Name: Hydroquinone Based Synthesis of Gold Nanorods (Video) | JoVE Journal
Uploaded: 2016-08-10T14:00:00.000+00:00
Duration: 8 min 55 s
Description: Learn hydroquinone-based synthesis of gold nanorods with controlled aspect ratios for nanomaterials research. This reproducible protocol enables efficient preparation for biomedical imaging and drug delivery applications.

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5 minutes

Funding for this research was provided by the Italian Ministry of Health under the frame of EuroNanoMed II (European Innovative Research &amp; Technological Development Projects in Nanomedicine, project title: ''InNaSERSS'').

1. Synthesis of Gold Nanorods
Note: Use highly purified water throughout.
<ol>
	<li>Preparation of the gold seeds
	<ol>
		<li>Dissolve 364.4 mg of hexadecyltrimethylammonium bromide (CTAB) in 5 ml water, under ultrasonication at 40 &#176;C until the solution becomes clear. Let the CTAB solution cool down to room temperature.</li>
		<li>Separately, prepare 5 ml of tetrachloroauric acid (HAuCl4) in water (0.5 mM).</li>
		<li>Add the HAuCl4 solution to the CTAB solution under vigorous magnetic stirring, maintaining the temperature constant at 27 &#176;C.</li>
		<li>Prepare 600 &#181;l of sodium borohydride (NaBH4) solution in water (10 mM) at 4 &#176;C. Add this solution to the mixture under vigorous stirring. Check if the solution&#39;s color immediately changes from yellow to brownish.</li>
		<li>Stir the suspension for 20 min before use. Store the seeds suspension for no longer than 24 hr at room temperature.</li>
		<li>Check the dimensions of the seeds using an UV-Vis spectrophotometer. Ensure that the seeds are small enough (about 2 nm) to be used in the preparation of gold nanorods by UV-visible spectroscopy. 
		Note: Spectrum must be similar to what is reported in Figure 1. Larger seeds identified by the presence of a plasmonic peak around 505-520 nm must not be used because they are likely to produce spherical nanoparticles.</li>
	</ol>
	</li>
	<li>Preparation of the &#34;growth solution&#34; of gold nanorods.
	<ol>
		<li>Dissolve 182.2 mg CTAB together with 22 mg hydroquinone in 5 ml water at 40 &#176;C using ultrasonication. Cool down the solution to 27 &#176;C.</li>
		<li>Prepare 200 &#181;l of 4 mM silver nitrate (AgNO3) solution.</li>
		<li>Separately, prepare 5 ml of 1 mM solution of tetrachloroauric acid (HAuCl4).</li>
		<li>First add the silver nitrate solution prepared in step 1.2.2. Then, add the HAuCl4 solution prepared in step 1.2.3 to the solution of CTAB and hydroquinone prepared in step 1.2.1 under magnetic stirring.</li>
		<li>Immediately after, add under magnetic stirring 12 &#181;l of the seeds suspension previously prepared according to the protocol reported at step 1.1 and let the reaction start. Check if the suspension changes color in about 30 min.</li>
		<li>Control the formation of the nanorods by checking the UV-visible spectrum of the suspension, as described in section 4, every 5 min. Proceed until the spectrum is stable. To allow the complete formation of the nanorods, leave the suspension under stirring for further 30 min (Figure 2).</li>
		<li>Divide the suspension in tubes (1 ml of suspension for each tube) and centrifuge at 10,000 x g for 10 min. Gold nanorods form a dark precipitate at the bottom of the tube.</li>
		<li>Resuspend the precipitate of each tube in 1 ml of water. Mix together the content of the tubes and store the suspension of gold nanorods at room temperature.</li>
		<li>Characterize the obtained nanorods by UV-visible spectroscopy and transmission electron microscopy as described in section 4 (Figure 3).</li>
	</ol>
	</li>
</ol>
2. Tuning the Aspect Ratio of Nanorods by Varying the Concentration of Ag+ Ions
<ol>
	<li>Prepare a silver nitrate solution with a concentration of 4 mM, dissolving 3.4 mg AgNO3 in 5 ml of water.</li>
	<li>Prepare in three different vials the solution with CTAB and hydroquinone as described in section 1.2.1 and add respectively 100 &#181;l, 150 &#181;l or 200 &#181;l of silver nitrate solution.</li>
	<li>Add the HAuCl4 solution prepared according to step 1.2.3 and proceed with the preparation of gold nanorods as described from point 1.2.5.</li>
	<li>Characterize the obtained nanorods by UV-visible spectroscopy and transmission electron microscopy. Vials with lower amounts of Ag+ will result in shorter nanorods (aspect ratio of 2 and 2.2 respectively) (Figure 4).</li>
</ol>
3. Tuning the Aspect Ratio of Nanorods by Varying the Concentration of CTAB
<ol>
	<li>Prepare different batches of gold nanorods with different concentrations of CTAB in the &#34;growth solution&#34;. Use concentrations from 10 mM to 100 mM to produce gold nanorods having different size and aspect ratio. The concentrations of CTAB used in each experiment are summarized in Table 1 with the corresponding amount of milligrams used. Dissolve the different amounts of CTAB always with 22 mg of hydroquinone in 5 ml of water.</li>
	<li>Add 200 &#181;l of silver nitrate solution (prepared according to step 1.2.2) and 5 ml of HAuCl4 solution (prepared according how described in step 1.2.3) in each vial under magnetic stirring.</li>
	<li>Add 12 &#181;l of seeds suspension and observe the change in color of the final mixture.</li>
	<li>Stop the stirring when the suspension&#39;s color and the UV-visible spectrum are stabilized; the reaction time depends on the CTAB concentration in the growth solution.</li>
	<li>Centrifuge at 10,000 x g for 10 min and resuspend in water.</li>
	<li>Characterize the obtained nanorods by UV-visible spectroscopy and transmission electron microscopy. A lower concentration of CTAB will result in shorter nanorods while a higher concentration will give longer but larger nanorods. On contrary the aspect ratio of the nanorods will be higher in the range around 40-50 mM and will decrease both at lower and higher concentrations (Figure 5 and Figure 6).</li>
</ol>
4. Characterization of Gold Nanorods
<ol>
	<li>UV-visible Spectroscopy
	<ol>
		<li>Dilute 100 &#181;l of nanorods solution with 400 &#181;l of water in a plastic micro-cuvette and acquire the UV-visible absorption spectrum (wavelength range between 400 and 840 nm) according to manufacturer&#39;s protocol.</li>
		<li>Collect UV-visible spectra (wavelength range between 400 and 840 nm) of the growth solution every 5 min in order to study the kinetics of the reaction.</li>
	</ol>
	</li>
	<li>Transmission Electron Microscopy (TEM)
	<ol>
		<li>Collect TEM images of each sample of nanorods, for the measurement of the size and of the aspect ratio of the obtained nanorods. Prepare the samples by placing a drop of suspension (4 &#181;l) onto an ultra-thin Formvar-coated 200-mesh copper grids and leave to dry in air at 4 &#176;C. Analyze the sample at TEM using an accelerating voltage of 200 kV according to manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>

<ol><li>Zhou, W., Gao, X., Liu, D., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gold+Nanoparticles+for+In+Vitro+Diagnostics%5BTitle%5D)+AND+%22Chem+Rev%22%5BJournal%5D)">Gold Nanoparticles for In Vitro Diagnostics.</a> Chem Rev. 115 (19), 10575-10636 (2015).</li>
<li>Bao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gold+nanoprisms+as+optoacoustic+signal+nanoamplifiers+for+in+vivo+bioimaging+of+gastrointestinal+cancers%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">Gold nanoprisms as optoacoustic signal nanoamplifiers for in vivo bioimaging of gastrointestinal cancers.</a> Small. 9 (1), 68-74 (2013).</li>
<li>Han, G., Ghosh, P., Rotello, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functionalized+gold+nanoparticles+for+drug+delivery%5BTitle%5D)+AND+%22Nanomedicine%22%5BJournal%5D)">Functionalized gold nanoparticles for drug delivery.</a> Nanomedicine. 2 (1), 113-123 (2007).</li>
<li>Choi, W. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+regression+in+vivo+by+photothermal+therapy+based+on+gold-nanorod-loaded%2C+functional+nanocarriers%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Tumor regression in vivo by photothermal therapy based on gold-nanorod-loaded, functional nanocarriers.</a> ACS Nano. 5 (3), 1995-2003 (2011).</li>
<li>Langille, M. R., Personick, M. L., Zhang, J., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+Rules+for+the+Shape+Evolution+of+Gold+Nanoparticles+%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Defining Rules for the Shape Evolution of Gold Nanoparticles .</a> J. Am. Chem. Soc. 134 (35), 14542-14554 (2012).</li>
<li>Lohse, S. E., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Quest+for+Shape+Control%3A+A+History+of+Gold+Nanorod+Synthesis%5BTitle%5D)+AND+%22Chem.+Mater%22%5BJournal%5D)">The Quest for Shape Control: A History of Gold Nanorod Synthesis.</a> Chem. Mater. 25 (8), 1250-1261 (2013).</li>
<li>Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+clearer+vision+for+in+vivo+imaging%5BTitle%5D)+AND+%22Nat.+Biotech%22%5BJournal%5D)">A clearer vision for in vivo imaging.</a> Nat. Biotech. 19 (4), 316-317 (2001).</li>
<li>Sau, T. K., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Seeded+High+Yield+Synthesis+of+Short+Au+Nanorods+in+Aqueous+Solution%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Seeded High Yield Synthesis of Short Au Nanorods in Aqueous Solution.</a> Langmuir. 20 (15), 6414-6420 (2004).</li>
<li>Ratto, F., Matteini, P., Rossi, F., Pini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size+and+shape+control+in+the+overgrowth+of+gold+nanorods%5BTitle%5D)+AND+%22J.+Nanopart.+Res%22%5BJournal%5D)">Size and shape control in the overgrowth of gold nanorods.</a> J. Nanopart. Res. 12, 2029-2036 (2010).</li>
<li>Morasso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+size+and+aspect+ratio+in+hydroquinone-based+synthesis+of+gold+nanorods%5BTitle%5D)+AND+%22J.+Nanopart.+Res%22%5BJournal%5D)">Control of size and aspect ratio in hydroquinone-based synthesis of gold nanorods.</a> J. Nanopart. Res. 17, 330-337 (2015).</li>
<li>Vigderman, L., Zubarev, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-yield+synthesis+of+gold+nanorods+with+longitudinal+SPR+peak+greater+than+1200+nm+using+hydroquinone+as+a+reducing+agent%5BTitle%5D)+AND+%22Chem.+Mater%22%5BJournal%5D)">High-yield synthesis of gold nanorods with longitudinal SPR peak greater than 1200 nm using hydroquinone as a reducing agent.</a> Chem. Mater. 25 (8), 1450-1457 (2013).</li>
<li>Walsh, M. J., Barrow, S. J., Tong, W., Funston, A. M., Etheridge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Symmetry+breaking+and+silver+in+gold+nanorod+growth%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Symmetry breaking and silver in gold nanorod growth.</a> ACS Nano. 9 (1), 715-724 (2015).</li>
</ol>

The authors have nothing to disclose.

The protocol presented here applies hydroquinone, an aromatic molecule characterized by a weak reduction potential, to produce gold nanorods. There are two main advantages of the present protocol toward the most commonly employed synthetic route based on the use of ascorbic acid: the first is that hydroquinone is able to almost quantitatively reduce the gold ions allowing the production of higher amount of gold nanorods.11 The latter is given by the fact that it requires a lower amount of CTAB and a subsequent...

Gold nanoparticles (AuNPs) are one of the most widespread and promising nanostructures to be used in biomedical applications. Their use is essential in many point-of-care in vitro diagnostics products.1 They have been proposed as an effective tool for a number of other different applications: as a contrast agent in imaging studies,2 as a drug delivery system3 and as drugs for light-induced thermotherapy (or photothermal therapy).4 The great potential of AuNPs has driven, in the last twenty years, intense research on the development of new synthesis that is able to increase the control on the size and shape obtained.5 This is because different kinds of AuNPs are in fact more suitable than others for specific applications.
Among the different gold nanostructures, gold nanorods (AuNRs) have emerged as one of the most interesting systems. AuNRs are characterized by two plasmonic peaks associated with the oscillation of electrons along the longitudinal and the transverse axes, respectively.6 It is particularly important that the position of the most intense longitudinal peak can be tuned precisely between 620 and 800 nm, depending on the aspect ratio of the rods. This region matches the biological window,7 where the human tissues almost do not absorb light, allowing the development of a number of in vivo photonic applications involving AuNPs.
Despite a huge interest in this kind of nanostructures, the synthetic protocols for the preparation of AuNRs suffer from several limitations. In most of the cases, nanorods are prepared according to a two-step method developed by Sau and coworkers.8 In their protocol, nanorods are synthesized by reducing gold ions using ascorbic acid in presence of preformed gold seeds, silver ions and a large amount of hexadecyl trimethylammonium bromide (CTAB), a cationic linear surfactant.
The drawback of this protocol is that the reduction yield of gold ions is relatively low (about 20%) 9 and that a high amount of CTAB, an expensive reagent that accounts for more than half of the total cost for the reagents in the synthesis, is needed. The development of a new and more efficient synthetic route is thence considered to be an important need, allowing the spreading of biomedical approaches based on AuNRs.
In the first part of the present paper, we present an optimized protocol for the preparation of AuNR having an aspect ratio of about three. The synthesis is based on the use of hydroquinone as a mild reducing agent and it allows the preparation of AuNR with an almost quantitative reduction of gold ions, making use of a reduced amount of CTAB.10 This protocol for the preparation of the AuNRs is based on a two-step approach where gold seeds are used in a &#34;growth solution&#34;.
In the second part, we show how to finely tune the size and aspect ratio of the obtained AuNR in two ways. The first way, similar to the standard protocol based on ascorbic acid, is to vary the amount of silver ions present in the &#34;growth solution&#34;. The second way is based on the variation of the amount of CTAB that can be reduced down to a concentration of 10 mM (close to the critical micellar concentration reported by the supplier) to obtain well defined short nanorods.

<table><tbody><tr><td>Gold(III) chloride trihydrate</td><td>Sigma Aldrich</td><td>520918</td><td></td></tr><tr><td>Hydroquinone</td><td>Sigma Aldrich</td><td>H17902</td><td></td></tr><tr><td>Silver Nitrate</td><td>Sigma Aldrich</td><td>209139</td><td>toxic</td></tr><tr><td>Sodium Borohydride</td><td>Sigma Aldrich</td><td>480886</td><td></td></tr><tr><td>Hexadecyltrimethylammonium bromide (CTAB)</td><td>Sigma Aldrich</td><td>H5882</td><td>Acute Tox. (oral). In this study we tested three different batches of CTAB (H5882) from Sigma Aldrich. Two of them were marked as made in China while one as made in India. In our experience only the batches marked as made in China were effective for the preparation of AuNR.</td></tr><tr><td>Spectrophotometer</td><td>Thermo scientific&amp;nbsp;</td><td>Nanodrop 2000C</td><td></td></tr><tr><td>TEM</td><td>JEOL</td><td>2100</td><td></td></tr></tbody></table>

hydroquinone based synthesis of gold nanorods

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.

UV visible spectra of the gold seeds can be seen in Figure 1. UV visible spectra acquired at different times after the injection of the gold seeds are presented in Figure 2. UV visible spectra and transmission electron microscopic (TEM) images of the obtained gold nanorods are shown in Figure 3. UV visible spectra and transmission electron microscopic (TEM) images of gold nanorods with different aspect ratio obtained by varying the amount...

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 ...

The overall goal of this procedure is to efficiently prepare gold nanorods with an aspect ratio between two and three. This method can help prepare gold nanorod in a reproducible and convenient way compared to other synthetic protocol in the scientific literature. The main advantages in the use of hydroquinone as the reducing agent are the dramatic increase in the gold ion reduction rate, and the lower use of cetyltrimethylammonium bromide, with substantially lower cost.This protocol consists of two steps. First is most spherical gold seeds are prepared using sodium borohydride. Secondly, gold nanorods are prepared from a seed using a seed-grow solution.To begin preparation of the gold seeds, first dissolve 364.4 milligrams of CTAB in five milliliters of water at 40 degrees Celsius using ultrasonication. Remove the sample from the sonicator once the solution becomes clear, and allow it to cool to room temperature before proceeding. Next, prepare five milliliters of a 0.5 millimolar solution of tetrochlorauric acid in water and add it to the vigorously stirring CTAB solution at a constant temperature of 27 degrees Celsius.Subsequently, make 600 microliters of a sodium borohydride solution in water at four degrees Celsius, and add this to the rapidly stirred CTAB mixture at 27 degrees Celsius. Check that the color immediately changes from yellow to brownish. Stir the resulting suspension for an additional 20 minutes.To determine the size of the seeds, first pipette 100 microliters of the seed suspension into 400 microliters of water in a plastic microcuvette. Next, transfer the microcuvette to a UV-visible spectrometer and analyze it at a wavelength range of 400 to 840 nanometers. For the seeds to be small enough to use, there should be no pronounced plasmonic peak in the range between 505 to 520 nanometers.Start preparation of the growth solution by dissolving CTAB and hydroquinone in five milliliters of water at 40 degrees Celsius using ultrasonication. Allow the solution to cool to 27 degrees Celsius before proceeding. Separately, prepare 200 microliters of a four millimolar aqueous silver nitrate solution and five milliliters of a one millimolar aqueous tetrachloroauric acid solution.Next, add the silver nitrate solution, followed by the tetrachloroauric acid solution, to the magnetically stirred CTAB solution. Once combined, immediately add 12 microliters of the previously prepared gold seed suspension. After 30 minutes, check to see that the color of the suspension has changed to brown.Sample the suspension every five minutes by pipetting 100 microliters of it into 400 microliters of water in a plastic microcuvette, and analyze the sample by UV-visible spectroscopy. In the UV-visible spectrum, a shift to the lower wavelength of the far red shifted plasmonic peak stabilizes after about 30 minutes indicating the reaction is complete. Divide the suspension into one milliliter aliquots in centrifuge tubes and centrifuge them.The gold nanorods form a dark precipitate at the bottom of the tubes. Subsequently re-suspend each of the precipitates by pipetting and sonicating them in one milliliter of water. Combine all the suspensions into one container and store at room temperature for later use.Analyze the gold nanorod suspension by TEM at 200 kilovolts, according to the manufacturer's protocol, and by UV-visible spectroscopy, at a wavelength range of 400 to 840 nanometers. To vary the nanorods'aspect ratio using silver ions, first prepare a four millimolar solution of silver nitrate in water. Next, set up three vials, each containing five milliliters of the CTAB and hydroquinone solution.Add 100 microliters, 150 microliters, or 200 microliters of the silver nitrate solution to the appropriate vial. Continue by adding five milliliters of the one millimolar tetrachloroauric acid solution to each vial. Complete the aspect ratio tuning process by adding 12 microliters of the gold seeds, sampling the reaction, and isolating the gold nanorods by centrifugation as previously described.To start, make CTAB solutions of different concentrations by adding the appropriate amount of CTAB according to Table One, to vials containing 22 milligrams of hydroquinone in five milliliters of water. Subsequently, add 200 microliters of the four millimolar silver nitrate solution and five milliliters of the one millimolar tetrachloroauric acid solution to each magnetically stirred vial of CTAB solution. Add 12 microliters of the gold seeds suspension to the vials and observe the change in color from blue to brown.Analyze the suspension by UV-visible spectroscopy. Discontinue stirring when the suspension's color and the UV-visible spectrum are stabilized. Prepare the sample for centrifugation.Centrifuge the particles and then re-suspend the precipitate of gold nanorods in one milliliter of water for final analysis by TEM and UV-visible spectroscopy. The lengths of the gold nanorods formed by different amounts of silver ions in the growth solution were measured using TEM. The images show that the length of the nanorods increases proportionally to the amount of silver ions used.Analysis of TEM images and UV-visible spectra, showed that when CTAB concentrations increased, the nanorods'length also increased. The nanorods'aspect ratios peaked at a CTAB concentration of 50 millimoles, because the rods'width grew more rapidly than their length at CTAB concentrations greater than 50 millimoles. Once mastered, this technique can be done in half a day if it is performed properly.While attempting this procedure, it's important to remember to always use clean glassware and to control the temperature of the growth solution in order to obtain reproducible results. Beginners may struggle with this method, as temperature, stirring speed, and the reagent reaction order, all need to be controlled in order to obtain gold nanorods of a desired size and aspect ratio. After watching this video you should have a good understanding of how to prepare a gold nanorod using hydroquinone as a reducing agent.Don't forget, working with chemicals can be extremely hazardous, and precautions such as wearing gloves, lab coat, and goggles, should always be taken while performing these procedures.

hydroquinone-based-synthesis-of-gold-nanorods

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Hydroquinone Based Synthesis of Gold Nanorods

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