Name: a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants
Uploaded: 2016-01-01T14:00:00
Description: 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

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.

Note: The following protocol describes the SPE performed on 100 ml water sample with C18 sorbent and elution with 50% v/v methanol. The enriched sample is diluted to reach 10% v/v methanol before analysis with an enzyme linked immunosorbent assay (ELISA) kit.
1. Preparing the Reagents
<ol>
	<li>Prepare the water samples
	<ol>
		<li>Prior to any other step, filter each 100 ml water sample with 0.2 &#181;m pore size filters.</li>
		<li>Spike the sample with desired concentration by diluting ap...

<ol>
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A new method for the preparation of water samples followed by analysis using immunoassay was proposed. The instrument enables to perform solid phase extraction in an automated and user-friendly way.
The filtration of the water sample prior to its injection into the system is critical. Any particulates still present in the solution would potentially cause clogging of the fluidic network and obstruct the SPE column. Another important step is the preparation of the SPE column. The amount of parti...

Sample preparation is an important step in any analytical process. In particular, removal of matrix effects, diminution of interferences, and enrichment of the analyte are necessary to obtain precise results and reach low limits of detection. Endocrine disrupting compounds (EDCs) are of particular concern due to their action on the living organisms even when present at very low levels in the environment. The natural hormone 17&#946;-estradiol is present on the EU water pollution Watch List and prone to be added to the list of priority substances regulated under the European Water Framework Directive. Solid phase extraction (SPE) is commonly applied for the analysis of small pollutants in water, with both chemical 1-5 (chromatography, mass spectrometry) and immunological 6-9 detection methods. The latter gained interest in the field of environmental monitoring, as immunoassays are available in large variety of formats, are specific to the target analyte, and reach low limits of detection.6, 7, 10, 11 Various enzyme linked immunosorbent assays (ELISA) are commercially available and enable to analyze multiple samples at once on a multi-well plate. The procedure consists in successive reaction steps that can take a few hours. The final product of reaction can be detected optically to determine the concentration of the target molecule based on a calibration curve.
Classical SPE procedures include sorbent pre-conditioning, sample extraction, washing, elution, and concentration by evaporation of the eluent. The solvent used for dilution of this extract is chosen depending on the detection method. For immunological methods, the amount of organic solvent influences strongly the sensitivity of the method.12
In addition to the recovery and the pre-concentration performances, the method also needs to be simple and cost efficient. Automation of the procedure helps to reduce human-related errors. In our previous work 13 we introduced our prototype for automated SPE, and our method was applied to the analysis of the natural hormone 17&#946;-estradiol in sea water samples. With the present video we would like to highlight the technical advantages of our method compared to traditional off-line and on-line SPE, and its particular compatibility with detection by immuno-reactions. We describe the protocol applied to water samples for the detection of 17&#946;-estradiol. SPE is performed with octadecyl-silica (C18) sorbent phase and elution is performed with diluted methanol.

<table border="0" cellpadding="0" cellspacing="0" width="609">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Filter membrane 0.2 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">GNWP04700</td>
			<td style="width:167px;">For sample filtration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon membrane 11 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">NY1104700</td>
			<td>For SPE column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable biopsy punch 5 mm</td>
			<td style="width:107px;">Medical Budget</td>
			<td style="width:119px;">39302439</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nucleodur C18 ec&#160;</td>
			<td style="width:107px;">Macherey Nagel</td>
			<td style="width:119px;">713550.01</td>
			<td>50 &#956;m particle diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic sea water</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">SSWS500-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol standard</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;"></td>
			<td>300 ng/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol ELISA kit&#160;</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;">ADI-900-008</td>
			<td>96 wells, range 30 - 3000 ng/L</td>
		</tr>
	</tbody>
</table>

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

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

Reproducibility of sorbent packing was evaluated by drying and weighting the pipetted sorbent in glass vials and the result is shown in Figure 1. Reproducibility of the time of injection was tested for 100 ml samples, as shown in Figure 2. Concentration in initial and pre-concentrated spiked samples were determined by using a commercial ELISA kit for 17&#946;-estradiol and are shown in Figure 3.
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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.

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

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