Funding from the National Science Foundation through the NUE in Engineering and the LEADER Consortium Programs is gratefully acknowledged.

1. Synthesis of Colloidal AgNPs
The reaction mechanism for the Creighton method (slightly modified, inexpensive)22 is described in great detail in the Supporting information of reference Pavel et.al together with the undesired hydrolysis side-reaction of NaBH4 at room temperature or higher.23
<ol>
	<li>Clean all glassware for 12-24 hr in a 10% HNO3 bath, then for 4-12 hr in a 1.25 M NaOH in 40% ethanol bath, and finally autoclave. Glassware should be rinsed thoroughly a minimum of five times with ultrapure water (17 M&Omega; or higher) after the acid and base bath steps.</li>
	<li>Prepare 300 ml of a 2 mM NaBH4 solution and 100 ml of a 1 mM AgNO3 solution using autoclaved water cooled at 10 &deg;C. The lower temperatures will prevent the side-reaction of NaBH4.</li>
	<li>Add 300 ml of 2 mM NaBH4 solution to a 500 ml Erlenmeyer reaction flask containing a stir bar and wrap the flask with aluminum foil to prevent silver oxidation. Place the flask in an ice bath on a stir plate and stir the solution at 325 rpm for 10 min.</li>
	<li>Prime a 25 ml burette by rinsing with a full column of ultrapure water. After priming, fill the burette with AgNO3 solution and wrap with aluminum foil.</li>
	<li>In a dark room, add 50 ml of 1 mM AgNO3 solution at a rate of 1 drop sec-1 to the NaBH4 solution with continuous stirring (Figure 1A). Cover the middle section of the apparatus with a "foil tent" to minimize light exposure during the AgNO3 addition. The AgNO3 addition will require 30-40 min. Replenish the ice bath periodically.</li>
	<li>After the AgNO3 addition is complete, replenish the ice bath and continue stirring the colloidal solution for an additional 45-50 min. The formation of colloidal AgNPs is signaled by a change in color from colorless to a golden yellow, which is characteristic of the surface plasmon resonance maximum of AgNPs (Figure 1B).</li>
	<li>Once the reaction is completed, refrigerate the colloid. Colloidal AgNP batches may be combined after one week if the colloid has remained consistent, i.e., the colloidal solution has not aggregated and the batch has been characterized using UV-Vis absorption spectrophotometry and Raman spectroscopy to identify possible aggregation or contaminants.</li>
</ol>
2. Characterization of Colloidal AgNPs
A Cary 50 UV-VIS-NIR spectrophotometer (Varian Inc.) and a LabRamHR 800 Raman system (Horiba Jobin Yvon, Inc.) equipped an Olympus BX41 confocal Raman microscope, were utilized for AgNP characterization. The Cary WinUV software, LabSpec v.5 and Origin 8.0 software were employed for the data collection and analysis.
Note: The acquisition parameters will have to be optimized for other instrumentation models.
Determination of Surface Plasmon Resonance of Colloidal AgNPs via UV-Vis Spectrophotometry
<ol>
	<li>Fill a 1 cm3 disposable cuvette with Creighton colloid and ultrapure water in a 1:10 volume ratio. Fill another 1 cm3 cuvette with ultrapure water for a blank baseline correction. Wipe the outside of both cuvettes with a Kimwipe.</li>
	<li>Set the spectrophotometer to absorbance mode from a Y minimum of -0.5 to a Y maximum of 1.0. Set the X scanning window to 200-800 nm and select a fast scan rate of 4,800 nm min-1 with baseline correction.</li>
	<li>Insert the cuvette filled with water into the instrument and run a baseline scan. Repeat if necessary until a non-zero baseline control is achieved.</li>
	<li>Replace the blank cuvette with the sample cuvette and initiate an absorbance scan for the collection of the UV-Vis absorption spectrum of the colloidal sample (Figure 1C).</li>
</ol>
Purity Test of Colloidal AgNPs via Raman spectroscopy
Due to the time limitation of the video demonstration (10-15 min video) and the space limitation of the protocol text (maximum 3 pages), this experimental section will not be videotaped.
<ol start="5">
	<li>Set the instrument parameter settings as follows: excitation source (632.8 nm He-Ne), filter (no filter, laser power at the sample ~ 17 mW), confocal hole (300 &mu;m), spectrometer (730 cm-1), holographic grating (600 groves/mm), objective lens (50x long working distance air objective), exposure time (30 s), and accumulation cycles (5).</li>
	<li>Use a clean pipette to fill a 2 ml quartz cuvette with colloid and gently insert the plug. Use a Kimwipe to clean off fingerprints, smudges or colloid from the surface of the cuvette. Significantly lower the microscope stage. Select the 50x objective lens and place the cuvette onto the stage.</li>
	<li>Focus the laser beam on the AgNP colloid directly underneath the inner wall of the cuvette using the video mode of the instrument and the Olympus camera. Turn off room lights and acquire Raman spectrum (Figure 1D).</li>
</ol>
3. Size-selection and Concentration of Colloidal AgNPs via Tangential Flow Ultrafiltration (TFU)
A KrosFlo II Research filtering system (Spectrum Laboratories, Rancho Dominguez, CA) was used to limit the AgNP polydispersity and to concentrate them (Figure 2). The three steps of the TFU process were: (1) Size-selection of AgNPs and AgNP-aggregates of 50-nm in diameter and larger using a 50-nm MidiKros polysulfone module (460 cm2), 2) Size selection and concentration of AgNPs of 1-20 nm in diameter using a 100-kD MidiKros filter (200 cm2), and (3) Further volume reduction using a 100-kD MicroKros polysulfone filter (20 cm2) (Figure 3).
Step 1
<ol>
	<li>Connect the size 17 MasterFlex feeding tubing to the peristaltic pump according to Figure 2A. A Y-junction and a tubing junction will be needed for the set-up. Attach tubing to the 50-nm MidiKros module. Be sure to secure tubing to filter using zip ties. Select tubing size 17 using SIZE button.</li>
	<li>Select counterclockwise pump direction using DIR button. Make sure MODE button is on INT.</li>
	<li>Lower the pump rate to below 300 ml min-1 before starting pump. The pump rate should be adjusted according to the size of the used tubing. It should be a small setting to permit the operator to promptly react to potential leaks but large enough to still have an effect of priming the system. In order to create the vacuum needed to draw colloid from the reservoir into the tubing and filter, cut the tubing that leads from the bottom section of the filter to the top portion of the Y-junction in the middle of the tubing.</li>
	<li>Place the section of tubing that leads from the filter into the reservoir bottle and clamp off the bottom section of tubing near the Y-junction with a clamp or by hand. Turn on the pump. The created vacuum should begin siphoning the colloid.</li>
	<li>Once the liquid is flowing freely through the tube, turn off the pump, join the broken section of tubing with a tubing junction and secure with zip ties. Turn on the pump again and continue filtration.</li>
	<li>Check the tubing circuit for leaks. If a leak is found, fix the leak by adjusting the fitting or re-securing with a zip tie. Once the tubing system is leak free, the pump flow rate may be increased to no greater than 700 ml min-1. This pump rate value should be optimized according to tubing size to avoid tubing failure. Continue filtration until the liquid in the reservoir bottle is depleted to nearly nothing.</li>
	<li>Once the filtration is complete, collect the filtrate that contains AgNPs of 50-nm diameter and smaller. The retentate may be saved for further analysis according to the specific AgNP application.</li>
</ol>
Step 2
<ol start="8">
	<li>Rinse the tubing with 2% HNO3 and ultrapure water prior to installing the 100-kD MidiKros filter using the same setup as for the 50-nm module.</li>
	<li>Repeat step 3.3 using the 100-kD MidiKros module.</li>
	<li>Once the filtration is complete, collect the contents of the tubing and the filter (100-kD retentate). The volume should be approximately 50 ml.</li>
</ol>
Step 3
<ol start="11">
	<li>Connect the size 14 MasterFlex tubing and the 100-kD MicroKros FILTER to the peristaltic pump according to Figure 2B. Secure all junctions with zip ties. Select tubing size 14 on the pump using the SIZE button and lower the pump rate to 30 ml min-1.</li>
	<li>Begin the filtration process. Check the tubing circuit for leaks. If a leak is found, fix the leak by adjusting the fitting or re-securing with a zip tie.</li>
	<li>Once the tubing system is leak free, the pump flow rate may be increased to no greater than 90 ml min-1. Continue filtration until the liquid remaining in the reservoir bottle contains a minimal amount of concentrate.</li>
	<li>The remaining contents of the tubing and filter can be collected into the reservoir bottle by removing the feeding tube from the bottle while the pump is still running. Once the tubing and filter contents are in the reservoir bottle, the pump may be turned off.</li>
</ol>
4. Quantification of Silver Amount in Colloidal AgNPs by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES)
Each colloidal sample was chemically digested and the amount of silver was quantified by ICP-OES using an A 710E spectrometer (Varian Inc.). A linear regression calibration curve for silver (Figure 4) was constructed using eight silver standards (0, 3, 7, 10, 15, 25, 50, and 100 &mu;g L-1), which were prepared from a 10,000 &mu;g ml-1 silver standard for trace metal analysis (Ultra Scientific).
<ol>
	<li>Chemically digest samples using HNO3. The representative samples are the original colloid (step 1), 50-nm filtrate (step 1), 100-kD retentate (step 2), and final 100-kD retentate (step 3) (Figure 3).</li>
	<li>The samples should be diluted with 2% HNO3 using the following volume ratios: 1:1000 for the original colloid, 1:1000 for the 50-nm filtrate, 1:25,000 for the first 100-kD retentate, and 1:250,000 for the final 100-kD retentate. To prevent silver leaching, all samples should be stored in low-density polypropylene containers.</li>
	<li>Set the ICP-OES instrument parameters as follows: wavelength for Ag (328.068 nm), power (1.20 kW), plasma flow (15.0 L min-1), auxiliary flow (1.50 L min-1), and nebulizer pressure (200 kPa).</li>
	<li>Each sample should be measured in triplicate with a replicate time of 10 s. Between-measurement stabilization time of 15 sec and a 30 sec sample uptake delay should be used. A method blank should be introduced between every sample to reduce potential cross-contamination.</li>
</ol>
5. Size Distribution of Colloidal AgNPs via Transmission Electron Microscopy (TEM)
A Phillips EM 208S TEM was used to visualize the colloidal AgNPs. Electron micrographs were captured using a high resolution Gatan Bioscan camera and analyzed in ImageJ software.24
<ol>
	<li>Dilute the 100-kD retentate sample with ultrapure water (1:100 volume ratio). Deposit 20 &mu;l of the original colloid and the diluted 100-kD retentate (step 3) on 300-mesh formvar-coated gold grids (Electron Microscopy Sciences). Allow the grids to dry in a desiccator. View within one day.</li>
	<li>Set the accelerating potential of the TEM instrument at 70 kV to visualize AgNPs. Capture electron micrographs (Figure 5) using the high-resolution camera and save as tagged image files format (tiff).</li>
</ol>
6. Representative Results
Synthesis and Characterization of Colloidal AgNPs
Four liters of Creighton colloidal AgNPs were successfully synthesized using the setup displayed in Figure 1A. The final colloid had a characteristic golden yellow color (Figure 1B).22, 23 The UV-Vis absorption spectrum of this colloid had a typical sharp, symmetrical surface plasmon peak (SPR) at 394 nm (Figure 1C). The Raman spectrum of the original Creighton colloid and the final 100-kD retentate presented only three vibrational modes, namely the bending (1640 cm-1) and symmetric and asymmetric stretching modes of H2O (3245 cm-1 and 3390 cm-1, respectively) (Figure 1D).
TFU of Colloidal AgNPs
The TFU setup and the schematic of the 3-step TFU process are depicted in Figures 2 and 3, respectively. In step 1, a 50-nm filter (460 cm2) was utilized to size-select and to remove AgNPs and AgNP-aggregates of 50-nm diameter and larger from the original colloid (about 100 ml of 50-nm retentate). This step was also accompanied by a small volume reduction from 4 L of original colloid down to 3.9 L of 50-nm filtrate. No backwashing or flow disruption step was used. The largest volume reduction (i.e., water removal) was obtained in step 2, when the 50-nm filtrate was subsequently run through a 100-kD filter (200 cm2). The resulting 100-kD retentate had a total volume of 50 ml. Most of the synthesis byproducts and excess reagents were eliminated in this step through the water solvent (3.850 ml of 100-kD filtrate). Further, AgNP concentration was achieved by the addition of a third filtration step to the previously reported procedure.19 In this step 3, a 100-kD filter of a smaller surface area (20 cm2) reduced the 100-kD retentate volume to 4.0 ml. The TEM measurements will demonstrate that this final 100-kD retentate consists mostly of lowly aggregated AgNPs of 1-20 nm in diameter.
ICP-OES and TEM of Colloidal AgNPs
A linear regression calibration curve (Figure 4) for silver was constructed from eight standards (0, 3, 7, 10, 15, 25, 50, and 100 &mu;g L-1). The amount of silver in each of the four representative colloidal samples was then determined from the ICP-OES calibration curve through extrapolation: original colloid (15.2 ppm, Figure 3A), 50-nm filtrate (14.1 ppm, Figure 3B), first 100-kD retentate (683.1 ppm, Figure 3C) and final 100-kD retentate (8,538.9 ppm, Figure 3D). The actual yield of 15.2 ppm is very close to the typical theoretical yield of 15.4 ppm for the Creighton reaction. The extreme concentration of AgNPs (4 ml of 8,538.9 ppm) was reflected by a dramatic change in color from golden yellow for the original colloid to dark brown for the final 100-kD retentate (Figure 3, insets of vial pictures). The quality of the filters was found to be critical to the TFU process, in particular to step 1. The final retentate concentrations ranged from 3,390.1 ppm to 9,333.3 ppm depending on the condition of the filters (heavily used versus brand new). If the membrane pores become compromised, AgNPs that have diameters less than 50-nm will also be retained and will subsequently decrease the overall amount of AgNPs that is collected in the filtrate. Optimization of the filtration process to include pressure monitoring and proper cleaning can increase the life span of the filters.
Representative TEM micrographs of the original Creighton colloid and the final 100-kD retentate (step 3) are shown in Figure 5A and 5C, respectively. In their unaggregated state, AgNPs appear as black round areas on a lighter grey background. Approximately 800 AgNPs were identified in the TEM micrographs of each of the two samples and were analyzed using the Image J software. One particle was defined by a complete and enclosed perimeter. An area threshold value was set at 1.0 nm2 according to the resolution of the TEM micrographs. The AgNP counts and area data were then exported into Microsoft Excel and the AgNP diameters were extrapolated. The average AgNP diameter in the original colloid and the final 100-kD retentate were determined to be 9.3 nm and 11.1 nm, respectively. The diameter measurements of the AgNPs were then exported to Origin 8.0 software and a TEM size histogram was constructed for each sample (Figure 5B and 5D).
<img alt="Figure 1" src="/files/ftp_upload/4167/4167fig1.jpg" /> 
Figure 1. A) Synthesis setup, B) Characteristic color, C) UV-Vis absorption spectrum, and D) Raman spectrum of Creighton colloidal AgNPs.
<img alt="Figure 2" src="/files/ftp_upload/4167/4167fig2.jpg" /> 
Figure 2. TFU experimental setup for A) steps 1 and 2: I) Reservoir containing Creighton colloidal AgNPs. II) Reservoir for filtrate collection. III) Y-junction in tubing. IV) Peristaltic pump head. V) Either 50-nm or 100-kD Midi Kros filter. B) step 3: I) Reservoir containing Creighton colloidal AgNPs. II) Reservoir for filtrate collection. III) 100-kD Micro Kros filter.
<img alt="Figure 3" src="/files/ftp_upload/4167/4167fig3.jpg" /> 
Figure 3. Flowchart depicting the TFU process. The blue-shaded boxes mark the colloidal suspensions of AgNPs collected for further analysis. Vial photographs show A) Original colloid batch, B) 50-nm filtrate collected after processing the original colloid through the 50-nm filter (460 cm2), C) first 100-kD retentate obtained after volume reduction using the 100-kD Midi Kros filter (200 cm2), and D) final 100-kD retentate resulting from the volume reduction using the 100-kD Micro Kros filter (20 cm2). The 100-kD filtrate looks like water.
<img alt="Figure 4" src="/files/ftp_upload/4167/4167fig4.jpg" /> 
Figure 4. ICP-OES linear calibration constructed using eight silver standards: 0, 3, 7, 10, 15, 25, 50, and 100 &mu;g L-1.
<img alt="Figure 5" src="/files/ftp_upload/4167/4167fig5.jpg" /> 
Figure 5. TEM micrographs of A) original Creighton AgNPs and C) final 100-kD retentate (scale bar is 100 nm). TEM size histograms constructed by analyzing approximately 800 AgNPs for B) original Creighton AgNPs, and D) final 100-kD retentate. The inset in Figure 5B shows the expanded 41-75 nm size range for comparison purposes. <a href="https://www.jove.com/files/ftp_upload/4167/4167fig5large.jpg" target="_blank"> Click here to view larger figure</a>.

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Optical+Properties+of+Metal+Nanoparticles:+The+Influence+of+Size,+Shape,+and+Dielectric+Environment.">The Optical Properties of Metal Nanoparticles: The Influence of Size, Shape, and Dielectric Environment.</a> J. Phys. Chem. B. 107, 668-677 (2003).</li><li>&#346;ileikait&#267;, A., Prosy&#269;evas, I., Pui&#353;o, J., Juraitis, A., Guobien&#267;, A. <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+Produced+by+Chemical+Reduction+of+Silver+Salt+Solution.">Analysis of Silver Nanoparticles Produced by Chemical Reduction of Silver Salt Solution.</a> Mater. Sci. (Medziagotyra). 12, 287-291 (2006).</li><li>Lewis, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+catalysis+by+colloids+and+clusters.">Chemical catalysis by colloids and clusters.</a> Chem. Rev. 93, 2693-2730 (1993).</li><li>Li, Y., Wu, Y., Ong, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+Synthesis+of+Silver+Nanoparticles+Useful+for+Fabrication+of+High-Conductivity+Elements+for+Printed+Electronics.">Facile Synthesis of Silver Nanoparticles Useful for Fabrication of High-Conductivity Elements for Printed Electronics.</a> J. Am. Chem. Soc. 127, 3266-3267 (2005).</li><li>Sun, 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=Shape-Controlled+Synthesis+of+Gold+and+Silver+Nanoparticles.">Shape-Controlled Synthesis of Gold and Silver Nanoparticles.</a> Science. 298, 2176-2179 (2002).</li><li>Han, X., Zhao, B., Ozaki, Y. <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+scattering+for+protein+detection.">Surface-enhanced Raman scattering for protein detection.</a> Analytical and Bioanalytical Chemistry. 394, 1719-1727 (2009).</li><li>Pavel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+SERS+Detection+of+Small+Proteins+Modified+to+Act+as+Bifunctional+Linkers.">Label-Free SERS Detection of Small Proteins Modified to Act as Bifunctional Linkers.</a> J. Phys. Chem. C. 112, 4880-4883 (2008).</li><li>Ladner, D. A., Steele, M., Weir, A., Hristovski, K., Westerhoff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalized+nanoparticle+interactions+with+polymeric+membranes.">Functionalized nanoparticle interactions with polymeric membranes.</a> J. Hazard. Mater. , (2011).</li></ol>

No conflicts of interest declared.

UV-Vis Absorption Spectrophotometry and Raman Spectroscopy of Colloidal AgNPs
It is well known that the number of surface plasmon resonance peaks in the absorption spectrum of a colloid decreases as the symmetry of the AgNPs increases. Additionally, AgNP aggregation leads to the appearance of broader or red-shifted peaks.25,26 The presence of a single, sharp and symmetrical SPR peak at 394 nm is indicative of small, spherical AgNPs of moderate aggregation and size distribution.
<...

<table border="1">
 <tr>
 <td>Silver nitrate 			(AgNO3)</td>
 <td>Acros Organics 			Inc.</td>
 <td>CAS: 7761-88-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium 			borohydride (NaBH4)</td>
 <td>Acros Organics 			Inc.</td>
 <td>CAS: 			16940-66-2</td>
 <td></td>
 </tr>
 <tr>
 <td>Nitric acid 			(HNO3, 			Optima)</td>
 <td>Fisher 			Scientific Inc.</td>
 <td>A467-1</td>
 <td>Trace 			metal grade for ICP 			analysis</td>
 </tr>
 <tr>
 <td>10,000 &mu;g 			ml-1 			silver standard, EnviroConcentrate</td>
 <td>Ultra 			Scientific</td>
 <td>US-IAA-047</td>
 <td></td>
 </tr>
 <tr>
 <td>KrosFlo 			Research IIi Tangential Flow Filtration System</td>
 <td>Spectrum 			Laboratories Inc.</td>
 <td><a href="http://www.spectrumlabs.com/pshop/RequestPrice/index.html?LiFrom=%2Ffiltration%2FKR2SystemO.html;FrName=Research+IIi+System;Pn=SYR2-U20-01N;" target="_blank">SYR2-U20-01N</a></td>
 <td></td>
 </tr>
 <tr>
 <td>0.05 			&mu;m PS (0.5 mm) 460 cm2</td>
 <td>Spectrum 			Laboratories Inc.</td>
 <td><a href="http://www.spectrumlabs.com/filtration/MidiKros.html?Pn=X30S-900-02N;" target="_blank">X30S-900-02N</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Midi 100 kD PS 			200 cm2</td>
 <td>Spectrum 			Laboratories Inc.</td>
 <td>X3-100S-901-02N</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro100 kD PS 			20 cm2</td>
 <td>Spectrum 			Laboratories Inc.</td>
 <td>X1AB-300-10N</td>
 <td></td>
 </tr>
 <tr>
 <td>MasterFlex 			C-Flex tubing L/S Size 17 </td>
 <td>Cole-Palmer 			Instrument Co.</td>
 <td>06424-17</td>
 <td></td>
 </tr>
 <tr>
 <td>MasterFlex 			C-Flex tubing L/S Size 14 </td>
 <td>Cole-Palmer 			Instrument Co.</td>
 <td>06424-14</td>
 <td></td>
 </tr>
 <tr>
 <td>Cary 50 			UV-VIS-NIR spectrophotometer</td>
 <td>Varian Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>LabRam HR 800 			system</td>
 <td>Horiba Jobin 			Yvon Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Varian 710ES 			ICP-OES</td>
 <td>Varian Inc.</td>
 <td></td>
 <td></td>
 </tr>
</table>
Table 1. Specific reagents and equipment.

tangential flow ultrafiltration: a  &ldquo;green&rdquo; method for the size selection and concentration of colloidal silver nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to their powerful antimicrobial properties.3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7 and size-based isolation4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 &mu;g ml-1 down to 10 ml of 198.7 &mu;g ml-1) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 &mu;g ml-1) were synthesized using the well-established Creighton method22,23 by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2 and 20 cm2) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 &mu;g ml-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.

Watch this Scientific Journal Video about Tangential Flow Ultrafiltration: A  â€œGreenâ€ Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles at JoVE.com

Tangential Flow Ultrafiltration: A  &amp;ldquo;Green&amp;rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 &mu;g ml-1 down to 4 ml of 8,539.9 &mu;g ml-1) with minimal aggregation.

Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to ...

tangential-flow-ultrafiltration-green-method-for-size-selection

Research

JoVE Journal

Biology

17.7K Views.  Wright State University. Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 μg ml-1 down to 4 ml of 8,539.9 μg ml-1) with minimal aggregation.

Video: Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...