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>

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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. 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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 border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Name of Material/ Equipment</td>
			<td style="width:101px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:247px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:253px;">PYREX 50mL Round Bottom Boiling Flask with Short Neck &#38; 24/40 [ST] Joint</td>
			<td>Corning</td>
			<td style="width:139px;">4320A-50</td>
			<td>Thermal decomposition reaction</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:253px;">PYREX 41 x 300mm 5-Bulb Allihn Condenser with 24/40 [ST] Outer/Inner Joints</td>
			<td>Corning</td>
			<td style="width:139px;">2480-300</td>
			<td>Thermal decomposition reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Silicone Oil</td>
			<td>Fisher</td>
			<td style="width:139px;">S159-500</td>
			<td>Oil bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Hot Plate Stirrer</td>
			<td>Corning</td>
			<td style="width:139px;">PC-351</td>
			<td>Heat the reacton with stirring function</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Thermometer</td>
			<td>ThermoWorks</td>
			<td style="width:139px;">221-092</td>
			<td>Measure temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:253px;">Iron (III) Acetylacetonate</td>
			<td>Fisher</td>
			<td style="width:139px;">AC11913-0250</td>
			<td rowspan="7" style="width:247px;">Materials for primary hybrid nanoparticles synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Oleic Acid 99%</td>
			<td>Fisher</td>
			<td style="width:139px;">A195-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Gold (III) Acetate</td>
			<td>Fisher</td>
			<td style="width:139px;">AA3974206</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Hexane</td>
			<td>Fisher</td>
			<td style="width:139px;">H292-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Phenyl Ether 99%</td>
			<td>Fisher</td>
			<td style="width:139px;">AC13060-0025</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-Hexadecanediol 90%</td>
			<td>Sigma</td>
			<td style="width:139px;">213748-50G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Oleylamine 70%</td>
			<td>Sigma</td>
			<td style="width:139px;">O7805-100G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Sodium Dodecyl Sulfate</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:139px;">BP166-100</td>
			<td rowspan="2" style="width:247px;">Cluster synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Sodium Citrate Dihydrate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:139px;">W302600</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Monoclonal Anti-EGF Receptor Antibody</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:139px;">E2156</td>
			<td style="width:247px;">Cell labeling specificity test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Monoclonal Anti-HER2 Antibody</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:139px;">AMAB90627</td>
			<td style="width:247px;">Cell labeling specificity test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Sodium Periodate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:139px;">311448</td>
			<td style="width:247px;">Oxidate Fc region of antibodies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Dithiolaromatic PEG6-CONHNH2</td>
			<td style="width:101px;">SensoPath Technologies</td>
			<td style="width:139px;">SPT-0014B</td>
			<td style="width:247px;">Heterofunctional linker for antibody conjugation to nanoclusters</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Methoxy-PEG-thiol, 5k</td>
			<td style="width:101px;">Creative PEGworks</td>
			<td style="width:139px;">PLS-604</td>
			<td style="width:247px;">Passivate the remaining gold surface after antibody conjugation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane</td>
			<td style="width:101px;">Millipore</td>
			<td style="width:139px;">UFC801008</td>
			<td style="width:247px;">Protien purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">HEPES</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:139px;">H3375</td>
			<td style="width:247px;">Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">PBS, 1X Solution</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:139px;">BP2438-20</td>
			<td style="width:247px;">Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">UV&#8722;vis Spectroscopy</td>
			<td style="width:101px;">BioTek&#160;</td>
			<td style="width:139px;">Synergy HT</td>
			<td style="width:247px;">Obtain spectrum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Separation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transmission Electron Microscope</td>
			<td>FEI</td>
			<td>TECNAI G2 F20 X-TWIN</td>
			<td>Obtain morphology of nanostructures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Upright microscope</td>
			<td>Leica</td>
			<td>DM6000</td>
			<td>Obtain dark-field images</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>Branson</td>
			<td>1510</td>
			<td>Sonication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Carbon Film 300 Mesh Grid</td>
			<td>EMS</td>
			<td style="width:139px;">CF300-Cu</td>
			<td>TEM imaging</td>
		</tr>
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			<td height="21" style="height:21px;width:253px;">96-well Plate</td>
			<td>Corning</td>
			<td style="width:139px;">09-761-145</td>
			<td>UV-vis reading plate</td>
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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. 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.

Video: Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Synthesis of Hierarchical ZnO/CdSSe Heterostructure Nanotrees

A two-step chemical vapor deposition procedure is here employed to prepare tree-like hierarchical ZnO/CdSSe hetero-nanostructures. The structures are composed of CdSSe branches grown on ZnO nanowires that are vertically aligned on a transparent sapphire substrate. The morphology was measured via scanning electron microscopy. The crystal structure was determined by X-ray powder diffraction analysis. Both the ZnO stem and CdSSe branches have a predominantly wurtzite crystal structure. The mole ratio of S and Se in the CdSSe branches was measured by energy dispersive X-ray spectroscopy. The CdSSe branches result in strong visible light absorption. Photoluminescence (PL) spectroscopy showed that the stem and branches form a type-II heterojunction. PL lifetime measurements showed a decrease in the lifetime of emission from the trees when compared to emission from individual ZnO stems or CdSSe branches and indicate fast charge transfer between CdSSe and ZnO. The vertically aligned ZnO stems provide a direct electron transport pathway to the substrate and allow for efficient charge separation after photoexcitation by visible light. The combination of the abovementioned properties makes ZnO/CdSSe nanotrees promising candidates for applications in solar cells, photocatalysis, and opto-electronic devices.

Here, we prepare and characterize novel tree-like hierarchical ZnO/CdSSe nanostructures, where CdSSe branches are grown on vertically aligned ZnO nanowires. The resulting nanotrees are a potential material for solar energy conversion and other opto-electronic devices.

A two-step chemical vapor deposition procedure is here employed to prepare tree-like hierarchical ZnO/CdSSe hetero-nanostructures. The structures are ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

Synthesis and Isolation of the MOF Product

To begin, cool the cold trap of the Schlenk line by placing a Dewar flask filled with liquid nitrogen around it. Use a towel to cover the top of the Dewar flask. This will slow down the evaporation of the liquid nitrogen during the experiment, open the bubbler to a light flow of inert gas, either nitrogen or argon.Roll a piece of weighing paper into a cone to use as a solid addition funnel and place it in the tap opening of the 10-milliliter flask. Ensure the bottom of the cone is inserted far enough that it extends past the hose attachment. Weigh the tetrakis triphenylphosphine palladium-0 into the 10-milliliter flask.Repeat this step for triphenylphosphine. After disposing the weighing paper cone, screw the polytetrafluoroethylene, or PTFE tap, onto the 10-milliliter flask. Similarly, weigh the tetratopic phosphine linker tin-1 into a separate 10-milliliter flask.To place the reagents under an inert atmosphere, connect hose from the Schlenk line to each of the 10-milliliter flasks. Open the PTFE tap just enough that the vessel is open to the hose. Open both the flasks to the vacuum and wait for five minutes.Close the tap on each flask and then close each hose to the vacuum. Switch the hoses to the inert gas and then slowly open the tap on each flask to backfill it with inert gas. Repeat these steps two more times for a total of three cycles.Remove the PTFE tap and replace it with a septum for each flask under a positive pressure of inert gas sufficient to prevent air from entering the flask. Use a syringe and needle to transfer 1.5 milliliters of dry and deoxygenated toluene into the flask containing the tetrakis triphenylphosphine palladium-0 and triphenylphosphine. Then, transfer 1.5 milliliters of dry and deoxygenated methylene chloride to the flask.Swirl the flask until all the solids have dissolved. Next, transfer three milliliters of dry and deoxygenated methylene chloride into the flask containing the tetratopic phosphine linker tin-1 and swirl the flask until all the solid has dissolved. Transfer the entire tin-1 linker solution into the flask containing the tetrakis triphenylphosphine palladium-0 and triphenylphosphine and swirl the solution for 30 seconds to thoroughly mix it.Replace the septum with the PTFE tap under a positive pressure of inert gas and seal the flask. Sonicate the reaction solution at 40 kilohertz for an additional 30 seconds. Place the sealed flask into a preheated oil bath at 60 degrees Celsius and leave it for 24 hours without agitating it.To isolate the MOF product, remove the flask from the oil bath and allow it to cool to room temperature. Set up a vacuum filtration apparatus using a small Buchner funnel and filter paper. Remove the PTFE tap from the flask and then use a pipette to transfer the total volume of the suspension to the filter.Rinse the solid with two milliliters of deoxygenated 3:1 methylene chloride/toluene solution. Repeat the step two more times and allow the solid to dry on the filter paper for three minutes. Scrape the solid into a pre-weighed vial and then weigh the vial to obtain the yield of the low-valent MOF.The synthesis of three-dimensional low valent MOFs using tetratopic phosphine ligands as linkers, palladium-0 and platinum-0 as nodes, and triphenylphosphine as a modulator is shown here. The central atom E can be silicon or tin.