microm), medium (MD, 2-6 microm), or large (LD, >6 microm). Blastocyst rates per oocyte were 22% (Experiment 1) and 15% (Experiment 2) higher (P < 0.05) for fructose than glucose. For Experiment 1, numbers of MD were lower for PES, DNP, and PES + DNP than control and FCS (P < 0.05). LD were lower for PES and DNP than control, and higher for FCS than all other treatments (P < 0.05). For Experiment 2, MD were lower (P < 0.05) for PES, and PES + DNP than C1, C3, and control. For LD, PES was lower (P < 0.05) than control, C1, and C3, but not different from PES + DNP. The only effect of hexose on lipids was that fructose resulted in fewer MD (P < 0.01) in Experiment 2. In conclusion, fructose produced more blastocysts than glucose, and PES reduced lipid accumulation.

Redox proteomics in the mussel, Mytilus edulis.

Pollutants (e.g. PAHs, metals) cause oxidative stress (OS) by forming reactive oxygen species. Redox proteomics provides a means for identifying protein-specific OS effects in Mytilus edulis. Groups of mussels were sampled from a clean site in Cork Harbour, Ireland and exposed to 1 mM H2O2 in holding tanks. Protein extracts of gill and digestive gland were separated by two dimensional electrophoresis and similar protein expression profiles were found. Effects of OS on disulphide bridge patterns were investigated in diagonal gels by separating proteins in non-reducing conditions followed by a second reducing dimension. Immunoprecipitation selected carbonylated and glutathionylated proteins. These methodologies can contribute to redox proteomic studies of pollutant responses in marine organisms.

Determination of trace levels of dinitrophenolic compounds in environmental water samples using hollow fiber supported liquid membrane extraction and high performance liquid chromatography.

A hollow fiber supported liquid membrane extraction method for the liquid chromatographic determination of dinitrophenolic compounds at ppt levels has been developed. Different variables affecting the extraction process, such as extraction time, shaking speed, acceptor pH, acceptor buffer concentration, salt content and humic acids have been studied. Enrichment factors up to 7000 times were obtained. Validation of the method included calibration experiments and studies of the linearity of the responses in different matrices. Good linearity was obtained in the environmental matrices evaluated. Detection limits range from 6.0 to 8.0 ng/L, and the relative standard deviations do not exceed 7% in terms of repeatability.

Protein carbonylation and heat shock response in Ruditapes decussatus following p,p'-dichlorodiphenyldichloroethylene (DDE) exposure: a proteomic approach reveals that DDE causes oxidative stress.

Protein carbonylation and levels of heat shock proteins (hsp; 60, 70 and 90 kDa) were measured in gill, mantle and digestive gland of Ruditapes decussatus following exposure to p,p'-dichlorodiphenyldichloroethylene (DDE). Heat shock response was measured by immunoblotting using antibodies specific to heat shock proteins (hsps). Densitometry analysis of individual bands revealed no difference between control and treated samples except appearance of hsp90 in DDE-treated mantle. Carbonylated protein content was determined following 2,4-dinitrophenylhydrazine derivatization and two-dimensional electrophoresis coupled with western blotting. Immunoblotting with dinitrophenol-specific antibody revealed extensive differences in both extent and number of carbonylated proteins in mantle and digestive gland in response to DDE while gill was unaffected. These results demonstrate for the first time that DDE causes tissue-specific formation of reactive oxygen species in clams.

Pediatric fatality following ingestion of dinitrophenol: postmortem identification of a "dietary supplement".

Dinitrophenol, a chemical currently used as an insecticide, is known to uncouple mitochondrial oxidative phosphorylation. A component of explosives, it has also been used in the past as a food coloring and clothing dye. In the 1930s, physicians prescribed it for weight loss, but this practice was discontinued when reports of cataracts, deaths, and other adverse outcomes came to light. We describe in our report the overdose and fatality of a teenager who purchased the product as a weight loss dietary supplement by mail order. We also describe a laboratory method that allowed postmortem determination of the dinitrophenol concentration in the victim's serum. Her death, despite prompt medical treatment, underscores the danger of dinitrophenol. The easy accessibility and apparent resurgent interest in dinitrophenol as a weight loss agent is extremely timely and troubling.

Rapid determination of mono and dinitrophenols by DPP, in the presence of lead and cadmium and using concentrated CaCl2 electrolyte.

The contamination of drinking water and industrial wastewaters is a critical environmental problem. The nitrophenol, dinitrophenol, cadmium, and lead contaminants are classified as hazardous compounds. Their rapid determination may be obtained using differential pulse polarography with concentrated electrolyte. CaCl2, which is very soluble to levels exceeding 5 mol l(-1), allows separation of coalescent peaks at 0.1 mol l(-1). A systematic study undertaken from 0.1 to 5 mol l(-1) shows good separation of lead and cadmium from the organic compounds, and optimization of the electrolyte concentration according to the objective is described. Preconcentration of real samples is necessary because pollution levels are usually very low.

Differential pulse adsorptive stripping voltammetric determination of dinoseb and dinoterb at a modified electrode.

A sensitive adsorptive stripping voltammetric method for the determination of dinitrophenolic herbicides, dinoseb (DSB) and dinoterb (DTB) at a bare carbon paste electrode (CPE) and a clay modified carbon paste electrode (CMCPE) was developed. A systematic study of various experimental conditions, such as the pH, accumulation variables and composition of a modifier on the adsorptive stripping response, were examined by using differential pulse voltammetry. A significant improvement was observed in the sensitivity by using the present method with CMCPE. When CMCPE was used, a linear response was obtained over the concentration range 2 x 10(-10) to 3 x 10(-7) M and 6 x 10(-10) to 6 x 10(-7) M with lower detection limits of 1 x 10(-10) M and 5.4 x 10(-10) M for dinoseb and dinoterb, respectively, at an accumulation time of 100 s. The interference from other herbicides and ions on the stripping signals of both compounds was also evaluated. The described method was applied to estimate of the dinoseb and dinoterb in environmental samples.

Toxicity of 2,6-Di-tert-butyl-4-Nitrophenol (DBNP).

U.S. Navy submarines reported a yellowing of metal surfaces on their internal surfaces. The yellowing was initially identified on the painted steel bulkheads but further examination indicated that it was not limited to steel surfaces and included bedding, thread tape, Formica, plastisol covered hand-wheels, and aluminum lockers. Crew members also reported to the medical department that their skin turned yellow when they came in contact with these contaminated surfaces and requested information on the effects of exposure. Studies conducted by General Dynamics' Electric Boat Division (EBD) determined that the agent was 2,6-Di-tertbutyl-4-Nitrophenol (DBNP). 2,6-Di-butylphenol (DBP) is an antioxidant additive used in lubricating oils and hydraulic fluids. In the enclosed atmosphere of a submarine, the oil mist could be spread throughout the boat by venting the lube oil to the atmosphere. Submarines use electrostatic precipitators (ESP) to clean the air of particulate materials. During passage through the ESP, oil mist containing DBP is nitrated to DBNP, which is then moved throughout the boat in the ventilation system. Analysis of the EBD data indicated 24-hour exposure concentrations to be in the range of <3.0 to 122 ppb in the laboratory and submarine settings. Submarine crews may be exposed to these concentrations for as many as 24 hours/ day for 90 days during underway periods. Toxicity studies regarding the oral and dermal uptake of DBNP were conducted. From the literature the lethal dose to 50 percent of the population (LD50) of DBNP (rat) was reported by Vesselinovitch et al. in 1961 to be 500 mg/kg. Our studies indicated that the LD50 is in the range of 80 mg/kg in the rat. Our work also includes dermal absorption studies, which indicated that DBNP is not well absorbed through intact skin. Within this study, no no-observable adverse effect level (NOAEL) or lowest observable adverse effect level (LOAEL) was identified. Calculation of a reference dose was completed using standard methods based on the LD50 as a numerator with several uncertainty and modifying factors. EBD's determination of airborne concentrations aboard submarines fall in the range of these anticipated allowable concentrations and could indicate significant chronic exposures. No adverse effects from DBNP exposures have been reported to date.

Rapid and sensitive determination of 4-nitrophenol, 3-methyl-4-nitrophenol, 4,6-dinitro-o-cresol, parathion-methyl, fenitrothion, and parathion-ethyl by liquid chromatography with electrochemical detection.

Liquid chromatography with electrochemical detection has been used to determine various nitropesticides, DNOC, fenitrothion, and parathion (methyl and ethyl), and some of their main metabolites, 4-nitrophenol for parathion (methyl and ethyl) and 3-methyl-4-nitrophenol for fenitrothion, by using indirect detection. Analysis of them in river water samples has been performed without a preconcentration step. The recovery efficiencies of the tested compounds yielded values between 96 and 112% at the fortification level of 0.5 ppb in a river water sample, and their relative standard deviations were between 1 and 15%. The detection limits of these compounds ranged between 0.05 and 0.14 ppb.

Use of a stopped-flow pneumatic mixing module to analyze dinitrophenol pesticides. Simultaneous determination of dinoseb and dinobuton.

A kinetic study of the degradation of dinobuton in an alkaline medium has been undertaken by using a stopped-flow pneumatic system. A semiautomatic method for determining dinobuton is proposed for the first time. A detection limit of 0.40 microg mL(-)(1) was calculated. Simultaneous determination of dinobuton and dinoseb can be made by combination of equilibrium and kinetic measurements. The proposed method has been applied to analyze binary mixtures of dinobuton and dinoseb samples with the amount of both components simulating the composition of one undegraded and several degraded dinobuton samples. Also, the procedure has been tested in the analysis of a commercial formulation of dinobuton and the results validated with high-performance liquid chromatography (HPLC).

[The determination of toxic compounds with a dinitrophenol structure in the forensic chemical study of cadaveric material]. / Opredelenie toksicheskikh soedinenii dinitrofenol'noi struktury pri sudebno-khimicheskom issledovanii trupnogo materiala.

A method for forensic chemical assessment of dinitrophenol toxic compounds has been developed. Acetone was used as the isolating agent. The isolates were purified by extraction followed by adsorption in a column packed with silica gel. The analyzed substances are analyzed by chromogenic reactions, from UV and visible spectra in alkali and DMPA, and by thin-layer and high-pressure chromatography. The method allows the detection of 47-83% dinitrophenols in artificial mixtures with liver tissue (with 50 mg of analyzed substance per 100 g of the object). The detection minimum for this group of compounds is 0.5 to 1.0 mg per 100 g of cadaveric material.

[The determination of toxic compounds with a dinitrophenol structure in biological fluids]. / Opredelenie toksichnykh soedinenii dinitrofenol'noi struktury v biologicheskikh zhidkostiakh.

Describes the specific features of chromatographic behavior of numerous toxic compounds of dinitrophenol structure in a thin layer of silica gel and in columns packed with Silasorb-600 adsorbent for high-pressure liquid chromatography. Demonstrates the possibility of using thin-layer and high-pressure liquid chromatography for identification and measurements of the above compounds in the urine and blood.