This work was supported by a BU/CIMIT Applied Healthcare Engineering Predoctoral Fellowship, a National Science Foundation Broadening Participation Research Initiation Grant in Engineering (BRIGE), and the National Institutes of Health (R21EB0094930).

1. Preparation of Phase-shift Nanoemulsion (PSNE)
<ol>
 <li>Dissolve 11 mg DPPC and 1.68 mg DSPE-PEG2000 in chloroform</li>
 <li>Evaporate the organic solvent to form a dry lipid film in a glass round-bottom flask</li>
 <li>Dessicate the lipid film overnight</li>
 <li>Rehydrate the lipid film with 5.5 ml of phosphate-buffered saline (PBS)</li>
 <li>Heat solution in a 45 &deg;C water bath until lipid film dissolves, vortexing periodically</li>
 <li>Transfer lipid solution into 7 ml vial</li>
 <li>Sonicate lipid solution for 2 min at 20% amplitude</li>
 <li>Divide solution into two vials of 2.5 ml each (discard remaining 0.5 ml)</li>
 <li>Add 2.5 ml PBS to each vial</li>
 <li>Place each vial in a 0 &deg;C ice-water bath</li>
 <li>Add 50 &mu;l DDFP to each vial</li>
 <li>Sonicate each vial in the ice-water bath using the following settings: 25% amplitude, pulsed mode (10 sec on, 50 sec off), 60 sec total on time</li>
 <li>Transfer PSNE solutions to 20 ml scintillation vials</li>
 <li>Add 5 ml PBS to each vial, resulting in 10 ml final volume</li>
 <li>Assemble extruder following directions provided by manufacturer
 <ol>
 <li>Rinse each part with deionized water</li>
 <li>Place	 the stainless steel support disc in the center of the filter support base</li>
 <li>Place the stainless steel mesh on top of the stainless steel support disc</li>
 <li>Using tweezers, place an extruder drain disc membrane (shiny side up) on the stainless steel mesh</li>
 <li>Using tweezers, place the extruder filter (shiny side up) on the drain disc membrane</li>
 <li>Carefully place the small O-ring on the filter and place the thermobarrel and extruder top above the support base</li>
 <li>Partially tighten each wing-nut first, then completely tighten the wing-nuts by hand in an alternating fashion</li>
 <li>Connect the extruder to a nitrogen gas line</li>
 <li>To prime the extruder, pipette 10 ml deionized water into the top sample port, cap the opening, and tighten the vent valve</li>
 <li>Slowly open the nitrogen gas line to increase the pressure, forcing the sample through the membranes, and collect the sample from the outlet tubing</li>
 <li>After use, disassemble in reverse order, rinse the extruder parts with deionized water, and discard the membrane filter and membrane drain disc</li>
 </ol>
 </li>
 <li>For 100 nm droplets only, pre-condition PSNE by extruding 10 times through 200 nm filter</li>
 <li>Extrude PSNE 16 times through 100 nm or 200 nm filter to obtain narrow size distribution</li>
</ol>
2. Preparation of Polyacrylamide Hydrogel Containing PSNE
<ol>
 <li>Prepare 24% BSA solution by diluting 1.2 g BSA powder in 5 ml deionized water</li>
 <li>Prepare 10% APS solution by diluted 0.1 g APS powder in 1 ml deionized water</li>
 <li>In the following order, mix 2.1 ml acrylamide solution, 1.2 ml Tris buffer, 0.1 ml 10% APS, 4.5 ml 24% BSA solution, and 3.6 ml deionized water in plastic chamber</li>
 <li>Heat to 40 &deg;C and place under vacuum for 1 hr</li>
 <li>Add 480 &mu;l of PSNE and thoroughly mix by gently swirling the plastic chamber.</li>
 <li>Add 12 &mu;l TEMED and place the chamber in a 12 &deg;C water bath for 2 hr</li>
</ol>
3. Representative Results
A schematic of the setup for ultrasound experiments with tissue-mimicking hydrogel phantoms is shown in Figure 1. This protocol results in lipid-coated perfluorocarbon droplets with a narrow size distribution that are stable in solution for at least a week. The size distribution measured with dynamic light scattering (90Plus Particle Size Analyzer, Brookhaven Instruments, Holtsville, NY) is shown in Figure 2 for PSNE extruded using 100 and 200 nm filters. The PSNE effective diameter over time, measured using dynamic light scattering, is listed in Table 1, demonstrating that PSNE are stable for at least a week. B-mode images of PSNE before and after vaporization in a polyacrylamide hydrogel are shown in Figure 3. Also, a lesion formed by 15 sec of HIFU-mediated heating in a polyacrylamide hydrogel containing albumin and PSNE is shown in Figure 4. The asymmetric shape of the lesion is a result of prefocal heating that occurs due to the presence of the bubble cloud in the ultrasound path. It is important to note that prefocal heating and lesion formation due to scatter from bubbles can be minimized by reducing the transmitted acoustic power.
<img src="/files/ftp_upload/4308/4308fig1.jpg" alt="Figure 1"/> 
 Figure 1. Schematic diagram of experimental setup for ultrasound experiments with tissue-mimicking hydrogels.
<img src="/files/ftp_upload/4308/4308fig2.jpg" alt="Figure 2"/> 
 Figure 2. Size distribution of PSNE extruded through 100 nm or 200 nm filters, measured using dynamic light scattering. The units of the ordinate axes are based on the intensity of scattered light from particles of a certain size relative to the total scattered light intensity from the sample.
<img src="/files/ftp_upload/4308/4308fig3.jpg" alt="Figure 3"/> 
 Figure 3. B-mode images (a) before and (b) after PSNE vaporization in a polyacrylamide hydrogel. The arrow indicates the focal region where a bubble cloud was formed by PSNE vaporization.
<img src="/files/ftp_upload/4308/4308fig4.jpg" alt="Figure 4"/> 
 Figure 4. Images of polyacrylamide hydrogel containing albumin and PSNE (a) before and (b) after vaporization and sonication with HIFU, demonstrating lesion formation as a result of ultrasound-induced heating. The ultrasound center frequency was 3.3 MHz. The ultrasound signal consisted of an initial 30-cycle, 6.4 W pulse to vaporize PSNE, immediately followed by 15 sec of continuous ultrasound at 0.77 W. 
<table border="1">
 <tbody>
 <tr>
 <td rowspan="2">Days after extrusion</td>
 <td colspan="2">Extruded with 200 nm filter</td>
 <td colspan="2">Extruded with 100 nm filter</td>
 </tr>
 <tr>
 <td>Mean Dia. (nm)</td>
 <td>Std. Dev. (nm)</td>
 <td>Mean Dia. (nm)</td>
 <td>Std. Dev. (nm)</td>
 </tr>
 <tr>
 <td>1</td>
 <td>182.9</td>
 <td>4.9</td>
 <td>118.0</td>
 <td>0.9</td>
 </tr>
 <tr>
 <td>7</td>
 <td>177.7</td>
 <td>2.5</td>
 <td>124.8</td>
 <td>3.1</td>
 </tr>
 </tbody>
</table>
Table 1. Mean diameter and standard deviation of PSNE at one and seven days after extrusion with 100 nm and 200 nm filters.

<ol>
	<li>Hynynen, K., Darkazanli, A., Unger, E., Schenck, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-guided+noninvasive+ultrasound+surgery.">MRI-guided noninvasive ultrasound surgery.</a> Med. Phys. 20, 107-115 (1993).</li><li>Baban, D. F., Seymour, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+tumour+vascular+permeability.">Control of tumour vascular permeability.</a> Adv. Drug Deliv. Rev. 34, 109-119 (1998).</li><li>Maeda, H., Wu, J., Sawa, T., Matsumura, Y., Hori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vascular+permeability+and+the+EPR+effect+in+macromolecular+therapeutics:+a+review.">Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review.</a> J. Control. Release. 65, 271-284 (2000).</li><li>Schadlich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+accumulation+of+NIR+fluorescent+PEG-PLA+nanoparticles:+impact+of+particle+size+and+human+xenograft+tumor+model.">Tumor accumulation of NIR fluorescent PEG-PLA nanoparticles: impact of particle size and human xenograft tumor model.</a> ACS Nano. 5, 8710-8720 (2011).</li><li>Williams, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convertible+perfluorocarbon+droplets+for+cancer+detection+and+therapy.">Convertible perfluorocarbon droplets for cancer detection and therapy.</a> 2010 IEEE Ultrasonics Symposium. , (2010).</li><li>Martz, T. D., Sheeran, P. S., Bardin, D., Lee, A. P., Dayton, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+manufacture+of+phase-change+perfluorocarbon+droplets+using+microfluidics.">Precision manufacture of phase-change perfluorocarbon droplets using microfluidics.</a> Ultrasound Med. Biol. 37, 1952-1957 (2011).</li><li>Giesecke, T., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-mediated+cavitation+thresholds+of+liquid+perfluorocarbon+droplets+in+vitro.">Ultrasound-mediated cavitation thresholds of liquid perfluorocarbon droplets in vitro.</a> Ultrasound Med. Biol. 29, 1359-1365 (2003).</li><li>Sheeran, P. S., Luois, S., Dayton, P. A., Matsunaga, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+and+Acoustic+Studies+of+a+New+Phase-Shift+Agent+for+Diagnostic+and+Therapeutic+Ultrasound.">Formulation and Acoustic Studies of a New Phase-Shift Agent for Diagnostic and Therapeutic Ultrasound.</a> Langmuir. 27, 10412-10420 (2011).</li><li>Sheeran, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decafluorobutane+as+a+phase-change+contrast+agent+for+low-energy+extravascular+ultrasonic+imaging.">Decafluorobutane as a phase-change contrast agent for low-energy extravascular ultrasonic imaging.</a> Ultrasound Med. Biol. 37, 1518-1530 (2011).</li><li>Zhang, 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> The Application of Phase-Shift Nanoemulsion in High Intensity Focused Ultrasound: An In Vitro Study [Doctoral Dissertation]. , (2011).</li><li>Allen, T. M., Hansen, C., Martin, F., Redemann, C., Yau-Young, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+containing+synthetic+lipid+derivatives+of+poly(ethylene+glycol)+show+prolonged+circulation+half-lives+in+vivo.">Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo.</a> Biochim. Biophys. Acta. 1066, 29-36 (1991).</li><li>Klibanov, A. L., Maruyama, K., Beckerleg, A. M., Torchilin, V. P., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+amphipathic+poly(ethylene+glycol)+5000+to+prolong+the+circulation+time+of+liposomes+depends+on+the+liposome+size+and+is+unfavorable+for+immunoliposome+binding+to+target.">Activity of amphipathic poly(ethylene glycol) 5000 to prolong the circulation time of liposomes depends on the liposome size and is unfavorable for immunoliposome binding to target.</a> Biochim. Biophys. Acta. 1062, 142-148 (1991).</li><li>Klibanov, A. L., Maryama, K., Torchilin, V. P., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphipathic+polyethyleneglycols+effectively+prolong+the+circulation+time+of+liposomes.">Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes.</a> FEBS Lett. 268, 235-237 (1990).</li><li>Gabizon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+circulation+time+and+enhanced+accumulation+in+malignant+exudates+of+Doxorubicin+encapsulated+in+polyethylene-glycol+coated+liposomes.">Prolonged circulation time and enhanced accumulation in malignant exudates of Doxorubicin encapsulated in polyethylene-glycol coated liposomes.</a> Cancer Res. 54, 987-992 (1994).</li><li>Awasthi, V. D., Garcia, D., Goins, B. A., Philips, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulation+and+biodistribution+profiles+of+long-circulating+PEG-liposomes+of+various+sizes+in+rabbits.">Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits.</a> Int. J. Pharm. 253, 121-132 (2003).</li><li>Zhang, P., Porter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+study+of+a+phase-shift+nanoemulsion:+a+potential+nucleation+agent+for+bubble-enhanced+HIFU+tumor+ablation.">An in vitro study of a phase-shift nanoemulsion: a potential nucleation agent for bubble-enhanced HIFU tumor ablation.</a> Ultrasound Med. Biol. 36, 1856-1866 (2010).</li><li>Lafon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+phantom+for+use+in+high-intensity+focused+ultrasound+dosimetry.">Gel phantom for use in high-intensity focused ultrasound dosimetry.</a> Ultrasound Med. Biol. 31, 1383-1389 (2005).</li></ol>

No conflicts of interest declared.

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors.1 To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth.2 Thus, nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors, a process known as the enhanced permeability and retention (EPR) effect...

<table border="1">
 <tbody>
 <tr>
 <td>Common Name</td>
 <td>Manufacturer</td>
 <td>Cat. Number</td>
 <td>Full Name / Description</td>
 </tr>
 <tr>
 <td>DPPC</td>
 <td>Avanti Lipids, Alabaster, AL, USA</td>
 <td>850355P</td>
 <td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine</td>
 </tr>
 <tr>
 <td>DSPE-PEG2000</td>
 <td>Avanti Lipids, Alabaster, AL, USA</td>
 <td>880120P</td>
 <td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)</td>
 </tr>
 <tr>
 <td>DDFP</td>
 <td>Fluoromed, Round Rock, TX, USA</td>
 <td>CAS: 138495-42-8</td>
 <td>Dodecafluoropentane (C5F12)</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Sigma-Aldrich, St. Louis, MO, USA</td>
 <td>P2194</td>
 <td>Phosphate-buffered saline</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Sigma-Aldrich, St. Louis, MO, USA</td>
 <td>372978</td>
 <td>Chloroform</td>
 </tr>
 <tr>
 <td>Acrylamide</td>
 <td>Sigma-Aldrich, St. Louis, MO, USA</td>
 <td>A9926</td>
 <td>40% 19:1 acrylamide/bis-acrylamide</td>
 </tr>
 <tr>
 <td>Tris buffer</td>
 <td>Sigma-Aldrich, St. Louis, MO, USA</td>
 <td>T2694</td>
 <td>1M, pH 8, trizma hydrochloride and trizma base</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma-Aldrich, St. Louis, MO, USA</td>
 <td>A3059</td>
 <td>Bovine serum albumin</td>
 </tr>
 <tr>
 <td>APS</td>
 <td>Sigma-Aldrich, St. Louis, MO, USA</td>
 <td>A3678</td>
 <td>Ammonium persulfate solution</td>
 </tr>
 <tr>
 <td>TEMED</td>
 <td>Sigma-Aldrich, St. Louis, MO, USA</td>
 <td>87689</td>
 <td>N,N,N',N'-Tetramethylethylenediamine</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Sonicator (3 mm tip)</td>
 <td>Sonics &amp; Materials, Inc., Newtown, CT, USA</td>
 <td>Vibra-Cell</td>
 <td></td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>Thermo Fisher Scientific, Waltham, MA, USA</td>
 <td>Neslab EX-7</td>
 <td></td>
 </tr>
 <tr>
 <td>Extruder</td>
 <td>Northern Lipids, Burnaby, BC, Canada</td>
 <td>LIPEX</td>
 <td></td>
 </tr>
 <tr>
 <td>Extruder Filters</td>
 <td>Whatman, Piscataway, NJ, USA </td>
 <td>Nuclepore #110605 and #110606</td>
 <td></td>
 </tr>
 <tr>
 <td>Extruder Drain Disc</td>
 <td>Sterlitech Corporation, Kent, WA, USA</td>
 <td>#PETEDD25100</td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic chamber</td>
 <td>U.S. Plastic Corporation, Lima, OH, USA</td>
 <td>#55288, 1 3/16"x1 3/16"x2 7/16"</td>
 <td></td>
 </tr>
 </tbody>
</table>

synthesis of phase-shift nanoemulsions with narrow size distributions for acoustic droplet vaporization and bubble-enhanced ultrasound-mediated ablation

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Watch this Scientific Journal Video about Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation at Jo

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

High-intensity  focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To  enhance localized heating and improve thermal ablation in ...

synthesis-phase-shift-nanoemulsions-with-narrow-size-distributions

Research

JoVE Journal

Engineering

11.0K Views.  Boston University. Phase-shift nanoemulsioni (PSNE) può essere vaporizzato con ultrasuoni focalizzati ad alta intensità per migliorare il riscaldamento localizzato e migliorare la termoablazione nei tumori. In questa relazione, la preparazione di PSNE stabile con una distribuzione di dimensione stretta è descritto. Inoltre, l'impatto di PSNE vaporizzato su ultrasuoni-mediata ablazione è in tessuti che imitano fantasmi.

Video: Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

This paper discusses fluorescent core microcavity-based sensors that can operate in a microfluidic analysis setup. These structures are based on the formation of a fluorescent quantum-dot (QD) coating on the channel surface of a conventional microcapillary. Silicon QDs are especially attractive for this application, owing in part to their negligible toxicity compared to the II-VI and II-VI compound QDs, which are legislatively controlled substances in many countries. While the ensemble emission spectrum is broad and featureless, an Si-QD film on the channel wall of a capillary features a set of sharp, narrow peaks in the fluorescence spectrum, corresponding to the electromagnetic resonances for light trapped within the film. The peak wavelength of these resonances is sensitive to the external medium, thus permitting the device to function as a refractometric sensor in which the QDs never come into physical contact with the analyte. The experimental methods associated with the fabrication of the fluorescent-core microcapillaries are discussed in detail, as well as the analysis methods. Finally, a comparison is made between these structures and the more widely investigated liquid-core optical ring resonators, in terms of microfluidic sensing capabilities.

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

This paper discusses  fluorescent core microcavity-based sensors that can operate in a microfluidic  analysis setup. These structures are based on the ...

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.

The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material ...

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device.

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we ...

Characterization of Nanocrystal Size Distribution using Raman Spectroscopy with a Multi-particle Phonon Confinement Model

Analysis of the size distribution of nanocrystals is a critical requirement for the processing and optimization of their size-dependent properties. The common techniques used for the size analysis are transmission electron microscopy (TEM), X-ray diffraction (XRD) and photoluminescence spectroscopy (PL). These techniques, however, are not suitable for analyzing the nanocrystal size distribution in a fast, non-destructive and a reliable manner at the same time. Our aim in this work is to demonstrate that size distribution of semiconductor nanocrystals that are subject to size-dependent phonon confinement effects, can be quantitatively estimated in a non-destructive, fast and reliable manner using Raman spectroscopy. Moreover, mixed size distributions can be separately probed, and their respective volumetric ratios can be estimated using this technique. In order to analyze the size distribution, we have formulized an analytical expression of one-particle PCM and projected it onto a generic distribution function that will represent the size distribution of analyzed nanocrystal. As a model experiment, we have analyzed the size distribution of free-standing silicon nanocrystals (Si-NCs) with multi-modal size distributions. The estimated size distributions are in excellent agreement with TEM and PL results, revealing the reliability of our model.

We demonstrate how to determine the size distribution of semiconductor nanocrystals in a quantitative manner using Raman spectroscopy employing an analytically defined multi-particle phonon confinement model. Results obtained are in excellent agreement with the other size analysis techniques like transmission electron microscopy and photoluminescence spectroscopy.

Analysis of the size distribution of nanocrystals is a critical requirement for the processing and optimization of their size-dependent properties. The ...

Characterization of Full Set Material Constants and Their Temperature Dependence for Piezoelectric Materials Using Resonant Ultrasound Spectroscopy

During the operation of high power electromechanical devices, a temperature rise is unavoidable due to mechanical and electrical losses, causing the degradation of device performance. In order to evaluate such degradations using computer simulations, full matrix material properties at elevated temperatures are needed as inputs. It is extremely difficult to measure such data for ferroelectric materials due to their strong anisotropic nature and property variation among samples of different geometries. Because the degree of depolarization is boundary condition dependent, data obtained by the IEEE (Institute of Electrical and Electronics Engineers) impedance resonance technique, which requires several samples with drastically different geometries, usually lack self-consistency. The resonant ultrasound spectroscopy (RUS) technique allows the full set material constants to be measured using only one sample, which can eliminate errors caused by sample to sample variation. A detailed RUS procedure is demonstrated here using a lead zirconate titanate (PZT-4) piezoceramic sample. In the example, the complete set of material constants was measured from room temperature to 120 &#176;C. Measured free dielectric constants <img alt="Equation 1" src="/files/ftp_upload/53461/image001.jpg" /> and&#160;<img alt="Equation 2" src="/files/ftp_upload/53461/image002.jpg" /> were compared with calculated ones based on the measured full set data, and piezoelectric constants d15 and d33 were also calculated using different formulas. Excellent agreement was found in the entire range of temperatures, which confirmed the self-consistency of the data set obtained by the RUS.

This protocol describes the procedure of measuring the temperature dependence of the full set material constants of piezoelectric materials using resonant ultrasound spectroscopy (RUS).

During the operation of high power electromechanical devices, a temperature rise is unavoidable due to mechanical and electrical losses, causing the ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must be smaller than the incident wavelength of sound and the width of the fluidic channels are typically tens to hundreds of micrometers across, acoustofluidic devices typically use ultrasonic waves generated from a piezoelectric transducer pulsating at high frequencies (in the megahertz range). At characteristic frequencies that depend on the geometry of the device, it is possible to induce the formation of standing waves that can focus particles along desired fluidic streamlines within a bulk flow. Here, we describe a method for the fabrication of acoustophoretic devices from common materials and clean room equipment. We show representative results for the focusing of particles with positive or negative acoustic contrast factors, which move towards the pressure nodes or antinodes of the standing waves, respectively. These devices offer enormous practical utility for precisely positioning large numbers of microscopic entities (e.g., cells) in stationary or flowing fluids for applications ranging from cytometry to assembly.

Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must ...

Measuring Spray Droplet Size from Agricultural Nozzles Using Laser Diffraction

When making an application of any crop protection material such as an herbicide or pesticide, the applicator uses a variety of skills and information to make an application so that the material reaches the target site (i.e., plant). Information critical in this process is the droplet size that a particular spray nozzle, spray pressure, and spray solution combination generates, as droplet size greatly influences product efficacy and how the spray moves through the environment. Researchers and product manufacturers commonly use laser diffraction equipment to measure the spray droplet size in laboratory wind tunnels. The work presented here describes methods used in making spray droplet size measurements with laser diffraction equipment for both ground and aerial application scenarios that can be used to ensure inter- and intra-laboratory precision while minimizing sampling bias associated with laser diffraction systems. Maintaining critical measurement distances and concurrent airflow throughout the testing process is key to this precision. Real time data quality analysis is also critical to preventing excess variation in the data or extraneous inclusion of erroneous data. Some limitations of this method include atypical spray nozzles, spray solutions or application conditions that result in spray streams that do not fully atomize within the measurement distances discussed. Successful adaption of this method can provide a highly efficient method for evaluation of the performance of agrochemical spray application nozzles under a variety of operational settings. Also discussed are potential experimental design considerations that can be included to enhance functionality of the data collected.

We present protocols to be used in the measurement of spray droplet size from agricultural nozzles used in both aerial and ground based agrochemical applications. These methods presented were developed to provide consistent and repeatable droplet size data both inter- and intra-laboratory, when using laser diffraction systems.

When making an application of any crop protection material such as an herbicide or pesticide, the applicator uses a variety of skills and information to ...

A Pipette-Tip Based Method for Seeding Cells to Droplet Microfluidic Platforms

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and analyzing cells at the single-cell level by eliminating the influence of external factors on the cellular microenvironment. Encapsulation of cells in droplets is dictated by the Poisson distribution as a function of the number of cells present in each droplet and the average number of cells per volume of droplet. Primary cells, especially immune cells, or clinical specimens can be scarce and loss-less encapsulation of cells remains challenging. In this paper, we present a new methodology that uses pipette-tips to load cells to droplet-based microfluidic devices without the significant loss of cells. With various cell types , we demonstrate efficient cell encapsulation in droplets that closely corresponds to the encapsulation efficiency predicted by the Poisson distribution. Our method ensures loss-less loading of cells to microfluidic platforms and can be easily adapted for downstream single cell analysis, e.g., to decode cellular interactions between different cell types.

This article presents a protocol for seeding scarce population of cells using pipette-tips to droplet microfluidic devices in order to provide higher encapsulation efficiency of cells in droplets.

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and ...