Voltammetric immunosensor for the diagnosis of celiac disease based on the quantification of anti-gliadin antibodies

Antibodies against gliadin are used to detect celiac disease (CD) in patients. An electrochemical immunosensor for the voltammetric detection of human anti-gliadin antibodies (AGA) IgA and AGA IgG in real serum samples is proposed. The transducer surface consists of screen-printed carbon electrodes modified with a carbon nanotube/gold nanoparticle hybrid system, which provides a very useful surface for the amplification of the immunological interactions. The immunosensing strategy is based on the immobilization of gliadin, the antigen for the autoantibodies of interest, onto the nanostructured surface. The antigen–antibody interaction is recorded using alkaline phosphatase labeled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions (3-IP/Ag+) was used as the substrate. The analytical signal is based on the anodic redissolution of the enzymatically generated silver by cyclic voltammetry. The electrochemical behavior of this immunosensor was carefully evaluated assessing aspects as sensitivity, non-specific binding and matrix effects, and repeatability and reproducibility. The results were supported with a commercial ELISA test.

Lipid content and fatty acid profile of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles as affected by feed containing different amounts of plant protein sources

A growth trial with Senegalese Sole (Solea senegalensis Kaup, 1858) juveniles fed with diets containing increasing replacement levels of fishmeal by mixtures of plant protein sources was conducted over 12 weeks. Total fat contents of muscle, liver, viscera, skin, fins and head tissues were determined, as well as fatty acid profiles of muscle and liver (GC-FID analysis). Liver was the preferential local for fat deposition (5.5–10.8% of fat) followed by fins (3.4–6.7% fat). Increasing levels of plant protein in the diets seems to be related to increased levels of total lipids in the liver. Sole muscle is lean (2.4–4.0% fat), with total lipids being similar among treatments. Liver fatty acid profile varied significantly among treatments. Plant protein diets induced increased levels of C16:1 and C18:2 n -6 and a decrease in ARA and EPA levels. Muscle fatty acid profile also evidenced increasing levels of C18:2 n 6, while ARA and DHA remained similar among treatments. Substitution of fishmeal by plant protein is hence possible without major differences on the lipid content and fatty acid profile of the main edible portion of the fish – the muscle.

Analysis of polycyclic aromatic hydrocarbons in fish: optimisation and validation of microwave-assisted extraction

An accurate and sensitive method for determination of 18 polycyclic aromatic hydrocarbons (PAHs) (16 PAHs considered by USEPA as priority pollutants, dibenzo[a,l]pyrene and benzo[j]fluoranthene) in fish samples was validated. Analysis was performed by microwave-assisted extraction and liquid chromatography with photodiode array and fluorescence detection. Response surface methodology was used to find the optimal extraction parameters. Validation of the overall methodology was performed by spiking assays at four levels and using SRM 2977. Quantification limits ranging from 0.15–27.16 ng/g wet weight were obtained. The established method was applied in edible tissues of three commonly consumed and commercially valuable fish species (sardine, chub mackerel and horse mackerel) originated from Atlantic Ocean. Variable levels of naphthalene (1.03–2.95 ng/g wet weight), fluorene (0.34–1.09 ng/g wet weight) and phenanthrene (0.34–3.54 ng/g wet weight) were detected in the analysed samples. None of the samples contained detectable amounts of benzo[a]pyrene, the marker used for evaluating the occurrence and carcinogenic effects of PAHs in food.

Practical Quality Control: Comparision of Methods on the Quantification of Stationary Phases in Paper and Thin-Layer Chromatographic Systems

Introduction: Paper and thin layer chromatography methods are frequently used in Classic Nuclear Medicine for the determination of radiochemical purity (RCP) on radiopharmaceutical preparations. An aliquot of the radiopharmaceutical to be tested is spotted at the origin of a chromatographic strip (stationary phase), which in turn is placed in a chromatographic chamber in order to separate and quantify radiochemical species present in the radiopharmaceutical preparation. There are several methods for the RCP measurement, based on the use of equipment as dose calibrators, well scintillation counters, radiochromatografic scanners and gamma cameras. The purpose of this study was to compare these quantification methods for the determination of RCP. Material and Methods: 99mTc-Tetrofosmin and 99mTc-HDP are the radiopharmaceuticals chosen to serve as the basis for this study. For the determination of RCP of 99mTc-Tetrofosmin we used ITLC-SG (2.5 x 10 cm) and 2-butanone (99mTc-tetrofosmin Rf = 0.55, 99mTcO4- Rf = 1.0, other labeled impurities 99mTc-RH RF = 0.0). For the determination of RCP of 99mTc-HDP, Whatman 31ET and acetone was used (99mTc-HDP Rf = 0.0, 99mTcO4- Rf = 1.0, other labeled impurities RF = 0.0). After the development of the solvent front, the strips were allowed to dry and then imaged on the gamma camera (256x256 matrix; zoom 2; LEHR parallel-hole collimator; 5-minute image) and on the radiochromatogram scanner. Then, strips were cut in Rf 0.8 in the case of 99mTc-tetrofosmin and Rf 0.5 in the case of 99mTc-HDP. The resultant pieces were smashed in an assay tube (to minimize the effect of counting geometry) and counted in the dose calibrator and in the well scintillation counter (during 1 minute). The RCP was calculated using the formula: % 99mTc-Complex = [(99mTc-Complex) / (Total amount of 99mTc-labeled species)] x 100. Statistical analysis was done using the test of hypotheses for the difference between means in independent samples. Results:The gamma camera based method demonstrated higher operator-dependency (especially concerning the drawing of the ROIs) and the measures obtained using the dose calibrator are very sensitive to the amount of activity spotted in the chromatographic strip, so the use of a minimum of 3.7 MBq activity is essential to minimize quantification errors. Radiochromatographic scanner and well scintillation counter showed concordant results and demonstrated the higher level of precision. Conclusions: Radiochromatographic scanners and well scintillation counters based methods demonstrate to be the most accurate and less operator-dependant methods.

Sensors for the detection and quantification of bacterial contamination in water for human use

The deterioration of water quality by Cyanobacteria cause outbreaks and epidemics associated with harmful diseases in Humans and animals because of the toxins that they release. Microcystin-LR is one of the hepatotoxins most widely studied and the World Health Organization, recommend a maximum value of 1mgL 1 in drinking water. Highly specific recognition molecules, such as molecular imprinted polymers are developed to quantify microcystins in waters for human use and shown to be of great potential in the analysis of these kinds of samples. The obtained results were auspicious, the detection limit found, 1.5mgL 1, being of the same order of magnitude as the guideline limit recommended by the WHO. This technology is very promising because the sensors are stable and specific, and the technology is inexpensive and allows for rapid on-site monitoring.

A multiresidue method for the analysis of carbamate and urea pesticides from soils by microwave-assisted extraction and liquid chromatography with photodiode array detection

A multiresidue approach using microwave-assisted extraction and liquid chromatography with photodiode array detection was investigated for the determination of butylate, carbaryl, carbofuran, chlorpropham, ethiofencarb, linuron,metobromuron, and monolinuron in soils. The critical parameters of the developed methodology were studied. Method validation was performed by analyzing freshly and aged spiked soil samples. The recoveries and relative standard deviations reached using the optimized conditions were between 77.0 ± 0.46% and 120 ± 2.9% except for ethiofencarb (46.4 ± 4.4% to 105 ± 1.6%) and butylate (22.1 ± 7.6% to 49.2 ± 11%). Soil samples from five locations of Portugal were analysed.

Screening of carbamates and ureas in fresh and processed tomato samples using micowave-assisted extraction and liquid chromatography

An analytical method, based on microwave-assisted extraction and liquid chromatography with diode array detection, for the determination of six carbamate and three urea pesticides in fresh and processed tomato samples is described. Significant parameters affecting extraction efficiency were optimized. Under optimum microwave-assisted extraction conditions (20mL acetonitrile, for 10 minutes, at 60º C), pesticides were extracted with recoveries ranging from 57.6 to 102% (RSDs<7%). Quantification limits between 6.5 and 39.6 µg=kg were obtained. A total number of 28 different fresh tomato samples and 6 processed tomato products were analysed. Confirmation of suspicious samples was performed by LC-MS.

Quantification of endocrine disruptors and pesticides in water by gas chromatography–tandem mass spectrometry. Method validation using weighted linear regression schemes

Amulti-residue methodology based on a solid phase extraction followed by gas chromatography–tandem mass spectrometry was developed for trace analysis of 32 compounds in water matrices, including estrogens and several pesticides from different chemical families, some of them with endocrine disrupting properties. Matrix standard calibration solutions were prepared by adding known amounts of the analytes to a residue-free sample to compensate matrix-induced chromatographic response enhancement observed for certain pesticides. Validation was done mainly according to the International Conference on Harmonisation recommendations, as well as some European and American validation guidelines with specifications for pesticides analysis and/or GC–MS methodology. As the assumption of homoscedasticity was not met for analytical data, weighted least squares linear regression procedure was applied as a simple and effective way to counteract the greater influence of the greater concentrations on the fitted regression line, improving accuracy at the lower end of the calibration curve. The method was considered validated for 31 compounds after consistent evaluation of the key analytical parameters: specificity, linearity, limit of detection and quantification, range, precision, accuracy, extraction efficiency, stability and robustness.

Screening of grapes and wine for azoxystrobin, kresoxim-methyl and trifloxystrobin fungicides by HPLC with diode array detection

The Quinone outside Inhibitors (QoI) are one of the most important and recent fungicide groups used in viticulture and also allowed by Integrated Pest Management. Azoxystrobin, kresoxim-methyl and trifloxystrobin are the main active ingredients for treating downy and powdery mildews that can be present in grapes and wines. In this paper, a method is reported for the analysis of these three QoI-fungicides in grapes and wine. After liquid–liquid extraction and a clean-up on commercial silica cartridges, analysis was by isocratic HPLC with diode array detection (DAD) with a run time of 13 min. Confirmation was by solid-phase micro-extraction (SPME), followed by GC/MS determination. The main validation parameters for the three compounds in grapes and wine were a limit of detection up to 0.073mg kg-1, a precision not exceeding 10.0% and an average recovery of 93% ±38.

Biosensor based on multi-walled carbon nanotubes paste electrode modified with laccase for pirimicarb pesticide quantification

This study focused on the development of a sensitive enzymatic biosensor for the determination of pirimicarb pesticide based on the immobilization of laccase on composite carbon paste electrodes. Multi- walled carbon nanotubes(MWCNTs)paste electrode modified by dispersion of laccase(3%,w/w) within the optimum composite matrix(60:40%,w/w,MWCNTs and paraffin binder)showed the best performance, with excellent electron transfer kinetic and catalytic effects related to the redox process of the substrate4- aminophenol. No metal or anti-interference membrane was added. Based on the inhibition of laccase activity, pirimicarb can be determined in the range 9.90 ×10- 7 to 1.15 ×10- 5 molL 1 using 4- aminophenol as substrate at the optimum pH of 5.0, with acceptable repeatability and reproducibility (relative standard deviations lower than 5%).The limit of detection obtained was 1.8 × 10-7 molL 1 (0.04 mgkg 1 on a fresh weight vegetable basis).The high activity and catalytic properties of the laccase- based biosensor are retained during ca. one month. The optimized electroanalytical protocol coupled to the QuEChERS methodology were applied to tomato and lettuce samples spiked at three levels; recoveries ranging from 91.0±0.1% to 101.0 ± 0.3% were attained. No significant effects in the pirimicarb electro- analysis were observed by the presence of pro-vitamin A, vitamins B1 and C,and glucose in the vegetable extracts. The proposed biosensor- based pesticide residue methodology fulfills all requisites to be used in implementation of food safety programs.

Molinate quantification in environmental water by a glutathione-S-transferase based biosensor

A glutathione-S-transferase (GST)based biosensor was developed to quantify the thiocarbamate herbicide molinate in environmental water.The biosensor construction was based on GST immobilization onto a glassy carbon electrode via aminosilane–glutaraldehyde covalent attachment. The principle supporting the use of this biosensor consists of the GST inhibition process promoted by molinate. Differential pulse voltammetry was used to obtain a calibration curve for molinate concentration, ranging from 0.19 to 7.9 mgL -1 and presenting a detection limit of 0.064 mgL- 1. The developed biosensor is stable,and reusable during 15 days.The GST-based biosensor was successfully applied to quantify molinate in rice paddy field floodwater samples. The results achieved with the developed biosensor were in accordance with those obtained by high performance liquid chromatography. The proposed device is suitable for screening environmental water analysis and, since no sample preparation is required, it can be used in situ and in real-time measurements.

Determination of methiocarb and Its degradation products, methiocarb sulfoxide and methiocarb sulfone, in bananas using QuEChERS extraction

The present work describes the development of an analytical method for the determination of methiocarb and its degradation products (methiocarb sulfoxide and methiocarb sulfone) in banana samples, using the QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure followed by liquid chromatography coupled to photodiode array detector (LCPAD). Calibration curves were linear in the range of 0.5−10 mg L−1 for all compounds studied. The average recoveries, measured at 0.1 mg kg−1 wet weight, were 92.0 (RSD = 1.8%, n = 3), 84.0 (RSD = 3.9%, n = 3), and 95.2% (RSD = 1.9%, n = 3) for methiocarb sulfoxide, methiocarb sulfone, and methiocarb, respectively. Banana samples treated with methiocarb were collected from an experimental field. The developed method was applied to the analysis of 24 samples (peel and pulp) and to 5 banana pulp samples. Generally, the highest levels were found for methiocarb sulfoxide and methiocarb. Methiocarb sulfone levels were below the limit of quantification, except in one sample (not detected).

Laccase–Prussian blue film–graphene doped carbon paste modified electrode for carbamate pesticides quantification

A novel enzymatic biosensor for carbamate pesticides detection was developed through the direct immobilization of Trametes versicolor laccase on graphene doped carbon paste electrode functionalized with Prussianblue films (LACC/PB/GPE). Graphene was prepared by graphite sonication-assisted exfoliation and characterized by transmission electron microscopy and X-ray photoelectron spectro- scopy. The Prussian blue film electrodeposited onto graphene doped carbon paste electrode allowed considerable reduction of the charge transfer resistance and of the capacitance of the device.The combined effects of pH, enzyme concentration and incubation time on biosensor response were optimized using a 23 full-factorial statistical design and response surface methodology. Based on the inhibition of laccase activity and using 4-aminophenol as redox mediator at pH 5.0,LACC/PB/GPE exhibited suitable characteristics in terms of sensitivity, intra-and inter-day repeatability (1.8–3.8% RSD), reproducibility (4.1 and 6.3%RSD),selectivity(13.2% bias at the higher interference: substrate ratios tested),accuracy and stability(ca. twenty days)for quantification of five carbamates widely applied on tomato and potato crops.The attained detection limits ranged between 5.2×10−9 mol L−1(0.002 mg kg−1 w/w for ziram)and 1.0×10−7 mol L−1 (0.022 mg kg−1 w/w for carbofuran).Recovery values for the two tested spiking levels ranged from 90.2±0.1%(carbofuran)to 101.1±0.3% (ziram) for tomato and from 91.0±0.1%(formetanate)to 100.8±0.1%(ziram)for potato samples.The proposed methodology is appropriate to enable testing pesticide levels in food samples to fit with regulations and food inspections.

Adding value [valorization] to tannery fleshings: part II – effect of protein hydrolysates on leather dyeing

The tanning industry generates a high quantity of solid wastes. Therefore, there is a need to create valorization [added value] options for these wastes. The main objective of the present work was to study the effect of protein hydrolysates (HP) prepared from fleshings on leather dyeing. During previous studies it was found that the application of HP products, obtained from fleshings, in leather retannage intensified the colour of crust leather. In this work the CIELAB colour system was used to evaluate the effect of HP on retannage processes. The main conclusions of this study were: (i) HP can be used instead of a dicyanodiamide resin (Fortan DC) if the colour parameters of the standard procedure are to be maintained, and (ii) the replacement of an acrylic resin (Fortan A40) by glutaraldehyde-modified HP (GHP) results in a darker skin, and can therefore be interesting for the reduction of the quantity of dye used.

Carbon surfaces for the oxidative quantification of pravastatin: glassy-carbon vs. screen-printed carbon electrodes

The electrooxidative behavior of pravastatin (PRV) in aqueous media was studied by square-wave voltammetry at a glassycarbon electrode (GCE) and at a screen-printed carbon electrode (SPCE). Maximum peak current intensities in a pH 5.0 buffer were obtained at +1.3 V vs. AgCl/Ag and +1.0 V vs. Ag for the GCE and SPCE surface respectively. Validation of the developed methodologies revealed good performance characteristics and confirmed their applicability to the quantification of PRV in pharmaceutical products, without significant sample pretreatment. A comparative analysis between the two electrode types showed that SPCEs are preferred as an electrode surface because of their higher sensitivity and the elimination of the need to clean the electrode’s surface for its renewal, which frequently is, if not always, the rate-limiting step in voltammetric analysis.