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: Il protocollo seguente descrive la SPE effettuato su 100 ml del campione d&#39;acqua con C18 sorbente e eluizione con 50% v / v di metanolo. Il campione arricchito viene diluita a raggiungere il 10% v / v di metanolo prima analisi con un enzima linked immunosorbent assay (ELISA) Kit. 1. Preparazione dei reagenti <ol><li> Preparare i campioni di acqua <ol><li> Prima di qualsiasi altro passo, filtrare ogni campione di 100 ml di acqua con filtri a 0,2 micron di dimensione dei pori. </li><li> Additiva con concentrazione desiderata diluendo il volume appropriato di soluzione di riferimento in un volume di acqua. Ad esempio, preparare 100 ml di campione con 100 ng / L di E2 diluendo 3,3 ml di soluzione di riferimento E2 ad una concentrazione di 300 mg / L. Diluire questa soluzione dieci volte per ottenere un campione a spillo con 10 ng / L E2. Diluire quest&#39;ultima altri dieci volte per ottenere un campione di 100 ml con 1 ng / L E2. </li><li> Collocare il campione filtrato (non modificato o spillo) in una bottiglia di vetro con GFilo L45. Utilizzare l&#39;esempio lo stesso giorno. Prendere una piccola frazione da analizzare con il test ELISA per stimare concentrazione iniziale. </li></ol></li><li> Preparare 300 ml di eluente diluendo in metanolo (di-) acqua deionizzata al 50% v / v in un tubo stretto. </li></ol> 2. Preparazione della colonna SPE <ol><li> Preparare a 20 mg / ml sospensione di particelle adsorbenti ottadecil-silice aggiungendo primi 1.600 ml di metanolo e poi 400 ml di acqua demineralizzata a 40 mg di sorbente in fase inversa. Strettamente chiudere il coperchio e agitare con un vortice. </li><li> Installazione della membrana inferiore della colonna <ol><li> Seleziona una membrana di nylon con dimensione dei pori 11 micron e metterlo su un doppio strato di tessuto anti-polvere. Con un diametro del punzone 3 mm, tagliare due piccole parti nella membrana. </li><li> Afferrare una delle piccole membrane con TV a fascia pinza filtra e posizionarlo su un lato della colonna. </li><li> Avvitare il connettore a fondo piatto con il tubo e stringere. La membrana è now nel luogo e l&#39;assorbente può essere aggiunto in modo sicuro. Disegnare una freccia sul corpo della colonna di puntamento verso la fine dove è stata posta la membrana. </li></ol></li><li> Preparazione della colonna assorbente imballato <ol><li> Fissare la colonna a supporto con la freccia rivolta verso il basso. La colonna deve essere impostato più verticale possibile. </li><li> Attaccare un vuoto 10 ml siringa monouso all&#39;estremità del tubo della colonna, utilizzando i connettori Luer-Lock. </li><li> Preparare un micro-pipetta con una punta di 20-200 microlitri, impostare il volume di 100 l. Questo formato puntale viene adattata alle dimensioni della colonna sorbente essere preparati. La procedura sarebbe più difficile con punte per pipette più grandi, come quelli usati con 1 ml pipette. </li><li> Agitare la sospensione sorbente con un vortice, e rapidamente pipettare 100 microlitri al centro della colonna. Mentre l&#39;iniezione, aspirare delicatamente tutta la soluzione pipettato trogolo membrana utilizzando la siringa con l&#39;altra mano. A questo stl&#39;età, la soluzione di riempimento della siringa deve essere libera da particelle, e un letto particella può eventualmente essere osservata nella colonna. </li><li> Ripetere questa operazione altre 2 volte agitando la sospensione di scorta tra tutti fasi di pipettamento per garantire la sospensione omogenea delle particelle nella soluzione. La colonna risultante contiene 6 mg di assorbente. </li><li> Quando i 300 ml di sospensione sono stati caricati ed essiccato aspirando con la siringa, tenere la siringa in posizione e posizionare la seconda membrana di nylon sulla parte superiore usando le pinze con l&#39;altra mano. </li><li> Avvitare il secondo connettore con il tubo e smaltire la siringa. La colonna SPE è pronto all&#39;uso. </li></ol></li></ol> 3. Preparazione del sistema <ol><li> Serrare colonna SPE il dispositivo utilizzando i connettori Luer-Lock. La freccia deve puntare nella stessa direzione come quella mostrata sul dispositivo. </li><li> Collegare la bottiglia contenente il campione al dispositivo avvitando ilprevisto tappo di sicurezza GL45 su di esso. </li><li> Carico 200 ml di eluente nel serbatoio &#39;Eluente&#39;. </li><li> Carico 800 ml di di-acqua nel serbatoio &#39;diluizione&#39;. </li><li> Posizionare una bottiglia all&#39;uscita rifiuti per raccogliere l&#39;acqua elaborati durante fase di estrazione. Il formato non è importante, ma il volume deve essere sufficientemente grande per quanto riguarda il volume del campione. </li><li> Mettere una piccola fiala all&#39;uscita sensore con capacità minima volume di 1,5 ml. Un volume massimo di 1 ml sarà troppo piccolo come le bolle si formano in questo sbocco al momento del ritiro eluente arricchito e tampone di diluizione. </li><li> Verificare il regolatore di pressione è in posizione chiusa ruotando manualmente, reverse-orario fino ulteriore movimento non è possibile. </li></ol> 4. SPE con il Prototype <ol><li> Accendere il prototipo premendo il pulsante sul retro. </li><li> Preparare l&#39;interfaccia utente e selezionando il programma <ol><li> Aprire l&#39;interfaccia utente. Selezionarela porta di comunicazione del computer a cui il dispositivo è collegato dalla lista, e fare clic sul pulsante &#39;Avanti&#39;. </li><li> Immettere i valori per 580 pset e 30 per dpset. La pompa regolerà per mantenere la pressione nel sistema di pressione e serbatoi a 580 ± 30 mbar durante l&#39;esecuzione. </li><li> Selezionare la modalità automatica. </li><li> Nell&#39;angolo modalità automatica, caricare il file di programma nel &#39;percorso del file di configurazione&#39; scatola. </li></ol></li><li> Regolazione del regolatore di pressione <ol><li> Avviare la pompa. </li><li> Attivare manualmente il regolatore di pressione fino a quando il valore letto per preg è inferiore ma vicino a 320 mbar. </li><li> Fermare la pompa. </li></ol></li><li> Avvio della procedura di SPE <ol><li> Premere &#39;start&#39; nell&#39;angolo modalità automatica. La pompa si accenderà e le fasi di estrazione, eluizione e diluizione seguirà automaticamente l&#39;un l&#39;altro. L&#39;intera procedura per un campione di 100 ml viene eseguita in 50 min. </li><li>Verificare il valore di preg. Deve essere compreso nell&#39;intervallo 320 - 350 mbar durante la fase di estrazione per garantire una portata ottimale. </li><li> Chiudere la piccola fiala e conservare a 4-5 ° C al buio fino al momento dell&#39;analisi. Effettuare analisi nella seguente 30 ore per impedire la degradazione dell&#39;analita. </li><li> Smaltire l&#39;acqua trattata. </li></ol></li><li> Pulizia del sistema Nota: Al termine di ogni procedura di estrazione del sistema deve essere pulito per evitare la contaminazione incrociata. <ol><li> Preparare una soluzione di 10 ml di metanolo 70% v / v in una bottiglia di vetro GL 45. </li><li> Scollegare colonna SPE e collegare il tubo di connettori. </li><li> In modalità automatica, selezionare il file &#39;pulizia&#39; e avviarlo con impostazioni di pressione stessi. </li></ol></li></ol> 5. Individuazione di Estradiolo Concentrazione con ELISA <ol><li> Preparare i campioni di calibrazione con concentrazioni come indicato nel protocollo fornito con il kit ELISA utilizzato. Preparare uno calibrazione impostato con la stessa matrice del campione, e un set di calibrazione con metanolo 10% v / v. </li><li> Dispensare la quantità necessaria di campione nei pozzetti sulla piastra, secondo le istruzioni del produttore. Utilizzare campioni di calibrazione, campioni di acqua non modificati e spillo che sono stati filtrati, ma non trattati con SPE, e arricchito i campioni in metanolo 10% v / v. Utilizzare 3 pozzetti per campione per ridurre l&#39;errore associato con il test. </li><li> Seguire le indicazioni del protocollo fornito con il kit per l&#39;aggiunta dei reagenti, tempo di incubazione, lavaggio e ulteriore reazione con l&#39;enzima. </li><li> Leggere il segnale ottico in ogni pozzetto secondo le istruzioni del produttore corredo con uno strumento lettore di piastre e raccogliere i dati. </li><li> Utilizzare il software dello strumento lettore di piastre per adattarsi alle curve di calibrazione e determinare la concentrazione di E2 nel campione iniziale con stessa matrice, e nei campioni arricchiti con metanolo 10% v / v. </li></ol>

<ol>
	<li>Chang, H. S., Choo, K. H., Choi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methods+of+identification,+analysis+and+removal+of+endocrine+disrupting+compounds+(EDCs)+in+water.">The methods of identification, analysis and removal of endocrine disrupting compounds (EDCs) in water.</a> J. Hazard. Matter. 172, 1-12 (2009).</li><li>Azzouz, A., Souhail, B., Ballesteros, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+solid-phase+extraction+and+gas+chromatography-mass+spectrometry+determination+of+pharmaceuticals+and+hormones+in+water+samples.">Continuous solid-phase extraction and gas chromatography-mass spectrometry determination of pharmaceuticals and hormones in water samples.</a> J. Chromatogr. A. 1217, 2956-2963 (2010).</li><li>Tomsikova, H., Aufartova, J., Solich, P., Novakova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+analysis+of+female-steroid+hormones+in+environ-mental+samples.">High sensitivity analysis of female-steroid hormones in environ-mental samples.</a> Trends Anal. Chem. 34, 35-58 (2012).</li><li>Ciofi, L., Fibbi, D., Chiuminatto, U., Coppini, E., Checchini, L., Del Bubba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+on-line+solid+phase+extraction+coupled+to+high-performance+liquid+chromatography-tandem+mass+spectrometric+analysis+at+sub-ng/L+levels+of+selected+estrogens+in+surface+water+and+wastewater.">Fully automated on-line solid phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometric analysis at sub-ng/L levels of selected estrogens in surface water and wastewater.</a> J. Chromatogr. A. 1283, 53-61 (2013).</li><li>Robles-Molina, J., Lara-Ortega, F. J., Gilbert-Lopez, B., Garcia-Reyes, J. F., Molina-Diaz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-residue+method+for+the+determination+of+over+400+priority+and+emerging+pollutants+in+water+and+wastewater+by+solid-phase+extraction+and+liquid+chromatography-time-of-flight+mass+spectrometry.">Multi-residue method for the determination of over 400 priority and emerging pollutants in water and wastewater by solid-phase extraction and liquid chromatography-time-of-flight mass spectrometry.</a> J. Chromatogr. A. 1350, 30-43 (2014).</li><li>Huang, C. H., Sedlak, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+estrogenic+hormones+in+municipal+waste+water+effluent+and+surface+water+using+enzyme-linked+immunosorbent+assay+and+gas+chromatography/tandem+mass+spectrometry.">Analysis of estrogenic hormones in municipal waste water effluent and surface water using enzyme-linked immunosorbent assay and gas chromatography/tandem mass spectrometry.</a> Environ. Toxicol. Chem. 20, 133-139 (2001).</li><li>Hintemann, T., Schneider, C., Sch&#246;ler, H. F., Schneider, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+study+using+two+immunoassays+for+the+determination+of+estradiol+and+ethinylestradiol+in+the+aquatic+environment.">Field study using two immunoassays for the determination of estradiol and ethinylestradiol in the aquatic environment.</a> Water Res. 40, 2287-2294 (2006).</li><li>Farr&#233;, M., Kuster, M., Brix, R., Rubio, F., Lopez de Alda, M. J., Barcelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+an+estradiol+enzyme-linked+immunosorbent+assay+kit,+liquid+chromatography-tandem+mass+spectrometry,+and+ultra-performance+liquid+chromatography-quadrupole+time+of+flight+mass+spectrometry+for+part-per-trillion+analysis+of+estrogens+in+water+samples.">Comparative study of an estradiol enzyme-linked immunosorbent assay kit, liquid chromatography-tandem mass spectrometry, and ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry for part-per-trillion analysis of estrogens in water samples.</a> J. Chromatogr. A. 1160, 166-175 (2007).</li><li>Pu, C., Wu, Y. F., Yang, H., Deng, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+analysis+of+contraceptive+drug+levonorgestrel+in+waste+water+samples+by+a+newly+developed+indirect+competitive+enzyme-linked+immunosorbent+assay+(ELISA)+coupled+with+solid+phase+extraction.">Trace analysis of contraceptive drug levonorgestrel in waste water samples by a newly developed indirect competitive enzyme-linked immunosorbent assay (ELISA) coupled with solid phase extraction.</a> Anal. Chim. Acta. 628, 73-79 (2008).</li><li>Van Emon, J. M., Gerlach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+status+report+on+field-portable+immunoassay.">A status report on field-portable immunoassay.</a> Environ. Sci. Technol. 29 (7), 312A-317A (1995).</li><li>Farr&#233;, M., Brix, R., Barcel&#242;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+water+for+pollutants+using+biological+techniques+under+European+Union+funding+during+the+last+10+years.">Screening water for pollutants using biological techniques under European Union funding during the last 10 years.</a> Trends Anal. Chem. 24 (6), 532-545 (2005).</li><li>Schneider, C., Sch&#246;ler, H. F., Schneider, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+enzyme-linked+immunosorbent+assay+for+ethinylestradiol+using+a+long-chain+biotinylated+EE2+derivative.">A novel enzyme-linked immunosorbent assay for ethinylestradiol using a long-chain biotinylated EE2 derivative.</a> Steroids. 69, 245-253 (2004).</li><li>Heub, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+and+portable+solid+phase+extraction+platform+for+immuno-detection+of+17&#946;-estradiol+in+water.">Automated and portable solid phase extraction platform for immuno-detection of 17&#946;-estradiol in water.</a> J. Chrom. A. 1381, 22-28 (2015).</li><li>Petrovic, M., Eljarrat, E., Lopez de Alda, M. J., Barcelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+disrupting+compounds+and+other+merging+contaminants+in+the+environment:+a+new+survey+on+new+monitoring+strategies+and+occurrence+data.">Endocrine disrupting compounds and other merging contaminants in the environment: a new survey on new monitoring strategies and occurrence data.</a> Anal. Bioanal. Chem. 378, 549-562 (2004).</li><li>Richardson, S. D., Ternes, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+analysis:+Emerging+contaminants+and+current+issues.">Water analysis: Emerging contaminants and current issues.</a> Anal. Chem. 86, 2813-2848 (2014).</li></ol>

The authors have nothing to disclose.

È stato proposto un nuovo metodo per la preparazione dei campioni di acqua seguita da analisi mediante immunodosaggio. Lo strumento permette di eseguire l&#39;estrazione in fase solida in modo automatizzato e facile da usare. La filtrazione del campione di acqua prima della sua iniezione nel sistema è critica. Eventuali particelle ancora presenti nella soluzione potrebbe potenzialmente causare l&#39;intasamento della rete fluidica e ostacolare colonna SPE. Un altro passo importante è la p...

La preparazione del campione è un passo importante in ogni processo di analisi. In particolare, la rimozione degli effetti matrice, diminuzione delle interferenze e arricchimento dell&#39;analita sono necessari per ottenere risultati precisi e raggiungere limiti di rilevabilità. Sistema endocrino composti (IE) sono di particolare interesse per la loro azione su organismi viventi, anche quando presenti a livelli molto bassi per l&#39;ambiente. L&#39;ormone naturale 17β-estradiolo è presente sul inquinamento delle acque dell&#39;UE selezione e soggetto ad essere aggiunto alla lista delle sostanze prioritarie disciplinati dalla direttiva quadro sulle acque europee. Estrazione in fase solida (SPE) è comunemente applicato per l&#39;analisi di piccole inquinanti in acqua, sia chimica 1-5 (cromatografia, spettrometria di massa) e immunologici 6-9 metodi di rilevamento. Quest&#39;ultimo ha guadagnato interesse nel campo del monitoraggio ambientale, come saggi immunologici sono disponibili in un&#39;ampia varietà di formati, sono specifici per la taanalita rget, e raggiungere limiti di rilevabilità. 6, 7, 10, 11 Vari Enzyme Linked immunoassorbimento (ELISA) sono commercialmente disponibili e consentono di analizzare più campioni contemporaneamente su una piastra multi-pozzo. La procedura consiste in fasi successive di reazione che possono prendere un paio d&#39;ore. Il prodotto finale di reazione può essere rilevato otticamente per determinare la concentrazione della molecola bersaglio sulla base di una curva di calibrazione. Procedure SPE Classical includono sorbente pre-condizionamento, dell&#39;estrazione del campione, lavaggio, eluizione e concentrazione per evaporazione dell&#39;eluente. Il solvente utilizzato per la diluizione di questo estratto è scelto in funzione del metodo di rilevamento. Per i metodi immunologici, la quantità di solvente organico influenze fortemente la sensibilità del metodo. 12 Oltre al recupero e le prestazioni pre-concentrazione, il metodo deve anche essere semplice ed economicamente conveniente. Automazione dei SEGUIRE IL PROCEDIMENTOe aiuta a ridurre gli errori umani legati. Nel nostro lavoro precedente 13 abbiamo introdotto il nostro prototipo per SPE automatizzata, e il nostro metodo è stato applicato all&#39;analisi della ormone naturale 17β-estradiolo in campioni di acqua di mare. Con la presente il video che vorremmo evidenziare i vantaggi tecnici del nostro metodo rispetto alle tradizionali off-line e on-line SPE, e la sua particolare compatibilità con rilevamento da immuno-reazioni. Descriviamo il protocollo applicato ai campioni di acqua per la rilevazione di 17β-estradiolo. SPE viene eseguita con octadecil-silice (C18) Fase sorbente e eluizione viene eseguita con metanolo diluito.

<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

Si propone un nuovo metodo per l&#39;estrazione in fase solida (SPE) di campioni di acqua ambientale. Il prototipo sviluppato è conveniente e facile da usare, e consente di eseguire un rapido, automatizzato e semplice SPE. La soluzione pre-concentrata è compatibile con l&#39;analisi mediante immunodosaggio, con un basso contenuto di solvente organico. Un metodo è descritto per l&#39;estrazione e pre-concentrazione di ormone naturale 17β-estradiolo in 100 ml campioni di acqua. Fase inversa SPE viene eseguita con sorbente ottadecil-silice e eluizione avviene con 200 ml di metanolo al 50% v / v. Eluente viene diluito con l&#39;aggiunta di acqua per abbassare la quantità di metanolo. Dopo aver preparato manualmente colonna SPE, la procedura generale viene eseguita automaticamente entro 1 ora. Al termine del processo, la concentrazione di estradiolo è misurato utilizzando un commerciale immuno-adsorbente assay enzimatico (ELISA). 100 volte pre-concentrazione si ottiene e il contenuto di metanolo in solo il 10% v / v. Recuperi completi della molecolavengono raggiunti con 1 ng / L spillo campioni deionizzata e sintetici acqua di mare.

Riproducibilità dell&#39;imballaggio sorbente è stata valutata con l&#39;essiccazione e la ponderazione del sorbente pipettato in flaconcini di vetro ed il risultato è mostrato in Figura 1. Riproducibilità del tempo di iniezione è stato testato per 100 ml di campioni, come mostrato nella Figura 2. Concentrazione iniziale e pre concentrato spillo campioni sono stati determinati utilizzando un kit ELISA commerciale pe...

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.

Un metodo semplice per Automated fase solida estrazione di campioni di acqua per l&#39;analisi immunologiche di piccole inquinanti

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 Views.  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.

Video: 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 Selection Size per migliorare la qualità delle Biblioteche di sequenziamento di nuova generazione Preparato da campioni di acqua ambientale

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

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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.

Preparazione e test di impedenza a base di biochip fluidici con le cellule RTgill-W1 per valutare rapidamente campioni di acqua potabile per la tossicità

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.

Caratterizzazione e applicazione di passivi Campionatori per il monitoraggio dei pesticidi in acqua

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, un metodo migliorato per Inex<em&gt; In Situ</em&gt; Misura di particolato e sciolto composti elaborati da Alimentatori sospensioni attive

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.

Un metodo modificato EPA 1623 che utilizza flusso tangenziale a fibre cave di ultrafiltrazione e di calore passi dissociazione per rilevare 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 ...

Immunologia e infezioni

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.

Identificazione- e specie chimica di Polyfluorinated con un flusso di lavoro combinato mirati e Screening di mirati spettrometria di massa ad alta risoluzione

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.

Realizzazione di un dipolo-assistita estrazione in fase solida Microchip per Trace analisi dei metalli in campioni di acqua

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

Bioingegneria

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).

Piattaforma di microfluidica digitale basata su Electrowetting per l'analisi automatizzata di Immunosorbent legata all'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

Estrazione solido-liquido (lisciviazione)

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 ...

Didattica

JoVE Science Education

Chimica

Chimica organica

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

Estrazione di biomarcatori dai sedimenti - estrazione accelerata con solvente

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

Un metodo semplice per Automated fase solida estrazione di campioni di acqua per l'analisi immunologiche di piccole inquinanti

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