This work was funded by the European Union Seventh Framework Program FP7/2007-2013 under grant agreement no. 265721. The authors thank the RIKILT Institute for Food Safety (NL) for their support in this project.

Nota: O protocolo seguinte descreve a SPE realizada em amostra de 100 ml de água com sorvente C18 e eluição com 50% v / v de metanol. A amostra é diluída enriquecido para chegar a 10% v / v de metanol, antes da análise com um ensaio de imunoabsorção enzimática kit (ELISA). 1. Preparar os reagentes <ol><li> Prepare as amostras de água <ol><li> Antes de qualquer outra etapa, filtrar cada amostra de 100 ml de água com filtros de tamanho de poro de 0,2 um. </li><li> Pico a amostra com a concentração desejada por diluição volume apropriado da solução de referência para um volume de água. Por exemplo, preparar 100 mL de amostra de água com 100 ng / L de E2 através da diluição de 3,3 ml de solução de referência E2 a uma concentração de 300 ug / L. Diluir esta solução dez vezes para obter uma amostra enriquecida com 10 ng / L E2. Dilui-se esta última mais de dez vezes para se obter uma amostra de 100 ml com 1 ng / L E2. </li><li> Colocar a amostra filtrada (não modificada ou perfurantes) em um frasco de vidro com GLinha L45. Utilizar a amostra no mesmo dia. Pegue uma pequena fração de ser analisadas com o ELISA para estimar concentração inicial. </li></ol></li><li> Preparar 300 ul de eluente por diluição em metanol (di) água desionizada a 50% v / v em um tubo apertado. </li></ol> 2. Preparação da coluna de SPE <ol><li> Preparar uma solução 20 mg / ml, suspensão de partículas de octadecil-sílica absorventes, adicionando primeiros 1.600 ul de metanol e em seguida 400 ul de di-água a 40 mg de adsorvente de fase inversa. Feche bem a tampa e agitar com um vortex. </li><li> Instalação da membrana inferior na coluna <ol><li> Selecione uma membrana de nylon com tamanho de poros de 11 mm e coloque-a sobre uma dupla camada de tecido anti-poeira. Com um furador de diâmetro de 3 mm, cortar duas peças pequenas na membrana. </li><li> Pegar numa das pequenas membranas com uma extremidade plana fórceps de filtro e colocá-lo sobre um lado da coluna. </li><li> Parafuso do conector de fundo plano com o tubo e aperte. A membrana é now no lugar e o adsorvente pode ser adicionado de forma segura. Desenhar uma seta no corpo da coluna de apontamento para o fim em que a membrana foi colocada. </li></ol></li><li> Preparação da coluna de sorvente embalado <ol><li> Fixe a coluna no suporte com a seta apontada para o fundo. A coluna deve ser definido como na vertical quanto possível. </li><li> Anexar um vazio 10 ml seringa descartável para a extremidade do tubo da coluna, utilizando os conectores Luer-Lock. </li><li> Prepare uma micro-pipeta com uma ponta de 20-200 ï¿½, ajustar o volume para 100 l. Este formato de ponta de pipeta é adaptada ao tamanho da coluna de adsorvente a ser preparado. O procedimento seria mais difícil com pontas de pipeta maiores, como os usados ​​com pipetas de 1 ml. </li><li> Agita-se a suspensão sorvente com um vortex, e pipeta-se rapidamente 100 pi no centro sobre a coluna. Embora a injecção, aspirar suavemente pipetado toda a solução através da membrana usando a seringa com a outra mão. Neste sta idade, a solução de encher a seringa tem de estar livre de partículas, e um leito de partículas pode, eventualmente, ser observada na coluna. </li><li> Repetir este processo mais 2 vezes por agitação da suspensão de pasta entre todos os passos de pipetagem para garantir a suspensão homogénea das partículas em solução. A coluna resultante contém 6 mg de sorvente. </li><li> Quando os 300 ul de suspensão ter sido carregado e secou-se por aspiração com a seringa, manter a seringa na posição e colocar a segunda membrana de nylon sobre a parte superior, utilizando a pinça com a outra mão. </li><li> Aperte o segundo conector com tubo e descarte da seringa. A coluna de SPE está pronto para uso. </li></ol></li></ol> 3. Preparação do Sistema <ol><li> Aperte a coluna SPE no dispositivo usando os conectores Luer-Lock. A seta aponta para deve ser na mesma direcção que a mostrada no dispositivo. </li><li> Ligue o frasco contendo a amostra para o dispositivo por enroscamento afornecida tampa de segurança GL45 nele. </li><li> De carga de 200 mL de eluente no reservatório &quot;Eluente &#39;. </li><li> Carga de 800 ul de di-água no reservatório &quot;diluição&quot;. </li><li> Coloque uma garrafa na saída de resíduos para recolher a água processada durante a etapa de extração. O formato não é importante, mas o volume tem de ser suficientemente grande no que diz respeito ao volume da amostra. </li><li> Coloque um pequeno frasco na saída do sensor com a capacidade mínima volume de 1,5 ml. Um volume máximo de 1 ml será muito pequeno como bolhas se formarão neste saída ao coletar eluente enriquecido e tampão de diluição. </li><li> Verifique se o regulador de pressão está na posição fechada, rodando-a manualmente, inverter-horário até novo movimento não é possível. </li></ol> 4. SPE com o Prototype <ol><li> Ligue o protótipo pressionando o botão na parte de trás. </li><li> Preparando a interface do usuário e selecionar o programa <ol><li> Abra a interface do usuário. Selecionara porta de comunicação do computador ao qual o dispositivo está conectado a partir da lista e clique no botão &quot;Next&quot;. </li><li> Digite os valores para 580 pset e 30 para dpset. A bomba irá ajustar para manter a pressão no sistema pressurizado e reservatórios em 580 ± 30 mbar quando em execução. </li><li> Selecione o modo automatizado. </li><li> No canto modo automatizado, carregar o arquivo de programa no &quot;caminho do arquivo de configuração &#39;caixa. </li></ol></li><li> Ajustar o regulador de pressão <ol><li> Comece a bomba. </li><li> Gire manualmente o regulador de pressão até que o valor lido para preg é inferior mas próximo de 320 mbar. </li><li> Parar a bomba. </li></ol></li><li> Iniciar o procedimento de SPE <ol><li> Prima &#39;start&#39; no canto automatizado modo. A bomba irá ligar e as etapas de extração, eluição e diluição seguirá automaticamente o outro. Todo o procedimento para uma amostra de 100 mL é realizada em 50 min. </li><li>Verifique se o valor de preg. Ele deve estar na gama de 320 - 350 mbar durante a fase de extracção para assegurar uma taxa de fluxo óptima. </li><li> Feche o pequeno frasco e armazenar a 4-5 ° C no escuro até à análise. Realizar a análise em 30 horas a seguir para evitar a degradação da substância a analisar. </li><li> Elimine a água processada. </li></ol></li><li> Limpeza do sistema Nota: Após cada procedimento de extração do sistema precisa ser limpo para evitar a contaminação cruzada. <ol><li> Prepara-se uma solução de 10 ml de metanol a 70% v / v em um frasco de vidro de 45 GL. </li><li> Desligue a coluna SPE e ligue o tubo com conectores. </li><li> No modo automático, selecione o arquivo &#39;limpeza&#39; e iniciá-lo com as configurações mesma pressão. </li></ol></li></ol> 5. Detecção de Concentração de Estradiol com ELISA <ol><li> Preparar as amostras de calibração com concentrações, tal como indicado no protocolo fornecido com o kit de ELISA que é utilizado. Prepare uma calibração definido com a mesma matriz, como a amostra, e um conjunto de calibragem com metanol a 10% v / v. </li><li> Retirar a quantidade necessária de amostra nos poços na placa, de acordo com as instruções do fabricante. Use amostras de calibração, as amostras de água não modificados e perfurantes que foram filtrados, mas não processados ​​com a SPE, e amostras enriquecidas em metanol a 10% v / v. Use 3 poços por amostra para reduzir o erro associado com o ensaio. </li><li> Seguir as indicações do protocolo que é fornecido com o kit para a adição de reagentes, tempo de incubação, lavagem e posterior reacção com a enzima. </li><li> Ler o sinal óptico em cada poço de acordo com as instruções do fabricante do kit com um instrumento leitor de placas e recolher os dados. </li><li> Utilizar o software do instrumento leitor de placas para se ajustar às curvas de calibração e determinar a concentração de E2 na amostra inicial com mesma matriz, e nas amostras enriquecidas com metanol a 10% v / v. </li></ol>

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The authors have nothing to disclose.

Foi proposto um novo método para a preparação de amostras de água seguida por análise utilizando imunoensaio. O instrumento permite realizar extração em fase sólida de forma automatizada e fácil de usar. A filtração da amostra de água antes da sua injecção no sistema é crítico. Quaisquer partículas ainda presentes na solução seria, potencialmente, causar o entupimento da rede de fluidos e obstruir a coluna de SPE. Outro passo importante é a preparação da coluna de SPE. ...

A preparação da amostra é uma etapa importante em qualquer processo analítico. Em particular, a remoção dos efeitos de matriz, diminuição das interferências, e enriquecimento da substância a analisar são necessárias para obter resultados precisos e atingem baixos limites de detecção. Compostos de desregulação endócrina (EDC) são particularmente preocupantes devido à sua ação sobre os organismos vivos, mesmo quando presentes em níveis muito baixos no meio ambiente. O hormônio natural 17β-estradiol está presente na poluição da água UE Watch List e propenso a ser adicionado à lista de substâncias prioritárias regulamentados pela Directiva-Quadro da Água da UE. Extracção em fase sólida (SPE), é geralmente utilizado para a análise de pequenas poluentes na água, com tanto química 1-5 (cromatografia, espectrometria de massa) e métodos de detecção imunológicos 6-9. Este último ganhou interesse no campo da monitorização ambiental, como imunoensaios estão disponíveis em grande variedade de formatos, são específicas para a TArPegue analito, e atingir baixos limites de detecção. 6, 7, 10, 11 Vários ensaios imunossorventes ligados a enzimas (ELISA) estão comercialmente disponíveis e permitem analisar várias amostras ao mesmo tempo sobre uma placa multi-poços. O procedimento consiste em passos de reacção sucessivos que podem levar algumas horas. O produto final da reacção pode ser detectado opticamente para determinar a concentração da molécula alvo com base em uma curva de calibração. SPE procedimentos clássicos incluem sorvente pré-condicionado, extracção da amostra, a lavagem, a eluição, e concentração por evaporação do eluente. O solvente utilizado para a diluição deste extracto é escolhido dependendo do método de detecção. 12 Para os métodos imunológicos, a quantidade de solvente orgânico influências fortemente a sensibilidade do método. Para além da recuperação e os desempenhos de pré-concentração, o método também tem de ser simples e de custo eficiente. Automação do Procedure ajuda a reduzir erros humanos-relacionados. No nosso trabalho anterior 13 que introduzida a protótipo para SPE automatizado, e o nosso método foi aplicado para a análise da hormona natural 17β-estradiol nas amostras de água do mar. Com o presente vídeo, gostaríamos de destacar as vantagens técnicas do nosso método em comparação com off-line tradicional e on-line SPE, e em particular a sua compatibilidade com detecção por imuno-reacções. Nós descrevemos o protocolo aplicado a amostras de água para a detecção de 17β-estradiol. SPE é realizada com octadecil-silica (C18) de fase sorvente e a eluição é realizada com metanol diluído.

<table><tbody><tr><td>Filter membrane 0.2 &amp;mu;m pore size</td><td>Merck Millipore</td><td>GNWP04700</td><td>For sample filtration</td></tr><tr><td>Nylon membrane 11 &amp;mu;m pore size</td><td>Merck Millipore</td><td>NY1104700</td><td>For SPE column</td></tr><tr><td>Disposable biopsy punch 5 mm</td><td>Medical Budget</td><td>39302439</td><td></td></tr><tr><td>Nucleodur C18 ec&amp;nbsp;</td><td>Macherey Nagel</td><td>713550.01</td><td>50 &amp;mu;m particle diameter</td></tr><tr><td>Synthetic sea water</td><td>Sigma Aldrich</td><td>SSWS500-500ML</td><td></td></tr><tr><td>Methanol</td><td>VWR</td><td></td><td></td></tr><tr><td>17beta-estradiol standard</td><td>Enzo Life Science</td><td></td><td>300 ng/ml</td></tr><tr><td>17beta-estradiol ELISA kit</td><td>Enzo Life Science</td><td>ADI-900-008</td><td>96 wells, range 30 - 3,000 ng/L</td></tr></tbody></table>

a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants

Um novo método de extracção em fase sólida (SPE) de amostras de água do ambiente é proposto. O protótipo desenvolvido é amigável custo-eficiente e usuário, e permite realizar SPE rápido, automatizado e simples. A solução de pré-concentrado é compatível com a análise por imunoensaio, com um conteúdo de solvente orgânico de baixo. É descrito um método para a extracção e concentração da pré-hormona natural 17β-estradiol em 100 ml de amostras de água. SPE de fase reversa é efectuada com octadecil-sílica adsorvente e a eluição é feita com 200 ul de metanol a 50% v / v. Eluente é diluída por adição de di-água para baixar a quantidade de metanol. Depois de preparar manualmente a coluna SPE, o procedimento geral é realizada automaticamente dentro de 1 hora. No final do processo, a concentração de estradiol é medido usando um ensaio imuno-sorvente comercial ligado a enzima (ELISA). 100 vezes pré-concentração é conseguida e o teor de metanol em apenas 10% v / v. Recuperações de pleno direito da moléculasão alcançados com 1 ng / L amostras adicionadas desionizada e água do mar sintética.

A reprodutibilidade da embalagem sorvente foi avaliada por secagem e pesagem do sorvente pipetado em frascos de vidro e o resultado é mostrado na Figura 1. A reprodutibilidade do tempo de injecção foi testado para as amostras de 100 ml, como mostrado na Figura 2. A concentração in inicial e pré- concentrado cravado amostras foram determinadas usando um kit comercial de ELISA para 17β-estradiol e são mostrados <st...

Watch this Scientific Journal Video about A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants at JoVE.com

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Um método simples para a extração automatizada de fase sólida das amostras de água para análise imunológica de Pequenas Poluentes

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

a-simple-method-for-automated-solid-phase-extraction-water-samples

Universidad San Sebastian

Research

JoVE Journal

Environment

13.6K visualizações.  Centre Suisse d'Electronique et de Microtechnique. A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Vídeo: A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Automated Gel seleção de tamanho para melhorar a qualidade das bibliotecas de sequenciação de próxima geração Preparado a partir de amostras de água do ambiente

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Ambiente

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

Preparação e Teste de impedância baseado em Biochips fluídicas com células RTgill-W1 para avaliação rápida das amostras de água para a toxicidade

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Caracterização e aplicação de amostradores passivos de monitorização de pesticidas em água

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP, um método melhorado para Inex<em&gt; In Situ</em&gt; Medição de partículas e dissolvidos compostos Processado por alimentadores de suspensão ativa

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Uma variante do método EPA 1623 que usa fluxo tangencial ultrafiltração de fibra oca e Passos de calor de dissociação para detectar Waterborne<em&gt; Cryptosporidium</em&gt; E<em&gt; Giardia spp.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Imunologia e Infecção

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Identificação por- e espécies químicas perfluorados com um fluxo de trabalho combinada alvo e não alvo-triagem de alta resolução de espectrometria de massa

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

Fabricação de um dipolo-assistida Fase Sólida Extração Microchip para análise de metais traço em amostras de água

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Bioengenharia

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Plataforma de Microfluidos Digitais baseada em eletrowetting para ensaio imunosorbent automatizado ligado à enzima

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Solid-Liquid Extraction

Source: Laboratory of Dr. Jay Deiner &#8212; City University of New York
Extraction is a crucial step in most chemical analyses. It entails removing the analyte from its sample matrix and passing it into the phase required for spectroscopic or chromatographic identification and quantification. When the sample is a solid and the required phase for analysis is a liquid, the process is called solid-liquid extraction. A simple and broadly applicable form of solid-liquid extraction entails combining the solid with a solvent in which the analyte is soluble. Through agitation, the analyte partitions into the liquid phase, which may then be separated from the solid through filtration. The choice of solvent must be made based on the solubility of the target analyte, and on the balance of cost, safety, and environmental concerns.

Solid-Liquid Extraction: Principle, Procedure, Applications

Extração sólido-líquido

Source: Laboratory of Dr. Jay Deiner &#8212; City University of New York
Extraction is a crucial step in most chemical analyses. It entails removing the ...

Education

JoVE Science Education

Química

Química orgânica

Extraction of Biomarkers from Sediments - Accelerated Solvent Extraction

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The distribution of a group of organic biomarkers called glycerol-dialkyl glycerol-tetraethers (GDGTs), produced by a suite of archaea and bacteria, were found in modern sediments to change in a predictable manner in response to air or water temperature1,2. Therefore, the distribution of these biomarkers in a sequence of sediments of known age can be used to reconstruct the evolution of air and/or water temperature on decadal to millennial timescales (Figure 1). The production of long high-resolution records of past climates, called paleoclimatology, depends on the rapid analysis of hundreds, possibly thousands of samples. Older extraction techniques, such as sonication or Soxhlet, are too slow. However, the newer Accelerated Solvent Extraction technique was designed with efficiency in mind.
<img alt="Figure 1" src="/files/ftp_upload/10097/10097fig1.jpg" /> 
Figure 1. An example of a paleoclimate record showing changes in sea surface temperature (SST) in the eastern Mediterranean Sea during the past ~27,000 years3. This record comprises ~115 samples and is based on the isoprenoidal GDGT-based TEX86 SST proxy.

Accelerated Solvent Extraction of Total Lipid from Sediments

Extração de Biomarcadores de Sedimentos - Extração Acelerada por Solventes

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
The distribution of a group of organic biomarkers called glycerol-dialkyl ...

Um método simples para a extração automatizada de fase sólida das amostras de água para análise imunológica de Pequenas Poluentes

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