Hydroquinone Based Synthesis of Gold Nanorods

Summary

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.

Abstract

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.

Introduction

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 "growth solution".

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 "growth solution". 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.

Protocol

1. Synthesis of Gold Nanorods

Note: Use highly purified water throughout.

1. Preparation of the gold seeds
   1. Dissolve 364.4 mg of hexadecyltrimethylammonium bromide (CTAB) in 5 ml water, under ultrasonication at 40 °C until the solution becomes clear. Let the CTAB solution cool down to room temperature.
   2. Separately, prepare 5 ml of tetrachloroauric acid (HAuCl₄) in water (0.5 mM).
   3. Add the HAuCl₄ solution to the CTAB solution under vigorous magnetic stirring, maintaining the temperature constant at 27 °C.
   4. Prepare 600 µl of sodium borohydride (NaBH₄) solution in water (10 mM) at 4 °C. Add this solution to the mixture under vigorous stirring. Check if the solution's color immediately changes from yellow to brownish.
   5. Stir the suspension for 20 min before use. Store the seeds suspension for no longer than 24 hr at room temperature.
   6. 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.
2. Preparation of the "growth solution" of gold nanorods.
   1. Dissolve 182.2 mg CTAB together with 22 mg hydroquinone in 5 ml water at 40 °C using ultrasonication. Cool down the solution to 27 °C.
   2. Prepare 200 µl of 4 mM silver nitrate (AgNO₃) solution.
   3. Separately, prepare 5 ml of 1 mM solution of tetrachloroauric acid (HAuCl₄).
   4. 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.
   5. Immediately after, add under magnetic stirring 12 µl 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.
   6. 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).
   7. 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.
   8. 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.
   9. Characterize the obtained nanorods by UV-visible spectroscopy and transmission electron microscopy as described in section 4 (Figure 3).

2. Tuning the Aspect Ratio of Nanorods by Varying the Concentration of Ag⁺ Ions

1. Prepare a silver nitrate solution with a concentration of 4 mM, dissolving 3.4 mg AgNO₃ in 5 ml of water.
2. Prepare in three different vials the solution with CTAB and hydroquinone as described in section 1.2.1 and add respectively 100 µl, 150 µl or 200 µl of silver nitrate solution.
3. 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.
4. 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).

3. Tuning the Aspect Ratio of Nanorods by Varying the Concentration of CTAB

1. Prepare different batches of gold nanorods with different concentrations of CTAB in the "growth solution". 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.
2. Add 200 µl 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.
3. Add 12 µl of seeds suspension and observe the change in color of the final mixture.
4. Stop the stirring when the suspension's color and the UV-visible spectrum are stabilized; the reaction time depends on the CTAB concentration in the growth solution.
5. Centrifuge at 10,000 x g for 10 min and resuspend in water.
6. 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).

4. Characterization of Gold Nanorods

1. UV-visible Spectroscopy
   1. Dilute 100 µl of nanorods solution with 400 µl of water in a plastic micro-cuvette and acquire the UV-visible absorption spectrum (wavelength range between 400 and 840 nm) according to manufacturer's protocol.
   2. 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.
2. Transmission Electron Microscopy (TEM)
   1. 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 µl) onto an ultra-thin Formvar-coated 200-mesh copper grids and leave to dry in air at 4 °C. Analyze the sample at TEM using an accelerating voltage of 200 kV according to manufacturer's protocol.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gold(III) chloride trihydrate	Sigma Aldrich	520918
Hydroquinone	Sigma Aldrich	H17902
Silver Nitrate	Sigma Aldrich	209139	toxic
Sodium Borohydride	Sigma Aldrich	480886
Hexadecyltrimethylammonium bromide (CTAB)	Sigma Aldrich	H5882	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.
Spectrophotometer	Thermo scientific	Nanodrop 2000C
TEM	JEOL	2100