Ultrasensitive detection of ovarian cancer marker using immunoliposomes and gold nanoelectrodes

Mucin-16 (MUC16) is the established ovarian cancer marker used to follow the disease during or after treatment for epithelial ovarian cancer. The emerging science of cancer markers also demands for the new sensitive detection methods. In this work, we have developed an electrochemical immunosensor for antigen MUC16 using gold nanoelectrode ensemble (GNEE) and ferrocene carboxylic acid encapsulated liposomes tethered with monoclonal anti-Mucin-16 antibodies ( MUC16). GNEEs were fabricated by electroless deposition of the gold within the pores of polycarbonate track-etched membranes. Afterwards, MUC16 were immobilized on preformed self-assembled monolayer of cysteamine on the GNEE via cross-linking with EDC-Sulfo-NHS. A sandwich immunoassay was performed on MUC16 functionalized GNEE with MUC16 and immunoliposomes. The differential pulse voltammetry was employed to quantify the faradic redox response of ferrocene carboxylic acid released from immunoliposomes. The dose–response curve for MUC16 concentration was found between the range of 0.001–300 U mL−1. The lowest detection limit was found to be 5 × 10−4 U mL−1 (S/N = 3). We evaluated the performance of this developed immunosensor with commercial ELISA assay by comparing results obtained from spiked serum samples and real blood serum samples from volunteers.

Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode

Bacterial food poisoning is an ever-present threat that can be prevented with proper care and handling of food products. A disposable electrochemical immunosensor for the simultaneous measurements of common food pathogenic bacteria namely Escherichia coli O157:H7 (E. coli), campylobacter and salmonella were developed. The immunosensor was fabricated by immobilizing the mixture of anti-E. coli, anticampylobacter and anti-salmonella antibodies with a ratio of 1:1:1 on the surface of the multiwall carbon nanotube-polyallylamine modified screen printed electrode (MWCNT-PAH/SPE). Bacteria suspension became attached to the immobilized antibodies when the immunosensor was incubated in liquid samples. The sandwich immunoassay was performed with three antibodies conjugated with specific nanocrystal ( -E. coli-CdS, -campylobacter-PbS and -salmonella-CuS) which has releasable metal ions for electrochemical measurements. The square wave anodic stripping voltammetry (SWASV) was employed to measure released metal ions from bound antibody nanocrystal conjugates. The calibration curves for three selected bacteria were found in the range of 1 × 103 – 5 × 105 cells mL−1 with the limit of detection (LOD) 400 cells mL−1 for salmonella, 400 cells mL−1 for campylobacter and 800 cells mL−1 for E. coli. The precision and sensitivity of this method show the feasibility of multiplexed determination of bacteria in milk samples.

Analysing organochlorine pesticides in strawberry jams using GC-ECD, GC-MS/MS and QuEChERS sample preparation

This paper describes a comparison of adaptations of the QuEChERS (quick, easy, cheap, effective, rugged and safe) approach for the determination of 14 organochlorine pesticide (OCP) residues in strawberry jam by concurrent use of gas chromatography (GC) coupled to electron capture detector (ECD) and GC tandem mass spectrometry (GC-MS/MS). Three versions were tested based on the original QuEChERS method. The results were good (overall average of 89% recoveries with 15% RSD) using the ultrasonic bath at five spiked levels. Performance characteristics, such as accuracy, precision, linear range, limits of detection (LOD) and quantification (LOQ), were determined for each pesticide. LOD ranged from 0.8 to 8.9 microg kg-1 ; LOQ was in the range of 2.5–29.8 microg kg- 1; and calibration curves were linear (r2>0.9970) in the whole range of the explored concentrations (5–100 microg kg- 1). The LODs of these pesticides were much lower than the maximum residue levels (MRLs) allowed in Europe for strawberries. The method was successfully applied to the quantification of OCP in commercially available jams. The OCPs were detected lower than the LOD.

Optimization of QuEChERS method for the analysis of organochlorine pesticides in soils with diverse organic matter

A QuEChERS method has been developed for the determination of 14 organochlorine pesticides in 14 soils from different Portuguese regions with wide range composition. The extracts were analysed by GC-ECD (where GC-ECD is gas chromatography-electron-capture detector) and confirmed by GC-MS/MS (where MS/MS is tandem mass spectrometry). The organic matter content is a key factor in the process efficiency. An optimization was carried out according to soils organic carbon level, divided in two groups: HS (organic carbon>2.3%) and LS (organic carbon<2.3%). Themethod was validated through linearity, recovery, precision and accuracy studies. The quantification was carried out using a matrixmatched calibration to minimize the existence of the matrix effect. Acceptable recoveries were obtained (70–120%) with a relative standard deviation of ≤16% for the three levels of contamination. The ranges of the limits of detection and of the limits of quantification in soils HS were from 3.42 to 23.77 μg kg−1 and from 11.41 to 79.23 μg kg−1, respectively. For LS soils, the limits of detection ranged from 6.11 to 14.78 μg kg−1 and the limits of quantification from 20.37 to 49.27 μg kg−1. In the 14 collected soil samples only one showed a residue of dieldrin (45.36 μg kg−1) above the limit of quantification. This methodology combines the advantages of QuEChERS, GC-ECD detection and GC-MS/MS confirmation producing a very rapid, sensitive and reliable procedure which can be applied in routine analytical laboratories.

Extraction of ochratoxin A in bread samples by the QuEChERS methodology

A QuEChERS method for the extraction of ochratoxin A (OTA) from bread samples was evaluated. A factorial design (23) was used to find the optimal QuEChERS parameters (extraction time, extraction solvent volume and sample mass). Extracts were analysed by LC with fluorescence detection. The optimal extraction conditions were: 5 g of sample, 15 mL of acetonitrile and 3 min of agitation. The extraction procedure was validated by systematic recovery experiments at three levels. The recoveries obtained ranged from 94.8% (at 1.0 μg kg -1) to 96.6% (at 3.0 μg kg -1). The limit of quantification of the method was 0.05 μg kg -1. The optimised procedure was applied to 20 samples of different bread types (‘‘Carcaça’’, ‘‘Broa de Milho’’, and ‘‘Broa de Avintes’’) highly consumed in Portugal. None of the samples exceeded the established European legal limit of 3 μg kg -1.

Analysis and simulation of a-Si Ha-SiC HPINIP structures for color image detection

It is presented in this paper a study on the photo-electronic properties of multi layer a-Si: H/a-SiC: H p-i-n-i-p structures. This study is aimed to give an insight into the internal electrical characteristics of such a structure in thermal equilibrium, under applied Was and under different illumination condition. Taking advantage of this insight it is possible to establish a relation among-the electrical behavior of the structure the structure geometry (i.e. thickness of the light absorbing intrinsic layers and of the internal n-layer) and the composition of the layers (i.e. optical bandgap controlled through percentage of carbon dilution in the a-Si1-xCx: H layers). Showing an optical gain for low incident light power controllable by means of externally applied bias or structure composition, these structures are quite attractive for photo-sensing device applications, like color sensors and large area color image detector. An analysis based on numerical ASCA simulations is presented for describing the behavior of different configurations of the device and compared with experimental measurements (spectral response and current-voltage characteristic). (c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pesticide residues in Portuguese strawberries grown in 2009–2010 using integrated pest management and organic farming

Pesticides are among the most widely used chemicals in the world. Because of the widespread use of agricultural chemicals in food production, people are exposed to low levels of pesticide residues through their diets. Scientists do not yet have a total understanding of the health effects of these pesticide residues. This work aims to determine differences in terms of pesticide residue content in Portuguese strawberries grown using different agriculture practices. The Quick, Easy, Cheap, Effective, Rugged, and Safe sample preparation method was conducted and shown to have good performance for multiclass pesticides extraction in strawberries. The screening of 25 pesticides residue was performed by gas chromatography–tandem mass spectrometry. In quantitative validation, acceptable performances were achieved with recoveries of 70–120 and <12 % residual standard deviation for 25 pesticides. Good linearity was obtained for all the target compounds, with highly satisfactory repeatability. The limits of detection were in the range of 0.1–28 μg/kg. The method was applied to analyze strawberry samples from organic and integrated pest management (IPM) practices harvested in 2009–2010. The results showed the presence of fludioxonil, bifenthrin, mepanipyrim, tolylfluanid, cyprodinil, tetraconazole, and malathion when using IPM below the maximum residue levels.

Optimization and validation of organochlorine compounds in adipose tissue by SPE-gas chromatography

Scientific evidence has shown an association between organochlorine compounds (OCC) exposure and human health hazards. Concerning this, OCC detection in human adipose samples has to be considered a public health priority. This study evaluated the efficacy of various solid-phase extraction (SPE) and cleanup methods for OCC determination in human adipose tissue. Octadecylsilyl endcapped (C18-E), benzenesulfonic acid modified silica cation exchanger (SA), poly (styrene-divinylbenzene (EN) and EN/RP18 SPE sorbents were evaluated. The relative sample cleanup provided by these SPE columns was evaluated using gas chromatography with electron capture detection (GC–ECD). The C18-E columns with strong homogenization were found to provide the most effective cleanup, removing the greatest amount of interfering substance, and simultaneously ensuring good analyte recoveries higher than 70%. Recoveries>70% with standard deviations (SD)<15% were obtained for all compounds under the selected conditions. Method detection limits were in the 0.003–0.009 mg/kg range. The positive samples were confirmed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The highest percentage found of the OCC in real samples corresponded to HCB, o,p′-DDT and methoxychlor, which were detected in 80 and 95% of samples analyzed respectively. Copyright © 2012 John Wiley & Sons, Ltd.

Automatic vehicle detection and classification

This paper presents a proposal for an automatic vehicle detection and classification (AVDC) system. The proposed AVDC should classify vehicles accordingly to the Portuguese legislation (vehicle height over the first axel and number of axels), and should also support profile based classification. The AVDC should also fulfill the needs of the Portuguese motorway operator, Brisa. For the classification based on the profile we propose:he use of Eigenprofiles, a technique based on Principal Components Analysis. The system should also support multi-lane free flow for future integration in this kind of environments.

Interlaboratory validation of an environmental monitoring method for trace analysis of endocrine disrupting compounds

Environmental pollution continues to be an emerging study field, as there are thousands of anthropogenic compounds mixed in the environment whose possible mechanisms of toxicity and physiological outcomes are of great concern. Developing methods to access and prioritize the screening of these compounds at trace levels in order to support regulatory efforts is, therefore, very important. A methodology based on solid phase extraction followed by derivatization and gas chromatography-mass spectrometry analysis was developed for the assessment of four endocrine disrupting compounds (EDCs) in water matrices: bisphenol A, estrone, 17b-estradiol and 17a-ethinylestradiol. The study was performed, simultaneously, by two different laboratories in order to evaluate the robustness of the method and to increase the quality control over its application in routine analysis. Validation was done according to the International Conference on Harmonisation recommendations and other international guidelines with specifications for the GC-MS methodology. Matrix-induced chromatographic response enhancement was avoided by using matrix-standard calibration solutions and heteroscedasticity has been overtaken by a weighted least squares linear regression model application. Consistent evaluation of key analytical parameters such as extraction efficiency, sensitivity, specificity, linearity, limits of detection and quantification, precision, accuracy and robustness was done in accordance with standards established for acceptance. Finally, the application of the optimized method in the assessment of the selected analytes in environmental samples suggested that it is an expedite methodology for routine analysis of EDC residues in water matrices.

An ultrasound based computer-aided diagnosis tool for steatosis detection

Liver steatosis is a common disease usually associated with social and genetic factors. Early detection and quantification is important since it can evolve to cirrhosis. In this paper, a new computer-aided diagnosis (CAD) system for steatosis classification, in a local and global basis, is presented. Bayes factor is computed from objective ultrasound textural features extracted from the liver parenchyma. The goal is to develop a CAD screening tool, to help in the steatosis detection. Results showed an accuracy of 93.33%, with a sensitivity of 94.59% and specificity of 92.11%, using the Bayes classifier. The proposed CAD system is a suitable graphical display for steatosis classification.

Global and local detection of liver steatosis from ultrasound

Liver steatosis is a common disease usually associated with social and genetic factors. Early detection and quantification is important since it can evolve to cirrhosis. Steatosis is usually a diffuse liver disease, since it is globally affected. However, steatosis can also be focal affecting only some foci difficult to discriminate. In both cases, steatosis is detected by laboratorial analysis and visual inspection of ultrasound images of the hepatic parenchyma. Liver biopsy is the most accurate diagnostic method but its invasive nature suggest the use of other non-invasive methods, while visual inspection of the ultrasound images is subjective and prone to error. In this paper a new Computer Aided Diagnosis (CAD) system for steatosis classification and analysis is presented, where the Bayes Factor, obatined from objective intensity and textural features extracted from US images of the liver, is computed in a local or global basis. The main goal is to provide the physician with an application to make it faster and accurate the diagnosis and quantification of steatosis, namely in a screening approach. The results showed an overall accuracy of 93.54% with a sensibility of 95.83% and 85.71% for normal and steatosis class, respectively. The proposed CAD system seemed suitable as a graphical display for steatosis classification and comparison with some of the most recent works in the literature is also presented.

Quantificação de metais potencialmente tóxicos em mexilhão dourado (Limnoperna fortunei) por espectrometria de absorção atômica com fonte contínua e alta resolução empregando amostragem direta de sólidos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The adjusted effect of maternal body mass index, energy and macronutrient intakes during pregnancy, and gestational weight gain on body composition of full-term neonates

Objective - To evaluate the effect of prepregnancy body mass index (BMI), energy and macronutrient intakes during pregnancy, and gestational weight gain (GWG) on the body composition of full-term appropriate-for-gestational age neonates. Study Design - This is a cross-sectional study of a systematically recruited convenience sample of mother-infant pairs. Food intake during pregnancy was assessed by food frequency questionnaire and its nutritional value by the Food Processor Plus (ESHA Research Inc, Salem, OR). Neonatal body composition was assessed both by anthropometry and air displacement plethysmography. Explanatory models for neonatal body composition were tested by multiple linear regression analysis. Results - A total of 100 mother-infant pairs were included. Prepregnancy overweight was positively associated with offspring weight, weight/length, BMI, and fat-free mass in the whole sample; in males, it was also positively associated with midarm circumference, ponderal index, and fat mass. Higher energy intake from carbohydrate was positively associated with midarm circumference and weight/length in the whole sample. Higher GWG was positively associated with weight, length, and midarm circumference in females. Conclusion - Positive adjusted associations were found between both prepregnancy BMI and energy intake from carbohydrate and offspring body size in the whole sample. Positive adjusted associations were also found between prepregnancy overweight and adiposity in males, and between GWG and body size in females.

Desenvolvimento de metodologias analíticas para a determinação de resíduos de pesticidas em Vinhos do Douro e Vinhos do Porto

A produção de vinho é uma actividade de elevada importância ao nível económico, ambiental e social. Como tal, a protecção da vinha e/ou da uva através da utilização de pesticidas assume um papel fundamental nesta actividade, permitindo um aumento no rendimento da produção. No entanto, a transferência destes compostos da uva para o vinho é inevitável, ocorrendo, na maioria dos casos, em quantidades reduzidas. Apesar de, geralmente, a quantidade de pesticidas que pode ser transferida para o vinho não apresentar problemas relevantes para a saúde pública, o desenvolvimento de metodologias que permitam garantir um controlo rigoroso da qualidade do vinho é de elevada importância. O controlo deste produto, assim como de qualquer produto alimentar, não deve ser de certa forma virtual, levando a uma desconfiança crescente do consumidor. Ao longo deste trabalho foram desenvolvidos dois métodos para a determinação de pesticidas em diferentes tipos de vinhos, por SPME-GC-MS/MS. O primeiro método desenvolvido visa a determinação de 8 pesticidas organofosforados e o segundo método permite a determinação de 7 pesticidas, 4 organoclorados e 3 dicarboximidas. Foram estudadas quatro matrizes diferentes: vinho branco do Douro, vinho tinto do Douro, vinho branco do Porto e vinho tinto do Porto. As metodologias adoptadas permitiram a obtenção de bons resultados, apesar das condições adoptadas não serem as mais vantajosas para alguns dos compostos estudados. Dada a complexidade das matrizes, a detecção por espectrometria de massa mostrou ser fundamental para a identificação inequívoca de cada um dos pesticidas. Verificaram-se, de um modo geral, bons resultados ao nível da linearidade, para as gamas de concentrações escolhidas, para os dois métodos. Obtiveram-se também bons resultados para os limites de detecção e quantificação, cujos valores se situam abaixo dos limites máximos de resíduos para as uvas, impostos pela regulamentação europeia, para a maioria dos compostos. No caso dos organofosforados, os limites de detecção variam entre 0,05 μg/L e 13,00 μg/L para os pesticidas clorpirifos-metilo e metidatião, respectivamente. Os limites de quantificação variam entre 0,18 μg/L e 43,32 μg/L, também para os pesticidas clorpirifos-metilo e metidatião. No que se refere ao método para os pesticidas organoclorados e dicarboximidas, foram calculados vários limites de detecção para cada composto, em cada matriz estudada, a partir das rectas de calibração diárias, resultando num conjunto de valores com variações significativas entre cada um, para o mesmo pesticida e na mesma matriz. No entanto, apenas os limites de quantificação foram validados, recorrendo-se aos estudos de repetibilidade e precisão intermédia. Obtiveram-se os seguintes limites de quantificação: captana 52,10 μg/L; clortalonil 20,95 μg/L; dicofol 4,37 μg/L; folpete 93,60 μg/L; iprodiona 274,70 μg/L; procimidona 76,04 μg/L e vinclozolina 10,03 μg/L. Os pesticidas metidatião e captana apresentam-se como os compostos mais problemáticos, uma vez que os limites de quantificação obtidos não permitem a garantia do cumprimento dos limites máximos de resíduos regulamentados. Demonstra-se também que, apesar do efeito de matriz ser significativo na determinação de todos os compostos analisados, o mesmo pode ser atenuado, procedendo-se às calibrações nas respectivas matrizes. Observaram-se variações significativas ao nível da resposta do equipamento ao longo do tempo, quer pela alteração das condições operatórias, quer pela decomposição de alguns compostos ao longo do tempo. Este efeito, evidenciado nos estudos das precisões intermédias e repetibilidades, levou à adopção de um método de validação diferente para a determinação de pesticidas organoclorados e dicarboximidas, recorrendo-se então a calibrações diárias e sequências de trabalho mais pequenas.