This material is based upon work supported by the U.S. Department of Homeland Security under Grant Award Number 2010-ST-061-FD0001 through a grant awarded by the National Center for Food Protection and Defense at the University of Minnesota. Disclaimer: The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security or the National Center for Food Protection and Defense.

1. Silver Nanoparticle Synthesis15
<ol>
	<li>Dissolve 18 mg silver nitrate in 100 ml ultrapure water (18.2 &#937;U) and vortex for 5 sec.</li>
	<li>Dissolve 27 mg sodium citrate dihydrate in 1 ml water and vortex for 5 sec.</li>
	<li>Transfer all of the prepared silver nitrate solution to a conical flask containing a stirring bar and put the flask on a magnetic hot plate. Heat the flask under vigorous stirring with a stirring speed of 700 rpm at ~350 &#176;C (setting temperature on the plate).</li>
	<li>When...

<ol>
	<li>Albrecht, M. G., Creighton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anomalously+intense+Raman+spectra+of+pyridine+at+a+silver+electrode.">Anomalously intense Raman spectra of pyridine at a silver electrode.</a> J. Am. Chem. Soc. 99 (15), 5215-5217 (1977).</li><li>Schatz, G. C., Young, M. A., Van Duyne, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electromagnetic mechanism of SERS. Surface-enhanced Raman scattering. , 19-45 (2006).</li><li>Matijevic, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+properties+of+uniform+size+colloids.">Preparation and properties of uniform size colloids.</a> Chem. Mater. 5 (4), 412-426 (1993).</li><li>Nickel, U., zu Castell, A., P&#246;ppl, K., Schneider, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+silver+colloid+produced+by+reduction+with+hydrazine+as+support+for+highly+sensitive+surface-enhanced+Raman+spectroscopy.">A silver colloid produced by reduction with hydrazine as support for highly sensitive surface-enhanced Raman spectroscopy.</a> Langmuir. 16 (23), 9087-9091 (2000).</li><li>Khanna, P. K., Subbarao, V. V. V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosized+silver+powder+via+reduction+of+silver+nitrate+by+sodium+formaldehydesulfoxylate+in+acidic+pH+medium.">Nanosized silver powder via reduction of silver nitrate by sodium formaldehydesulfoxylate in acidic pH medium.</a> Mater. Lett. 57 (15), 2242-2245 (2003).</li><li>Henglein, A., Giersig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+colloidal+silver+nanoparticles:+capping+action+of+citrate.">Formation of colloidal silver nanoparticles: capping action of citrate.</a> J. Phys. Chem. B. 103 (44), 9533-9539 (1999).</li><li>Sun, X., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloidal+carbon+spheres+and+their+core/shell+structures+with+noble-metal+nanoparticles.">Colloidal carbon spheres and their core/shell structures with noble-metal nanoparticles.</a> Angew. Chem. Int. Edit. 43 (5), 597-601 (2004).</li><li>Lu, L., 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=Seed-mediated+growth+of+large,+monodisperse+core-shell+gold-silver+nanoparticles+with+Ag-like+optical+properties.">Seed-mediated growth of large, monodisperse core-shell gold-silver nanoparticles with Ag-like optical properties.</a> Chem. Commun. (2), 144-145 (2002).</li><li>Aslan, K., Wu, M., Lakowicz, J. R., Geddes, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+core-shell+Ag@SiO2+nanocomposites+for+metal-enhanced+fluorescence+and+single+nanoparticle+sensing+platforms.">Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms.</a> J. Am. Chem. Soc. 129 (6), 1524-1525 (2007).</li><li>Lu, Y., Yin, Y., Li, Z. Y., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+self-assembly+of+Au@+SiO2+core-shell+colloids.">Synthesis and self-assembly of Au@ SiO2 core-shell colloids.</a> Nano. Lett. 2 (7), 785-788 (2002).</li><li>Link, S., Wang, Z. L., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alloy+formation+of+gold-silver+nanoparticles+and+the+dependence+of+the+plasmon+absorption+on+their+composition.">Alloy formation of gold-silver nanoparticles and the dependence of the plasmon absorption on their composition.</a> J. Phys. Chem. B. 103 (18), 3529-3533 (1999).</li><li>He, L., 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=Rapid+Detection+of+Ricin+in+Milk+Using+Immunomagnetic+Separation+Combined+with+Surface+Enhanced+Raman+Spectroscopy.">Rapid Detection of Ricin in Milk Using Immunomagnetic Separation Combined with Surface Enhanced Raman Spectroscopy.</a> J. Food. Sci. 76 (5), N49-N53 (2011).</li><li>Zheng, J., He, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-Enhanced+Raman+Spectroscopy+for+the+Chemical+Analysis+of+Food.">Surface-Enhanced Raman Spectroscopy for the Chemical Analysis of Food.</a> Compr. Rev. Food. Sci. F. 13 (3), 317-328 (2014).</li><li>He, L., Haynes, C. L., Diez-Gonzalez, F., Labuza, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+a+foreign+protein+in+milk+using+IMS-SERS.">Rapid detection of a foreign protein in milk using IMS-SERS.</a> J. Raman. Spectrosc. 42 (6), 1428-1434 (2011).</li><li>Wei, W. Y., White, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+filter-based+approach+to+surface+enhanced+Raman+spectroscopy+for+trace+chemical+detection.">A simple filter-based approach to surface enhanced Raman spectroscopy for trace chemical detection.</a> Analyst. 137 (5), 1168-1173 (2012).</li><li>Cheng, M. L., Tsai, B. C., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+nanoparticle-treated+filter+paper+as+a+highly+sensitive+surface-enhanced+Raman+scattering+(SERS)+substrate+for+detection+of+tyrosine+in+aqueous+solution.">Silver nanoparticle-treated filter paper as a highly sensitive surface-enhanced Raman scattering (SERS) substrate for detection of tyrosine in aqueous solution.</a> Anal. Chim. Acta. 708 (1), 89-96 (2011).</li><li>Fierro-Mercado, P. M., Hern&#225;ndez-Rivera, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+filter+paper+substrate+for+SERS+trace+explosives+detection.">Highly sensitive filter paper substrate for SERS trace explosives detection.</a> Int. J. Spectrosc. 2012, 716527 (2012).</li><li>Tran, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnanogram+detection+of+dyes+on+filter+paper+by+surface-enhanced+Raman+scattering+spectrometry.">Subnanogram detection of dyes on filter paper by surface-enhanced Raman scattering spectrometry.</a> Anal. Chem. 56 (4), 824-826 (1984).</li><li>Wu, D., Fang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adsorption+behavior+of+p-hydroxybenzoic+acid+on+a+silver-coated+filter+paper+by+surface+enhanced+Raman+scattering.">The adsorption behavior of p-hydroxybenzoic acid on a silver-coated filter paper by surface enhanced Raman scattering.</a> J. Colloid Interface Sci. 265 (2), 234-238 (2003).</li><li>Wigginton, K. R., Vikesland, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold-coated+polycarbonate+membrane+filter+for+pathogen+concentration+and+SERS-based+detection.">Gold-coated polycarbonate membrane filter for pathogen concentration and SERS-based detection.</a> Analyst. 135 (6), 1320-1326 (2010).</li><li>Berthod, A., Laserna, J. J., Winefordner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+by+surface+enhanced+Raman+spectroscopy+on+silver+hydrosols+and+silver+coated+filter+papers.">Analysis by surface enhanced Raman spectroscopy on silver hydrosols and silver coated filter papers.</a> J Pharm Biomed Anal. 6 (6), 599-608 (1988).</li><li>Ackermann, K. R., Henkel, T., Popp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Online+Detection+of+Low-Concentrated+Drugs+via+a+SERS+Microfluidic+System.">Quantitative Online Detection of Low-Concentrated Drugs via a SERS Microfluidic System.</a> ChemPhysChem. 8 (18), 2665-2670 (2007).</li><li>Walter, A., M&#228;rz, A., Schumacher, W., R&#246;sch, P., Popp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+fast,+high+specific+and+reliable+discrimination+of+bacteria+on+strain+level+by+means+of+SERS+in+a+microfluidic+device.">Towards a fast, high specific and reliable discrimination of bacteria on strain level by means of SERS in a microfluidic device.</a> Lab. Chip. 11 (6), 1013-1021 (2011).</li><li>Lee, 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=Fast+and+sensitive+trace+analysis+of+malachite+green+using+a+surface-enhanced+Raman+microfluidic+sensor.">Fast and sensitive trace analysis of malachite green using a surface-enhanced Raman microfluidic sensor.</a> Anal. Chim. Acta. 590 (2), 139-144 (2007).</li><li>Guo, H., 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=Analysis+of+Silver+Nanoparticles+in+Antimicrobial+Products+Using+Surface-Enhanced+Raman+Spectroscopy+(SERS).">Analysis of Silver Nanoparticles in Antimicrobial Products Using Surface-Enhanced Raman Spectroscopy (SERS).</a> Environ. Sci. Technol. 49 (7), 4317-4324 (2015).</li><li>Narayanan, V. A., Begun, G. M., Stokes, D. L., Sutherland, W. S., Vo-Dinh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+Raman+and+surface+enhanced+Raman+scattering+(SERS)+spectra+of+some+fungicides+and+related+chemical+compounds.">Normal Raman and surface enhanced Raman scattering (SERS) spectra of some fungicides and related chemical compounds.</a> J. Raman. Spectrosc. 23 (5), 281-286 (1992).</li><li>Kang, J. S., Hwang, S. Y., Lee, C. J., Lee, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SERS+of+dithiocarbamate+pesticides+adsorbed+on+silver+surface;+thiram.">SERS of dithiocarbamate pesticides adsorbed on silver surface; thiram.</a> Bull. Korean. Chem. Soc. 23 (11), 1604-1610 (2002).</li><li>Li, Y. T., 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=Rapid+and+sensitive+in-situ+detection+of+polar+antibiotics+in+water+using+a+disposable+Ag-graphene+sensor+based+on+electrophoretic+preconcentration+and+surface-enhanced+Raman+spectroscopy.">Rapid and sensitive in-situ detection of polar antibiotics in water using a disposable Ag-graphene sensor based on electrophoretic preconcentration and surface-enhanced Raman spectroscopy.</a> Biosens. Bioelectron. 43, 94-100 (2013).</li><li>Clarke, S. J., Littleford, R. E., Smith, W. E., Goodacre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+monitoring+of+antibiotics+using+Raman+and+surface+enhanced+Raman+spectroscopy.">Rapid monitoring of antibiotics using Raman and surface enhanced Raman spectroscopy.</a> Analyst. 130 (7), 1019-1026 (2005).</li><li>Zheng, J., Pang, S., Labuza, T. P., He, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-quantification+of+surface-enhanced+Raman+scattering+using+a+handheld+Raman+spectrometer:+a+feasibility+study.">Semi-quantification of surface-enhanced Raman scattering using a handheld Raman spectrometer: a feasibility study.</a> Analyst. 138 (23), 7075-7078 (2013).</li><li>Zheng, J., Pang, S., Labuza, T. P., He, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+surface-enhanced+Raman+scattering+detection+using+a+handheld+and+a+bench-top+Raman+spectrometer:+A+comparative+study.">Evaluation of surface-enhanced Raman scattering detection using a handheld and a bench-top Raman spectrometer: A comparative study.</a> Talanta. 129, 79-85 (2014).</li></ol>

The authors have nothing to disclose.

One of the critical steps in this protocol is the Ag NPs synthesis, where uniform Ag NPs are the key for consistent results. The heating time and the concentrations of precursors must be precisely controlled. The average size of this AgNPs preparation is 80 nm, which was measured by the Zetasizer (data not shown). Another critical step is the salt aggregation where the salt concentration and aggregation time must be precisely controlled. In addition, the choice of membrane is also critical as the membrane with a smaller ...

Surface enhanced Raman spectroscopy (SERS) is a technique combining Raman spectroscopy with nanotechnology. The intensity of Raman scattering of analytes at noble metallic nano-surfaces is greatly enhanced by the localized surface plasmon resonance.1 Silver nanoparticles (Ag NPs) are by far the most widely used SERS substrates due to its high enhancement ability.2 Up to now, various synthetic methods of Ag NPs have been developed.3-6 Ag NPs can be used alone as effective SERS substrates, or combined with other materials and structures to enhance its sensitivity and/or functionality.7-11
SERS techniques have demonstrated great capacity for detection of various trace amount contaminants in food and environmental samples.12 Traditionally, there are two common ways for preparing a SERS sample: solution-based and substrate-based methods.13 The solution-based method uses NP colloids to mix with samples. Then the NP-analyte complex is collected using centrifugation, and deposited onto a solid support for Raman measurement after drying. The substrate-based method is usually applied by depositing several microliters of liquid sample onto the pre-fabricated solid substrate.14 However, neither of these two methods are effective and applicable for a large amount of sample volume. Several modifications of the SERS assays overcame the volume limits, such as the integration of a filter system15-21 or the incorporation of a microfluidic device.21-24 The modified SERS assays have shown great enhancement in sensitivity and feasibility for monitoring the chemical contaminants in large water samples.
Here we demonstrate the detailed protocol of fabrication and application of a syringe filter based SERS method to detect trace amount of pesticide ferbam and antibiotic ampicillin.

<table border="0" cellpadding="0" cellspacing="0" width="604">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:136px;">BP1760-5</td>
			<td style="width:131px;">N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Ferbam</td>
			<td style="width:128px;">Chem Service</td>
			<td style="width:136px;">N-11970-250MG</td>
			<td style="width:131px;">98+%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Silver nitrate</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:136px;">209139</td>
			<td style="width:131px;">99.0+%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Sodium citrate dehydrate</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:136px;">W302600</td>
			<td style="width:131px;">99+%</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:211px;">Sodium chloride</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:136px;">S7653</td>
			<td style="width:131px;">99.5+%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">EMD Millipore Durapore PVDF Membrane Filters</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:136px;">VVLP01300</td>
			<td style="width:131px;">0.10 &#181;m Pore Size, hydrophilic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Polycarbonate Filter Holders</td>
			<td style="width:128px;">Cole-Parmer</td>
			<td style="width:136px;">EW-29550-40</td>
			<td style="width:131px;">13 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Analog Vortex Mixer</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td>02-215-365</td>
			<td>N/A</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:211px;">Nutating Mixers</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td>05-450-213</td>
			<td style="width:131px;">N/A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">DXR Raman spectroscope</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:136px;">IQLAADGABFFAHCMAPB</td>
			<td style="width:131px;">Laser power: 1 mW Exposure time: 5 s</td>
		</tr>
	</tbody>
</table>

a filter-based surface enhanced raman spectroscopic assay for rapid detection of chemical contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

The major steps of this experiment were shown in the schematic diagram (Figure 1). Figure 2 demonstrated the importance to use the optimized volume of AgNPs in the membrane coating in order to reach the maximized sensitivity. 1 ml of Ag NPs provides the strongest signal when using ferbam, as compared to 0.5 ml (insufficient coating) or 2 ml (too much coating).
We were able to detect ferbam at 10...

Watch this Scientific Journal Video about A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants at JoVE.com

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

The overall goal of this filter-based Surface Enhanced Raman Spectroscopy method is to detect various target molecules at trace levels in liquid samples. Two advantages of this technique are the reduced costs in sample preparation time, more importantly, this technique provides higher sensitivity by using larger sample volumes, thus lowering the limit of detection. Generally, individuals new to this method will struggle because nano-particles may not be trapped in the pores of the membranewithout a proper aggregation step or they may not be uniformly distributed on the membraneTo begin, prepare 100 milliliters of fresh silver nitrate solution and heat it to boiling on a 350 degree Celsius hotplate with magnetic stirring at 700 RPM.Once boiling, immediately add one milliliter of freshly prepared sodium citrate solution and let the solution boil for 25 minutes. As the silver nano-particles form, the solution will turn greenish-brown. After 25 minutes, put the flask on an unheated stir plate and let it stir overnight at 700 RPM.In the morning, the mixture should be at a stable state, with a constant color and transparency. If desired, measure the absorbence of the nano-particles. Proceed by diluting the mixture with ultra-pure water to 100 milliliters.Then, use a zetasizer to measure the size of the silver nano-particles. For storage, transfer the silver nano-particles to a sealed container. Protect it from light by a foil wrap.The colloid will remain good for up to two months at four degrees Celsius. To make a SERS active filter membranefirst add one milliliter of five millimolar sodium chloride to one milliliter of prepared colloid. And mix them using a nutator at 20 RPM for 10 minutes.As the silver nano-clusters form, prepare a PVDF membranewith a zero point one micron pores. It is critical that the filter holder be completely dry and that the filter is in the correct orientation. After 10 minutes of mixing, filter the two milliliters of nano-clusters by pushing them through the membraneat a rate of about one drop per second.Push the solution through continuously without breaks. The result is a SERS active filter membraneCarefully detach the active filter membraneusing tweezers. Then, set it up to air dry for three minutes.Once dry transfer the SERS active membraneto a glass slide. Now, for Raman detection of the SERS substrate, set the instrument to a 780 nano-meter laser operating at five miliwatts. Set the exposure time to one second and the exposure strength to two.Using a 10 X subjective, focus on the sample. Make sure the software is also set to a 10 X subjective. Now, randomly select eight to 10 spots on the membraneand use the instrument to collect data from them.Then, open the spectral data in the manufacturer's software for analysis. To begin, make a serial dilution of 100 parts per million ferbam solution to obtain five milliliters of 10 parts per billion ferbam solution and 50%acitre-nitrile. Now, position the active membraneinto a filter holder with the nano-particle coating facing up.Then, load the five milliliter sample into a new syringe and attach the active filter. Now, push the sample through the filter at a continuous flow of about one drop per second. Once the solution is filtered, carefully detach the membraneand allow it to air dry for three minutes.Immediately proceed with Raman detection once the membraneis dry. Perform the data collection as before. Various volumes of silver nano-particles were tested using this method.The volume of silver nano-particles solution used to coat the membranethat provided the maximized sensitivity was one milliliter. Measuring the Raman shift shows that the SERS spectrum of 10 parts per billion of ferbam was detectable. The peak, at 1386, is due to the mixed vibration of cyanide and carbon sulfur double bond stretching and from symmetric methyl group deformation.The peak at 1516, is associated with methyl group and cyanide stretching and the peak at 561, is generated by sulfur bond stretching. A spectrum from one parts per million of ampicillin was also clearly detected. The peaks as 1594 and 1447 are from carbon double bond stretching and methylin ethyl group deformation respectively.The strong peak at 1001, is from the benzine ring vibration. The peak at 852 is associated with symmetric CNC stretching. By increasing the sample volume, the detection limit increased.The advantages of this filter based method include an adjustable volume and adjustable limit of detection. After watching this video you should have a good understanding of how to fabricate a uniform sensitive nanosubstrate using a filter membraneOnce mastered this technique can be done in 30 minutes if it is performed properly, following this procedure other methods like SEM or TEM can be performed in order to answer additional questions like the distribution of silver nano-particles on the membraneAfter its development, this technique paved the way for asserters in field of analytical chemistry to explore trace chemical targets in various food and environmental samples. Don't forget that working with pesticides and biological toxins can be extremely hazardous.A lab coat, respirator and safety goggles should always be worn while performing this procedure.

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JoVE Journal

Chemistry

9.3K visualizações.  University of Massachusetts Amherst. The overall goal of this filter-based Surface Enhanced Raman Spectroscopy method is to detect various target molecules at trace levels in liquid samples. Two advantages of this technique are the reduced costs in sample preparation time, more importantly, this technique provides higher sensitivity by using larger sample volumes, thus lowering the limit of detection. Generally, individuals new to this method will struggle because nano-particles may not be trapped in the pores of the membranewi...