This work was supported in part by the NIH grants R01 EB008101 and R01 CA103830.

1. Instrumentations and Glassware Preparation
<ol>
	<li>Wear appropriate protective equipment, i.e., a lab coat, disposable gloves, and eye protection.</li>
	<li>Connect a round-bottom flask to a condenser and immerse it in a silicone oil bath with a temperature monitoring by a thermometer. Place a source of heat (e.g., hot plate) under the oil bath (Figure 1). Use a thermometer capable of measuring the temperature higher than 260 &#176;C.</li>
</ol>
2. Synthesis of Primary Hybrid Magneto-plasmonic Nanoparticles
<ol>
	<li>Making Magnetic Core Nanoparticles
	<ol>
		<li>Add 353.2 mg (1 mmol) iron(III) acetylacetonate, 1 ml (2 mmol) oleic acid, 1 ml (2 mmol) oleylamine, 1.292 g (5 mmol) 1,2-hexadecanediol, and 10 ml phenyl ether to a round-bottom flask.</li>
		<li>Stir the mixture vigorously using a magnetic stir-bar and heat to 250-260 &#176;C for 1 hr under reflux. Then, wait for the solution to cool down to RT. Ensure the temperature is under 260 &#176;C to prevent boiling of the phenyl ether and prevent a burst of the reaction mixture from the round-bottom flask to the condenser. 
		CAUTION: The reaction mixture is extremely hot and the chemicals may cause irritation. Must operate under a fume hood and wear appropriate personal protection equipment. Ensure adequate ventilation for the oil bath. 
		NOTE: The oil bath is kept at 250-260 &#186;C temperature for 1&#160;hr during synthesis of the magnetic nanoparticles. In principle, a Pyrex glass dish can be used for this purpose. However, the maximum continuous temperature for Pyrex glass is ~260 &#186;C according to vendor information. Therefore, a metal container provides a safer option for the reaction as it can withstand a higher temperature and last longer during multiple runs.</li>
	</ol>
	</li>
	<li>Deposition a gold shell onto magnetic core nanoparticles
	<ol>
		<li>Add 411.5 mg (1.1 mmol) gold acetate, 0.25 ml (0.75 mmol) oleic acid, 1.5 ml (3.0 mmol) oleylamine, 775.3 mg (3 mmol) 1,2-hexadecanediol, and 15 ml phenyl ether to a round-bottom flask.</li>
		<li>Add 5 ml suspension of magnetic nanoparticles from step 2.1. Heat the reaction mixture to 180 &#176;C and keep under reflux for 1 hr. Wait for the solution to cool down to RT.</li>
		<li>Add 50 ml ethanol to precipitate the hybrid primary nanoparticles followed by centrifugation at 3,250 &#215; g for 15 min.</li>
		<li>Resuspend the precipitate in 25 ml hexane by using a bath sonicator. Add 25 ml ethanol to precipitate the primary hybrid nanoparticles. Centrifuge at 3,250 x&#160;g for 15 min and resuspend the precipitate in hexane. Repeat this step three times.</li>
		<li>Dry the precipitated primary hybrid nanoparticles in a vacuum desiccator O/N. Confirm that the particles are completely dry.</li>
	</ol>
	</li>
</ol>
3. Hybrid Magneto-plasmonic Nanoclusters Synthesis and Size Separation
<ol>
	<li>Resuspend 5 mg of dry primary hybrid nanoparticles in 1 ml hexane by using a bath sonicator. Sonicate the nanoparticle suspension until no visible precipitate is present.</li>
	<li>Add the solution from step 3.1 to 10 ml aqueous solution of sodium dodecyl sulfate (2.8 mg/ml) in a 20 ml glass vial with attached caps. Add the suspension of primary hybrid nanoparticles drop by drop to avoid mixing of the two phases before the next step.</li>
	<li>Sonicate the two-phase solution in a bath sonicator for 2 hr, followed by heating in a water bath at 80 &#176;C for 10 min. Wait for the solution to cool down to RT.
	<ol>
		<li>Fill water to the operating level line of the sonication bath. Center the glass vial in the sonication bath. An emulsion forms immediately between the two phases. Shake the two-phase solution by hand after beginning of the sonication; this facilitates mixing between the phase containing primary hybrid nanoparticles and the bottom aqueous phase. 
		NOTE: Be aware that the sonicator will heat up after 2 hr of operation.</li>
	</ol>
	</li>
	<li>Centrifuge the hybrid nanocluster suspension at 100 x&#160;g for 30 min. Collect both the precipitate and the supernatant. Resuspend the precipitate in 0.1 mM sodium citrate under 10 min sonication. The expected size of the nanoclusters is ~180 nm in diameter.</li>
	<li>Transfer the supernatant from step 3.3 to a new conical tube.</li>
	<li>Centrifuge the suspension from step 3.4 at 400 x&#160;g for 30 min. Collect both the precipitate and the supernatant. Resuspend the precipitate in 0.1 mM sodium citrate under 10 min sonication. The expected size of the nanoclusters is ~130 nm in diameter.</li>
	<li>Transfer the supernatant from step 3.5 to a new conical tube.</li>
	<li>Centrifuge the suspension from step 3.6 at 1,500 x&#160;g for 30 min. Collect the precipitate and resuspend in 0.1 mM sodium citrate under 10 min sonication. The expected size of the nanoclusters is ~90 nm in diameter.</li>
	<li>Add 300 &#956;l nanocluster suspension to a 96-well microplate reader for measuring a UV-Vis-NIR absorption spectrum. Drop 10 &#956;l nanocluster suspension onto carbon-coated copper grid for TEM imaging.</li>
</ol>
4. Conjugation of Monoclonal Antibodies to Nanoclusters
<ol>
	<li>Prepare 100 &#956;l monoclonal antibody solution (1 mg/ml) in PBS, pH 7.2, e.g., anti-Epidermal Growth Factor Receptor 2 (HER2) antibodies or anti-Epidermal Growth Factor Receptor 1 (EGFR) antibodies.</li>
	<li>Add the antibody solution from step 4.1 to 3.9 ml 4 mM HEPES, pH 7.2. Centrifuge the solution through a 10 k MWCO centrifugal filter at 3,250 x&#160;g for 20 min at 8 &#176;C. Resuspend the antibody in 4 mM HEPES, pH 7.2, to a final volume of 100 &#956;l. 
	NOTE: This step is carried out to replace the original media in the antibody solution with HEPES.</li>
	<li>Add 10 &#956;l of 100 mM NaIO4 to 100 &#956;l of antibody solution. Cover the reaction vial with an aluminum foil at RT and mix for 30 min using an orbital shaker.</li>
	<li>Quench the reaction by adding 500 &#956;l of 1x PBS.</li>
	<li>Add 2 &#956;l of 46.5 mM linker solution (dithiolaromatic PEG6-CONHNH2) to the antibody solution from step 4.4 and shake for 1 hr at RT.</li>
	<li>Filter the solution using a 10 k MWCO centrifuge filter at 3,250 x&#160;g for 20 min at 8 &#176;C. Resuspend the antibody in 1x PBS to a final volume of 100 &#956;l which leads to an antibody concentration of ~1 mg/ml.</li>
	<li>Mix 100 &#956;l nanocluster suspension at O.D. ~1.0 with 1 &#956;l modified antibodies from step 4.6 (1 mg/ml) for 120 min at RT.</li>
	<li>Add 10 &#956;l of 10-3 M 5 kDa thiol PEG and shake for 15 min at RT.</li>
	<li>Centrifuge the solution at 830 x&#160;g for 3 min. Discard the supernatant and resuspend the sediment in 100 &#956;l 2% w/v 5 kDa PEG in PBS, pH 7.2.</li>
	<li>Measure the absorbance spectrum of the antibody-conjugated nanoclusters and compare to the absorbance spectrum of the bare nanoclusters. Expect a couple of nanometer red shift after the conjugation.</li>
	<li>If the nanoparticles aggregate as shown by a significant shift with an increase in O.D. in the red-NIR region, increase the concentration of thiol PEG to 5 x&#160;10-3 M. Also, increase the incubation time with thiol PEG to 30 min and decrease the centrifugal speed in 200 x&#160;g increments.</li>
	<li>For cancer cell labeling test, add the antibody conjugated nanoparticles from step 4.9 to cancer cell suspension in either medium or 1x PBS (1 ml ~106 cells) and mix for 60 min.</li>
</ol>

<ol>
	<li>Bigall, N. C., Parak, W. J., Dorfs, 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,+magnetic+and+plasmonic&#8212;Hybrid+multifunctional+colloidal+nano+objects.">Fluorescent, magnetic and plasmonic&#8212;Hybrid multifunctional colloidal nano objects.</a> Nano Today. 7, 282-296 (2012).</li><li>Gautier, J., Allard-Vannier, E., Herve-Aubert, K., Souce, M., Chourpa, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+strategies+of+hybrid+metallic+nanoparticles+for+theragnostic+applications.">Design strategies of hybrid metallic nanoparticles for theragnostic applications.</a> Nanotechnology. 24, 432002 (2013).</li><li>Wei, Q., Wei, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+with+dynamic+contrast+agents.">Optical imaging with dynamic contrast agents.</a> Chemistry. 17, 1080-1091 (2011).</li><li>Aaron, J. 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=Increased+optical+contrast+in+imaging+of+epidermal+growth+factor+receptor+using+magnetically+actuated+hybrid+gold/iron+oxide+nanoparticles.">Increased optical contrast in imaging of epidermal growth factor receptor using magnetically actuated hybrid gold/iron oxide nanoparticles.</a> Optics express. 14, 12930-12943 (2006).</li><li>Song, H. -. M., Wei, Q., Ong, Q. K., Wei, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmon-resonant+nanoparticles+and+nanostars+with+magnetic+cores:+synthesis+and+magnetomotive+imaging.">Plasmon-resonant nanoparticles and nanostars with magnetic cores: synthesis and magnetomotive imaging.</a> ACS nano. 4, 5163-5173 (2010).</li><li>Qu, M., 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=Magneto-photo-acoustic+imaging.">Magneto-photo-acoustic imaging.</a> Biomedical optics express. 2, 385-396 (2011).</li><li>Jin, Y., Jia, C., Huang, S. -. W., Donnell O&amp;39, M., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+nanoparticles+as+coupled+contrast+agents.">Multifunctional nanoparticles as coupled contrast agents.</a> Nature communications. 1, 41 (2010).</li><li>Wu, C. -. 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=Versatile+Immunomagnetic+Nanocarrier+Platform+for+Capturing+Cancer+Cells.">Versatile Immunomagnetic Nanocarrier Platform for Capturing Cancer Cells.</a> ACS. 7, 8816-8823 (2013).</li><li>Galanzha, E. I., 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=In+vivo+magnetic+enrichment+and+multiplex+photoacoustic+detection+of+circulating+tumour+cells.">In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells.</a> Nature nanotechnology. 4, 855-860 (2009).</li><li>Larson, T. A., Bankson, J., Aaron, J., Sokolov, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+plasmonic+magnetic+nanoparticles+as+molecular+specific+agents+for+MRI/optical+imaging+and+photothermal+therapy+of+cancer+cells.">Hybrid plasmonic magnetic nanoparticles as molecular specific agents for MRI/optical imaging and photothermal therapy of cancer cells.</a> Nanotechnology. 18, 325101 (2007).</li><li>Yu, 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=Dumbbell-like+bifunctional+Au-Fe3O4+nanoparticles.">Dumbbell-like bifunctional Au-Fe3O4 nanoparticles.</a> Nano letters. 5, 379-382 (2005).</li><li>Wang, 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=Monodispersed+core-shell+Fe3O4@Au+nanoparticles.">Monodispersed core-shell Fe3O4@Au nanoparticles.</a> The journal of physical chemistry. B. 109, 21593-21601 (2005).</li><li>Wang, H., Brandl, D. W., Le, F., Nordlander, P., Halas, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanorice:+a+hybrid+plasmonic+nanostructure.">Nanorice: a hybrid plasmonic nanostructure.</a> Nano letters. 6, 827-832 (2006).</li><li>Hu, X., 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=Trapping+and+Photoacoustic+Detection+of+CTCs+at+the+Single+Cell+per+Milliliter+Level+with+Magneto&#8208;Optical+Coupled+Nanoparticles.">Trapping and Photoacoustic Detection of CTCs at the Single Cell per Milliliter Level with Magneto&#8208;Optical Coupled Nanoparticles.</a> Small. 9, 2046-2052 (2013).</li><li>Truby, R. L., Emelianov, S. Y., Homan, K. 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The authors declare that they have no competing financial interests.

Critical steps in successful synthesis of magneto-plasmonic nanoclusters include making highly monodispersed primary gold shell/iron oxide core nanoparticles and directing self-assembly of the primary particles into nanoclusters. A molar ratio between the primary particles and surfactants play an important role in determining size distribution of the nanoclusters. Non-uniform size distribution of primary nanoparticles may cause formation of big aggregates during assembly of magneto-plasmonic nanoclusters. In addition, th...

Hybrid nanoparticles consisting of different materials with distinct physicochemical properties can open new opportunities in biomedical applications including multimodal molecular imaging, therapy delivery and monitoring, new screening and diagnostic assays1-3. The combination of plasmonic and magnetic properties in a single nanoparticle is of particular interest because it provides a very strong light scattering and absorption cross-sections associated with plasmon resonances and&#160;responsiveness to a magnetic field. For example, magneto-plasmonic nanoparticles were used to increase contrast in dark-field imaging of labeled cells by applying a temporal signal modulation via an external electromagnet3-5. More recently, a similar principle was applied in development of a new imaging modality &#8211; magneto-photoacoustic imaging, where magneto-plasmonic nanoparticles enable great improvements in&#160;contrast and signal-to-background ratio6,7. It was also shown that the hybrid nanoparticles can be used for simultaneous capture and detection of circulating tumor cells in whole blood and in vivo8,9.&#160;Furthermore, magneto-plasmonic nanoparticles are promising theranostic agents which can be used for molecular specific optical and MR imaging combined with photothermal therapy of cancer cells10.
Several approaches were explored for synthesis of magneto-plasmonic nanoparticles. For example, Yu et al. utilized decomposition and oxidation of Fe(CO)5 on&#160;gold nanoparticles to form dumbbell-like bifunctional Au&#8211;Fe3O4 nanoparticles11. Wang et al. have synthesized gold-coated iron oxide nanoparticle by using thermal decomposition method12. Some other approaches rely on coating polymer or amine functional molecules onto magnetic core nanoparticles followed by deposition of a gold shell onto the polymer surface to create the hybrid particles7,13. In addition, iron-oxide nanoparticles were attached to gold nanorods via electrostatic interactions or a chemical reaction14,15. Although these approaches yield magneto-plasmonic nanostructures, they compromise to some extent properties of the magneto-plasmonic combination such as optical absorbance in the near-infrared (NIR) window or a strong magnetic moment both of which are highly desirable in biomedical applications. For example,&#160;dumbbell Au-Fe3O4 nanoparticles have a plasmon resonance peak at 520 nm which limits their utility in vivo due to high tissue turbidity in this spectral range. Furthermore, the magneto-plasmonic nanoparticles produced by&#160;current protocols are limited to just one11 or few (less than 10)14,15 superparamagnetic moieties (e.g., iron oxide nanoparticles) that is significantly less than could be achieved in a densely packed nanostructure. For example, a densely packed 60 nm diameter spherical nanoparticle can contain on the order of a thousand of 6 nm superparamagnetic nanoparticles. Therefore, there is a great room for improving magnetic properties of the hybrid nanoparticles. Moreover, some of the previously described protocols are relatively complex and require careful optimization in order to avoid particle aggregation during synthesis14,15.
Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong NIR absorbance that addresses major limitations of the current art. The synthesis has its origins in oil-in-water microemulsion method16. It is based on assembly of nanoparticles of a desired size from a much smaller primary particles. This approach has been successfully used to produce nanostructures from a single material such as gold, iron oxide, and semiconductor primary particles16. We extended it to synthesis of magneto-plasmonic nanoparticles by, first, making 6 nm diameter gold shell/iron oxide core particles and, then, assembling the primary hybrid particles into the final spherical nanostructure. Assembling primary particles into nanoclusters not only allows enhancing the properties of constituent nanoparticles, such as achieving a stronger magnetic moment while preserving superparamagnetic properties, but also takes advantage of the interactions between individual nanoparticles thus&#160;creating&#160;new characteristics absent from the constituent nanoparticles, such as strong optical absorbance in the NIR window. This protocol yields hybrid nanoparticles with a high density of magnetic and plasmonic functionalities. After primary particles are synthetized, our method is essentially a simple one-pot reaction. The overall plasmon resonance strength and magnetic moment are determined by a&#160;number of primary particles and, therefore, can be easily optimized depending on an application. Furthermore, we also developed a procedure for antibody conjugation to the hybrid nanoparticles for various biomedical applications which require molecular specific targeting. Antibodies are attached through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

<table><tbody><tr><td>PYREX 50 ml round bottom boiling flask with short neck &amp;amp; 24/40 [ST] joint</td><td>Corning</td><td>4320A-50</td><td>Thermal decomposition reaction</td></tr><tr><td>PYREX 41 x 300 mm 5-bulb Allihn condenser with 24/40 [ST] outer/inner joints</td><td>Corning</td><td>2480-300</td><td>Thermal decomposition reaction</td></tr><tr><td>Silicone oil</td><td>Fisher</td><td>S159-500</td><td>Oil bath</td></tr><tr><td>Hot plate stirrer</td><td>Corning</td><td>PC-351</td><td>Heat the reacton with stirring function</td></tr><tr><td>Thermometer</td><td>ThermoWorks</td><td>221-092</td><td>Measure temperature</td></tr><tr><td>Iron(III) acetylacetonate</td><td>Fisher</td><td>AC11913-0250</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Oleic acid 99%</td><td>Fisher</td><td>A195-500</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Gold(III) acetate</td><td>Fisher</td><td>AA3974206</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Hexane</td><td>Fisher</td><td>H292-1</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Phenyl ether 99%</td><td>Fisher</td><td>AC13060-0025</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>1,2-Hexadecanediol 90%</td><td>Sigma</td><td>213748-50G</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Oleylamine 70%</td><td>Sigma</td><td>O7805-100G</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Fisher</td><td>BP166-100</td><td>Cluster synthesis</td></tr><tr><td>Sodium citrate dihydrate</td><td>Sigma</td><td>W302600</td><td>Cluster synthesis</td></tr><tr><td>Monoclonal anti-EGF receptor antibody</td><td>Sigma</td><td>E2156</td><td>Cell labeling specificity test</td></tr><tr><td>Monoclonal anti-HER2 antibody</td><td>Sigma</td><td>AMAB90627</td><td>Cell labeling specificity test</td></tr><tr><td>Sodium periodate</td><td>Sigma</td><td>311448</td><td>Oxidate Fc region of antibodies</td></tr><tr><td>Dithiolaromatic PEG6-CONHNH&lt;sub&gt;2&lt;/sub&gt;</td><td>SensoPath Technologies</td><td>SPT-0014B</td><td>Heterofunctional linker for antibody conjugation to nanoclusters</td></tr><tr><td>Methoxy-PEG-thiol, 5 k</td><td>Creative PEGworks</td><td>PLS-604</td><td>Passivate the remaining gold surface after antibody conjugation</td></tr><tr><td>Amicon Ultra-4 centrifugal filter unit with Ultracel-10 membrane</td><td>Millipore</td><td>UFC801008</td><td>Protein purification</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Buffer</td></tr><tr><td>PBS, 1x solution</td><td>Fisher</td><td>BP2438-20</td><td>Buffer</td></tr><tr><td>UV-Vis spectroscopy</td><td>BioTek&amp;nbsp;</td><td>Synergy HT</td><td>Obtain spectrum</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>Separation</td></tr><tr><td>Transmission Electron Microscope</td><td>FEI</td><td>TECNAI G2 F20 X-TWIN</td><td>Obtain morphology of nanostructures</td></tr><tr><td>Upright microscope</td><td>Leica</td><td>DM6000</td><td>Obtain dark-field images</td></tr><tr><td>Sonicator</td><td>Branson</td><td>1510</td><td>Sonication</td></tr><tr><td>Carbon film 300 mesh grid</td><td>EMS</td><td>CF300-Cu</td><td>TEM imaging</td></tr><tr><td>96-well plate</td><td>Corning</td><td>09-761-145</td><td>UV-Vis reading plate</td></tr></tbody></table>

synthesis of immunotargeted magneto-plasmonic nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

A scheme for synthesis of immunotargeted magneto-plasmonic nanoclusters is shown in Figure 2. First, magnetic Fe3O4 iron oxide nanoparticles are synthetized via thermal decomposition method. Then, a thin ca. 1 nm gold shell is deposited on the iron oxide core particles via thermal decomposition. The primary ca. 6 nm hybrid nanoparticles serve as seeds to create magneto-plasmonic nanoclusters by utilizing an oil-in-water microemulsion approach. The nanoclusters are ...

Watch this Scientific Journal Video about Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters at JoVE.com

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

synthesis-of-immunotargeted-magneto-plasmonic-nanoclusters

Research

JoVE Journal

Chemistry

15.2K Views.  University of Texas at Austin.  Hier beschrijven we een protocol voor de synthese van magneto-plasmon nanodeeltjes met een sterk magnetisch moment en een sterke nabij-infrarood (NIR) absorptie. Het protocol omvat ook antilichaam conjugatie aan de nanodeeltjes door de Fc eenheid voor diverse biomedische toepassingen die moleculaire specifieke targeting vereisen. 

Video: Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Bioengineering

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating

One of the most widely used methods for manufacturing colloidal gold nanospherical particles involves the reduction of chloroauric acid (HAuCl4) to neutral gold Au(0) by reducing agents, such as sodium citrate or sodium borohydride. The extension of this method to decorate iron oxide or similar nanoparticles with gold nanoparticles to create multifunctional hybrid Fe2O3-Au nanoparticles is straightforward. This approach yields fairly good control over Au nanoparticle dimensions and loading onto Fe2O3. Additionally, the Au metal size, shape, and loading can easily be tuned by changing experimental parameters (e.g., reactant concentrations, reducing agents, surfactants, etc.). An advantage of this procedure is that the reaction can be done in air or water, and, in principle, is amenable to scaling up. The use of such optically tunable Fe2O3-Au nanoparticles for hyperthermia studies is an attractive option as it capitalizes on plasmonic heating of gold nanoparticles tuned to absorb light strongly in the VIS-NIR region. In addition to its plasmonic effects, nanoscale Au provides a unique surface for interesting chemistries and catalysis. The Fe2O3 material provides additional functionality due to its magnetic property. For example, an external magnetic field could be used to collect and recycle the hybrid Fe2O3-Au nanoparticles after a catalytic experiment, or alternatively, the magnetic Fe2O3 can be used for hyperthermia studies through magnetic heat induction. The photothermal experiment described in this report measures bulk temperature change and nanoparticle solution mass loss as functions of time using infrared thermocouples and a balance, respectively. The ease of sample preparation and the use of readily available equipment are distinct advantages of this technique. A caveat is that these photothermal measurements assess the bulk solution temperature and not the surface of the nanoparticle where the heat is transduced and the temperature is likely to be higher.

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating

We describe the synthesis and properties of multifunctional Fe2O3-Au nanoparticles produced by a wet chemical approach and investigate their photothermal properties using laser irradiation. The composite Fe2O3-Au nanoparticles retain the properties of both materials, creating a multifunctional structure with excellent magnetic and plasmonic properties.

One of the most widely used methods for manufacturing colloidal gold nanospherical particles involves the reduction of chloroauric acid (HAuCl4) to ...

Engineering

Colloidal Synthesis of Nanopatch Antennas for Applications in Plasmonics and Nanophotonics

We present a method for colloidal synthesis of silver nanocubes and the use of these in combination with a smooth gold film, to fabricate plasmonic nanoscale patch antennas. This includes a detailed procedure for the fabrication of thin films with a well-controlled thickness over macroscopic areas using layer-by-layer deposition of polyelectrolyte polymers, namely poly(allylamine) hydrochloride (PAH) and polystyrene sulfonate (PSS). These polyelectrolyte spacer layers serve as a dielectric gap in between silver nanocubes and a gold film. By controlling the size of the nanocubes or the gap thickness, the plasmon resonance can be tuned from about 500 nm to 700 nm. Next, we demonstrate how to incorporate organic sulfo-cyanine5 carboxylic acid (Cy5) dye molecules into the dielectric polymer gap region of the nanopatch antennas. Finally, we show greatly enhanced fluorescence of the Cy5 dyes by spectrally matching the plasmon resonance with the excitation energy and the Cy5 absorption peak. The method presented here enables the fabrication of plasmonic nanopatch antennas with well-controlled dimensions utilizing colloidal synthesis and a layer-by-layer dip-coating process with the potential for low cost and large-scale production. These nanopatch antennas hold great promise for practical applications, for example in sensing, ultrafast optoelectronic devices and for high-efficiency photodetectors.

A protocol for the colloidal synthesis of silver nanocubes and fabrication of plasmonic nanoscale patch antennas with sub-10 nm gaps is presented.

We present a method for colloidal synthesis of silver nanocubes and the use of these in combination with a smooth gold film, to fabricate plasmonic ...

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing in combination with new nanofabrication techniques. A plasmon is a quantum of charge density oscillation that lends nanoscale metals such as gold and silver unique optical properties. In particular, gold and silver nanoparticles exhibit localized surface plasmon resonances-collective charge density oscillations on the surface of the nanoparticle-in the visible spectrum. Here, we focus on the fabrication of periodic arrays of anisotropic plasmonic nanostructures. These half-shell (or nanocup) structures can exhibit additional unique light-bending and polarization-dependent optical properties that simple isotropic nanostructures cannot. Researchers are interested in the fabrication of periodic arrays of nanocups for a wide variety of applications such as low-cost optical devices, surface-enhanced Raman scattering, and tamper indication. We present a scalable technique based on colloidal lithography in which it is possible to easily fabricate large periodic arrays of nanocups using spin-coating and self-assembled commercially available polymeric nanospheres. Electron microscopy and optical spectroscopy from the visible to near-infrared (near-IR) was performed to confirm successful nanocup fabrication. We conclude with a demonstration of the transfer of nanocups to a flexible, conformal adhesive film.

We demonstrate the fabrication of periodic gold nanocup arrays using colloidal lithographic techniques and discuss the importance of nanoplasmonic films.

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing ...

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

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