T.K.&#160;acknowledges&#160;support&#160;from the&#160;Slovenia Research Agency (ARRS, grants BI-US/13-14-040, and J3-6803). O.S. acknowledges&#160;support&#160;from National Institute of Health (NIH) grant RO1HL49277.

1. Preparation of Reagents
Caution: Always use caution when working with chemicals and solutions. Follow appropriate safety practices and wear gloves, glasses and a lab coat at all times. Be aware that nanomaterials may have additional hazards as compared to their bulk counterpart. 
Note: All chemical solutions are made as molal (gram moles per kg of solvent) rather than molar (gram moles per liter of solution).
<ol>
	<li>Preparation of gold chloride
	<ol>
		<li>Dissolve 1 g of gold(III) chloride trihydrate in 100 g of H2O to give 25 mM HAuCl4.</li>
	</ol>
	</li>
	<li>Preparation of borax (Na2B4O7&#183;10H2O)
	<ol>
		<li>Dissolve 3.81 g of borax into 100 g of H2O to give 0.1 molal borax (warm if necessary to ensure complete solution).</li>
	</ol>
	</li>
	<li>Preparation of sodium thiocyanate
	<ol>
		<li>Dissolve 8.1 g of sodium thiocyanate in 100 g of H2O to give 1 molal NaSCN.</li>
	</ol>
	</li>
	<li>Preparation of sodium carbonate
	<ol>
		<li>Dissolve 5.3 g of anhydrous sodium carbonate in 100 g of H2O to give 0.5 molal Na2CO3.</li>
	</ol>
	</li>
	<li>Preparation of glutathione
	<ol>
		<li>Dissolve 154 mg of reduced glutathione (GSH) per 1 ml of 0.5 molal Na2CO3 to give 0.5 molal GSH.</li>
	</ol>
	</li>
</ol>
2. Synthesis of Gold Oligoclusters
<ol>
	<li>Delay-time Synthesis of Gold Oligoclusters
	<ol>
		<li>Add 59.5 ml of H2O to a clean 125 ml Wheaton glass bottle containing a stir bar. Use any flat bottom clean glass container, but ensure that it is very clean.</li>
		<li>Add 7 ml of 0.1 molal borax and bring solution to a vigorous stir.</li>
		<li>Add 2.8 ml of ~25 mM HAuCl4 under vigorous mixing and wait desired delay time (addition of HAuCl4 begins the delay-time). Delay times will determine the size of the as synthesized oligoclusters as shown in Table 1.</li>
		<li>After desired delay time, add 700 &#181;l of 1 molal NaSCN under brief vigorous stirring (1,200 rpm for 30 sec).</li>
		<li>Remove stir bar and allow the reaction to go to completion O/N (size distribution of the oligoclusters can be further improved by allowing mixture to stir continuously O/N while the reaction goes to completion). Once reaction has come to completion the as-synthesized crude oligoclusters are stable for weeks.</li>
	</ol>
	</li>
	<li>Add-on Growth of Oligoclusters
	<ol>
		<li>Combine 10 ml of as-synthesized oligoclusters to 60 ml of HG. The ratio of as-synthesized oligoclusters to HG determines the size of resulting oligoclusters, increasing the relative amount of HG produces larger oligoclusters.</li>
		<li>Add 900 &#181;l of 1 molal NaSCN under brief vigorous stirring (1,200 rpm for 30 sec).</li>
		<li>Allow the reaction to go to completion O/N (size distribution of the oligoclusters can be further improved by allowing mixture to stir continuously O/N while the reaction goes to completion).</li>
	</ol>
	</li>
</ol>
3. GSH Derivatization and Concentration of Oligoclusters
<ol>
	<li>Add 70 ml of as-synthesized crude oligoclusters (or oligoclusters from the add-on method) to a 70 ml 30 kDa cutoff centrifugal filter.</li>
	<li>Spin for 15 min at 3,000 x g. This concentrates the particles down to a volume of ~250 &#181;l.</li>
	<li>Flip device over and recover retentate by spinning device for 3 min at 500 x g. Recovered volume should be ~250 &#181;l.</li>
	<li>Measure recovered volume using a micropipette.</li>
	<li>Add a volume of 0.5 molal glutathione (or other thiol) equal to 1/9th the recovered volume of concentrated oligoclusters (final concentration 50 mmolal GSH).</li>
	<li>Allow derivatization reaction to sit at RT for 5-10 min. Derivatization occurs quickly. Overly long times may dissolve particles.</li>
	<li>Dilute derivatized oligoclusters into 50 ml of Dulbecco&#8217;s phosphate buffered saline. (Other buffers or H2O can be chosen as the diluent/wash buffer at this step. The choice is usually determined by intended downstream application.)</li>
	<li>Add all of the diluted derivatized oligoclusters to 30 kDa cutoff centrifugal filter.</li>
	<li>Spin the centrifugal filter for 15 min at 3,000 x g.</li>
	<li>Flip device over and recover retentate by spinning device for 3 min at 500 x g. Recovered volume should be ~250 &#181;l. The recovered concentrated particles are ready for use and are stable for months at 4 &#176;C.</li>
</ol>
4. Analysis and Verification of Oligocluster Synthesis
<ol>
	<li>Gel electrophoresis of Oligoclusters
	<ol>
		<li>Electrophoresis of crude oligocluster preparation
		<ol>
			<li>Mix the as-synthesized oligocluster preparations 2:1 with load buffer containing 60% glycerol, ~0.15% bromophenol blue, and 150 mmolal GSH (from stock of 0.5 molal GSH dissolved in 0.5 molal Na2CO3).</li>
			<li>Load 30 &#181;l onto precast polyacrylamide gradient gel (any kDa) and run with Tris-Glycine Running buffer (25 mM Tris, 192 mM glycine; no SDS is used) for 26 min at constant voltage (200 V).</li>
		</ol>
		</li>
		<li>Electrophoresis of GSH Derivatized Oligoclusters
		<ol>
			<li>Dilute GSH-derivatized oligocluster preparation 1:3 with H2O (Typically 2 &#181;l of GSH-oligoclusters with 6 &#181;l H2O).</li>
			<li>Mix diluted GSH-derivatized oligoclusters 2:1 with load buffer containing 60% glycerol, ~0.15% bromophenol blue, and 150 mmolal sodium bicarbonate.</li>
			<li>Load 10 &#181;l onto precast polyacrylamide gradient gel (any kDa) and run with Tris-Glycine Running buffer (25 mM Tris, 192 mM glycine; no SDS is used) for 26 min at constant voltage (200 V).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>&#160;Transmission Electron Microscopy (TEM)
	<ol>
		<li>Preparing Oligoclusters for TEM
		<ol>
			<li>To wash oligoclusters dilute 20 &#181;l of concentrated oligoclusters with 0.5 ml of H2O and load into to a 0.5 ml 30 kDa cutoff centrifugal filter.</li>
			<li>Spin at 14,000 x g for 10 min.</li>
			<li>Remove filtrate and resuspend retentate with a fresh 0.5 ml of H2O.</li>
			<li>Repeat washing twice for a total of 3 washes.</li>
			<li>Dilute final retentate 500 fold in H2O (oligoclusters are ready for gridding at this point).</li>
		</ol>
		</li>
		<li>Gridding Oligoclusters
		<ol>
			<li>Glow discharge carbon-coated grid.</li>
			<li>Deposit 0.6 &#181;l of washed and diluted oligoclusters onto a carbon-coated glow discharged grid.</li>
			<li>Allow grid to air-dry for 10 min.</li>
			<li>Visualize oligoclusters by TEM at 100,000X magnification. Operate at 80 kV for images shown here.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

This manuscript provides a detailed protocol for bench top synthesis of monodisperse gold oligoclusters (Figure 3). The method is capable of producing a wide range of sizes by simply varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of the reducing agent, sodium thiocyanate. The addition of HAuCl4 to alkaline buffered aqueous solution results in the time dependent hydroxylation of HAuCl4 to hydroxylated gold (Na+[Au(OH4-x)Clx]-). This hydroxylation results in less HAuCl4 being available, though the hydroxylation does not go to completion as it is an equilibrium reaction. The nucleation and formation of de novo gold monomers can only be initiated by HAuCl4. Hydroxylated gold is only capable of adding on to existing gold nanoparticles, resulting in the formation of gold oligoclusters; our add-on method takes advantage of this 16. Oligoclusters formed with the delay-time method can be used as seeds upon which hydroxylated gold is deposited, thereby increasing the size of seeded oligoclusters. Seeded growth can be controlled by varying the ratio of hydroxylated gold (HG) vs. as-synthesized oligocluster (Figure 1). In both methods the size of particles can easily be predicted by choosing the right time delay (Figure 2A, B) or by choosing the right starting seeds and the right ratio of added hydroxylated gold (HG) (Figure 2C). Predictions for most useful particle sizes are presented (Table 1). The increasing size of GSH derivatized oligoclusters can be monitored by electrophoresis as larger particles migrate less and appear notably darker, the later resulting from the fact that the extinction coefficient of gold nanoparticles increase in proportion to particle size.
The add-on method has two limitations, the first of which is the large reaction volumes required at high HG:seed ratios. A second limitation to the add-on method originates from the aforementioned fact that the hydroxylation of HAuCl4 is an equilibrium reaction and does not go to completion. The incomplete hydroxylation of HAuCl4 has minimal influence on the add-on reaction when the concentration of oligocluster seeds remains high. When the concentration of oligocluster seeds are low, as is the case when using long delay-time seed and high HG:seed ratios, the influence of unhydroxylated HAuCl4 can become significant. Under these conditions HAuCl4 is able to nucleate the synthesis of new oligoclusters, resulting in heterogeneous populations of oligoclusters.
The as-synthesized oligoclusters produced by delay-time or add-on method are stable for weeks, only developing trace amounts of gold precipitate. Even after being concentrated 300 fold the oligoclusters remain stable and resist aggregation. The gold oligoclusters described here also have the additional benefit of being able to be concentrated without prior derivatization, thus allowing expensive derivatizing agents to be used in smaller volumes. After being derivatized with glutathione (GSH), clusters remained stable up to one year. GSH-derivatization also provides strong negative charge 13 that makes them resist aggregation when exposed to physiological buffers or animal plasma, thus making them suitable for in vivo experiments. Derivatization can be achieved with a wide variety of thiol group containing reagents.
The amenability of the oligoclusters to derivatization with other thiol containing molecules 17,18 allows convenient and easy modification of the surface monolayer, thus controlling surface chemistry and reactivity of oligoclusters. Other chemicals used in this protocol can be readily substituted for similar chemicals without impairing synthesis. This includes the substitution of borax with other alkaline buffers (e.g., carbonate) and sodium thiocyanate for other thiocyanate salts (e.g., KSCN).
The main attribute of this protocol is its simplicity, which must be emphasized. Only a milligram weight scale and magnetic stirrer is required to produce commercial quality gold oligoclusters which can be used for advanced biological and material applications. Broad applicability is aided by the broad range of sizes than can be produced and by monodispersity. Additionally, in house production is low cost.
The oligoclusters are particularly valuable for studies of permeability of basal membranes and blood barriers. They can be easily administrated with saline through different routes and tracked in vivo 19-21. Obtained tissue samples can be subsequently examined under an electron microscope 16,22. Besides permeability, bio distribution provides valuable pharmacological information and the administration of mixture of oligoclusters of different sizes gives valuable information about size dependent distribution of particles inside the body 23-25. Lastly, because of their unique structure they fail to manifest localized surface plasmon resonance (LSPR) perhaps making them ideal candidates for fluorescent labeling, which is not readily achievable in gold nanoparticles because interference between the LSPR and fluorophore results in almost complete quenching of fluorescence 26.

The use of gold nanoparticles as tools in both biomedical applications and basic research has grown tremendously over the past few decades. Few modern nanomaterials have been applied to so many diverse fields, finding their use in everything from solar panels to photothermal cancer treatment; from electrical to biological sensors; from chemical catalysis to drug delivery systems 1-7. The interests in gold nanoparticles as tools in these areas are driven by the unique properties gold nanoparticles possess which include special structural, optical and electronic properties 8.
There is an increasing use of gold nanoparticles 9,10 in biological and chemical assays. Despite the availability of many sources for the purchase of gold nanoparticles, they come at a considerable price when compared to the cost of in house synthesis. The high cost of commercially available nanoparticles makes in house synthesis desirable. Our procedure involves synthesis of oligomeric nanoclusters made by small 2-3 nm spherical gold subunits. Having all of the advantages of classical gold nanoparticles, oligomeric nanoclusters are preferred choice when it comes to permeability or filtration rates measurements because their modular structure mimics the structure of proteins.
Currently, the most common approaches to the in house synthesis of gold nanoparticles involve the reduction of gold chloride (HAuCl4) under aqueous conditions 11,12. Reduction of HAuCl4 with common reducing reagents, such as sodium borohydride (NaBH4) or sodium citrate, allows for the production of spherical nanoparticles 13. Gold nanoparticles synthesized by these methods are limited in their useful size range, because they become sensitive to the presence of salts in biological buffers as their core diameters increase. A method has previously been described for the synthesis of yellow nanoparticles of 2-3 nm diameter from the reduction of HAuCl4 with sodium thiocyanate under alkaline conditions 14,15.
Here, we describe a modification of that method which produces a grape-like oligocluster of the yellow nanoparticles without the need for additional capping agents. By simply varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, sodium thiocyanate, we are able to vary the resulting size of the gold particles from ~3 nm to ~25 nm. To produce larger particles, a simple add-on procedure can be used to grow these oligoclusters by the addition of hydroxylated gold (HG) to the as-synthesized oligoclusters in the presence of sodium thiocyanate. Using these two methods, we are able to reliably produce oligoclusters covering a range from ~3 nm to ~70 nm. The fact that this method allows well controlled synthesis of high quality gold oligoclusters under bench-top conditions with standard equipment and a limited number of reagents potentially extends the benefits of gold nanoparticles as a research tool to researchers with little or no expertise in chemical synthesis.

<table><tbody><tr><td>125 ml Wheaton glass bottles</td><td>Fisher Scientific</td><td>SC-06-404F</td><td></td></tr><tr><td>Borax (Na&lt;sub&gt;2&lt;/sub&gt;B&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;middot;10H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>S25537</td><td></td></tr><tr><td>Gold(III) Chloride trihydrate</td><td>Sigma Aldrich</td><td>G4022</td><td></td></tr><tr><td>Sodium thiocyanate</td><td>Sigma Aldrich</td><td>251410</td><td></td></tr><tr><td>Sodium carbonate</td><td>Sigma Aldrich</td><td>S7795</td><td></td></tr><tr><td>Glutathione</td><td>Sigma Aldrich</td><td>G4251</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Centricon Plus - 70</td><td>Millipore</td><td>UCF703008</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6014</td><td></td></tr><tr><td>CF200-Cu Carbon film on 200 mesh copper grids&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>71150</td><td></td></tr><tr><td>10x Tris/Glycine buffer</td><td>Bio-Rad</td><td>161-0734</td><td></td></tr><tr><td>Any kD Mini-PROTEAN TGX Gel</td><td>Bio-Rad</td><td>456-9033</td><td></td></tr></tbody></table>

a simple method for the size controlled synthesis of stable oligomeric clusters of gold nanoparticles under ambient conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

The syntheses of gold oligoclusters were analyzed by gel electrophoresis (Figure 1) and transmission electron microscopy (TEM) (Figure 2). The size of GSH-coated oligoclusters can be monitored by electrophoresis as larger particles migrate less and appear darker. In addition, the quality of any given size preparation can be inferred by the broadness of the band seen after electrophoresis (i.e., for a given size, preparations with narrower size distributions will produce tighter bands than preparations of the same size with broader size distributions). Figure 2 describes the relationship of time delay (delay-time method) or HG:seed (add-on method) to oligocluster size. Mean diameters calculated by TEM are used to determine delay-time and HG:seed dependence growth of oligoclusters for delay-time and add-on methods, respectively. A flow chart (Figure 3) outlining the procedure for both methods and a table (Table 1) providing predicted parameters to produce oligoclusters of the desired size are presented.
<img alt="Figure 1" src="/files/ftp_upload/53388/53388fig1.jpg" /> 
Figure 1. Polyacrylamide gradient gel electrophoresis of oligoclusters formed by the Delay-time and Add-on methods. Oligoclusters produced by delay-time and add-on methods were analyzed on gradient gel electrophoresis. Lanes 2-4: oligoclusters formed after different delay times (45, 135, and 405 sec) between making the HAuCl4 alkaline and the addition of NaSCN. Lanes 5-8: oligoclusters formed by the add-on method. Seed was formed by the delay-time method with 405 sec&#160;delay, indicated by &#8595;. Varying amounts of HG were used for add-on. The ratios of HG solution (1 mM in gold) to seed solution (1 mM in gold) used for preparing each sample are indicated, as 4xHG, 6xHG, 12xHG, and 24xHG. <a href="https://www.jove.com/files/ftp_upload/53388/53388fig1large.jpg" target="_blank">Please click here to download this file.</a>
<img alt="Figure 2" src="/files/ftp_upload/53388/53388fig2.jpg" /> 
Figure 2. Diameters of gold oligoclusters formed by the delay-time and add-on methods. Oligoclusters prepared by delay-time and add-on methods were analyzed by TEM. A) and B) are adapted with permission from ref.16, Copyright 2014 American Chemical Society. (A) Representative TEM images of 50 nm x 50 nm areas of grids prepared from samples made using the delay-time method. Diameter of the particles (Y axis) and the delay times used in their preparation (X axis) are indicated, both axis are logarithmic. The heavy black line (R2 = 0.973) is a best fit with empirical 3-parameter equation Ddelay-time = D0 + a(1 - e-bt), where Ddelay-time is the mean diameter of clusters in nm, D0 is minimum diameter of clusters (~3.5 nm); a is the maximum increase in core size caused by extending the delay time (~20 nm), and b = 0.0021 sec-1. (B) Diameters of the oligoclusters formed after different delay times before adding NaSCN (delay-time method) presented on a linear scale. (C) Diameters of the oligoclusters formed after addition (add-on method) of different amounts of HG onto preformed gold seeds formed by the delay-time method with 405 sec&#160;delay time. As shown by the heavy black line, it can be easily seen that the diameter of oligoclusters formed by the add-on method is <img alt="Equation 4" src="/files/ftp_upload/53388/53388eq4.jpg" />, where cHG and cSeeds are the concentrations of chloroauric acid used in making the solution of HG in add-on method and in making oligoclusters by the delay-time method, respectively. Similarly VHG and VSeeds are the corresponding volumes. <a href="https://www.jove.com/files/ftp_upload/53388/53388fig2large.jpg" target="_blank">Please click here to download this file.</a>
<img alt="Figure 3" src="/files/ftp_upload/53388/53388fig3.jpg" /> 
Figure 3. Wall chart diagram of Delay-time and Add-on methods for making gold oligoclusters of different sizes. Flow chart outlining the procedures for synthesizing gold oligoclusters of different sizes using either the delay-time or add-on methods. The alkaline solution of chloroauric acid is blue. The HG is red. The gold nanoparticle seeds and oligoclusters are black. <a href="https://www.jove.com/files/ftp_upload/53388/53388fig3large.jpg" target="_blank">Please click here to download this file.</a>
<table>
	<tbody>
		<tr>
			<td colspan="4" style="width:318px">Delay-time procedure</td>
			<td colspan="6">Add-on procedure</td>
		</tr>
		<tr>
			<td colspan="4"></td>
			<td colspan="6">predicted diameter (nm)</td>
		</tr>
		<tr>
			<td>delay time (sec)</td>
			<td>delay time (min)</td>
			<td>predicted diameter (nm)</td>
			<td>measured diameter &#177; s.d. (nm)</td>
			<td>4&#215;HG</td>
			<td>6&#215;HG</td>
			<td>12&#215;HG</td>
			<td>24&#215;HG</td>
			<td>100&#215;HG</td>
			<td>1000&#215;HG</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.02</td>
			<td>3.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.03</td>
			<td>3.6</td>
			<td>3.1&#177;1.3</td>
			<td>6.1</td>
			<td>6.9</td>
			<td>8.4</td>
			<td>10.5</td>
			<td>16.7</td>
			<td>36</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.05</td>
			<td>3.6</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4</td>
			<td>0.07</td>
			<td>3.7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5</td>
			<td>0.08</td>
			<td>3.7</td>
			<td>2.6&#177;1.1</td>
			<td>6.3</td>
			<td>7.1</td>
			<td>8.7</td>
			<td>10.8</td>
			<td>17.3</td>
			<td>37</td>
		</tr>
		<tr>
			<td>6</td>
			<td>0.10</td>
			<td>3.8</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7</td>
			<td>0.12</td>
			<td>3.8</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8</td>
			<td>0.13</td>
			<td>3.8</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>9</td>
			<td>0.15</td>
			<td>3.9</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10</td>
			<td>0.17</td>
			<td>3.9</td>
			<td></td>
			<td>6.7</td>
			<td>7.5</td>
			<td>9.2</td>
			<td>11.4</td>
			<td>18</td>
			<td>39</td>
		</tr>
		<tr>
			<td>11</td>
			<td>0.18</td>
			<td>4.0</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12</td>
			<td>0.20</td>
			<td>4.0</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>13</td>
			<td>0.22</td>
			<td>4.0</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14</td>
			<td>0.23</td>
			<td>4.1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15</td>
			<td>0.25</td>
			<td>4.1</td>
			<td>3.3&#177;1.5</td>
			<td>7.0</td>
			<td>7.9</td>
			<td>9.7</td>
			<td>12.0</td>
			<td>19</td>
			<td>41</td>
		</tr>
		<tr>
			<td>20</td>
			<td>0.33</td>
			<td>4.3</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25</td>
			<td>0.42</td>
			<td>4.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30</td>
			<td>0.50</td>
			<td>4.7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35</td>
			<td>0.58</td>
			<td>4.9</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40</td>
			<td>0.67</td>
			<td>5.1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>45</td>
			<td>0.75</td>
			<td>5.3</td>
			<td>6.4&#177;2</td>
			<td>9.1</td>
			<td>10.1</td>
			<td>12.5</td>
			<td>15.5</td>
			<td>25</td>
			<td>53</td>
		</tr>
		<tr>
			<td>60</td>
			<td>1.0</td>
			<td>5.9</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>75</td>
			<td>1.3</td>
			<td>6.4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>90</td>
			<td>1.5</td>
			<td>6.9</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>105</td>
			<td>1.8</td>
			<td>7.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>120</td>
			<td>2.0</td>
			<td>8.0</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>135</td>
			<td>2.3</td>
			<td>8.4</td>
			<td>11&#177;3</td>
			<td>14.4</td>
			<td>16.1</td>
			<td>20</td>
			<td>25</td>
			<td>39</td>
			<td>84</td>
		</tr>
		<tr>
			<td>165</td>
			<td>2.8</td>
			<td>9.4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>195</td>
			<td>3.3</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>225</td>
			<td>3.8</td>
			<td>11</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>255</td>
			<td>4.3</td>
			<td>12</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>285</td>
			<td>4.8</td>
			<td>13</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>315</td>
			<td>5.3</td>
			<td>13</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>345</td>
			<td>5.8</td>
			<td>14</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>375</td>
			<td>6.3</td>
			<td>14</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>405</td>
			<td>6.8</td>
			<td>15</td>
			<td>14&#177;5</td>
			<td>26</td>
			<td>29</td>
			<td>35</td>
			<td>44</td>
			<td>70</td>
			<td>150</td>
		</tr>
		<tr>
			<td>435</td>
			<td>7.3</td>
			<td>15</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>465</td>
			<td>7.8</td>
			<td>16</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>495</td>
			<td>8.3</td>
			<td>16</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>525</td>
			<td>8.8</td>
			<td>17</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>555</td>
			<td>9.3</td>
			<td>17</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>585</td>
			<td>9.8</td>
			<td>18</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>615</td>
			<td>10</td>
			<td>18</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>900</td>
			<td>15</td>
			<td>20</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1200</td>
			<td>20</td>
			<td>22</td>
			<td>20&#177;11</td>
			<td>37</td>
			<td>42</td>
			<td>51</td>
			<td>64</td>
			<td>102</td>
			<td>219</td>
		</tr>
		<tr>
			<td>1500</td>
			<td>25</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1800</td>
			<td>30</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2100</td>
			<td>35</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2400</td>
			<td>40</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2700</td>
			<td>45</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3000</td>
			<td>50</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3300</td>
			<td>55</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3600</td>
			<td>60</td>
			<td>23</td>
			<td>25&#177;11</td>
			<td>40</td>
			<td>45</td>
			<td>55</td>
			<td>69</td>
			<td>109</td>
			<td>235</td>
		</tr>
	</tbody>
</table>
Table 1. Oligocluster size prediction table. Predicted diameters of gold oligoclusters formed using either the delay-time or add-on methods. Predicted diameter for the delay-time method is calculated using an empirical formula for average oligocluster diameter&#160;Ddelay-time = D0 + a(1 - e-bt), where D is the mean diameter of gold oligoclusters in nm, D0 is the minimum diameter (3.5 nm), a is the maximum increase in core size (20 nm), and b is 0.0021 sec-1, as shown previously 16. Predicted diameter for the add-on method is calculated taking into account that new nanoparticles cannot form from HG, rather it is deposited uniformly around preformed spherical seeds, thus making them larger. No other assumption is necessary. It can be easily seen that the diameter of oligoclusters formed by the add-on method is <img alt="Equation 6" src="/files/ftp_upload/53388/53388eq6.jpg" />, where cHG and cSeeds are the concentrations of chloroauric acid used in making the solution of HG in add-on method and in making oligoclusters by the delay-time method, respectively. Similarly VHG and VSeeds are the corresponding volumes.

Watch this Scientific Journal Video about A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions at JoVE.com

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

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Research

JoVE Journal

Chemistry

22.0K Views.  University of North Carolina at Chapel Hill.  We beschrijven een eenvoudige werkwijze voor het produceren van zeer stabiele oligomere clusters van goud nanodeeltjes via de reductie chlorogoudzuur (HAuCl 4) met natrium thiocyanaat (NaSCN). De oligoclusters een smalle grootteverdeling en kunnen worden met uiteenlopende afmetingen en oppervlakte lagen. 

Video: A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

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.

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, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Bioengineering

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

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 ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because 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 ...

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 ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

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