Rapid purification of gold nanorods for biomedical applications

Graphical abstract


Method
The final reaction mixture for GNRs contains the desired GNRs, a variety of gold nanoparticles (NPs) of different shapes (spherical, cubic, etc.). To obtain an homogeneous population of GNRs, two cycles of centrifugation were carried out. After the synthesis, all reaction products (spheres, cubes, and rods) were first isolated and purified from the excess of cetyltrimethylammonium bromide (CTAB) through centrifugation at high rpm. CTAB needs to be removed as much as possible for many applications owing to its cytotoxicity [1]. Then, shape separation was afforded through a second cycle of centrifugation adapted from the method reported by Sharma et al. [2] and optimized for the size of interest.
Due to the large shape-dependent differences in the respective sedimentation velocities, in the course of centrifugation, spheres and cubes were sedimented at the bottom of the centrifuge tube while most GNRs remained in the supernatant solution. Therefore, an efficient shape separation can be achieved by tuning the centrifugation times and speeds.

Method details
Step 1: GNR synthesis

Procedure
GNRs, with AR of 3.9 AE 0.5 (width = 7.5 AE 0.8 nm, length = 28.6 AE 3.5 nm) were synthesized by a seedmediated method according to Nikoobakht et al. [3] and Ratto et al. [4] with slight modifications. After the synthesis, the structural and optical properties of GNRs were characterized by Transmission Electron Microscopy (TEM) and spectrophotometric analysis. TEM images were acquired on a JEOL 200FX with a tungsten filament operating at 200 kV. Optical extinction spectra were recorded using a Molecular Devices SpectraMax M2 plate reader.
Step 2: GNR separation from CTAB The GNRs were separated from excess CTAB and then purified from the by-products through centrifugation with an Eppendorf centrifuge 5810R using 2 ml centrifuge tubes. During centrifugation, temperature was kept at 27 8C to avoid the crystallization of CTAB that may affect the separation.
The shape separation is achieved through centrifugation according to the following steps: Isolation of all gold products of the reaction from the excess of cetyltrimethylammonium bromide through a first cycle of centrifugation. Optimization of the speed and the time of centrifugation for the separation of GNRs from the reaction byproducts. Shape separation of GNRs through a second cycle of centrifugation. The effectiveness of this procedure is documented.

Procedure
-Synthesized GNRs were placed in centrifuge tubes (1.5 ml of GNRs for each centrifuge tube) and centrifuged at the rate of 10,630 rpm (12,000 Â g) for 30 min. After centrifugation, precipitates can be clearly seen at the bottom of the centrifuge tubes.
-The supernatant was carefully removed with a pipet, leaving a volume of $50 ml solution with the precipitates. -The precipitates were resuspended by addition of 1.45 ml of Milli-Q water, leading to a final volume of 1.5 ml. -The resuspended GNRs were then combined (Sample A).
Step 3: Shape separation -The isolated products from the first centrifugation were placed in centrifuge tubes (1.5 ml for each centrifuge tube) and centrifuged at the rate of 6000 rpm (3824 g) for 15 min.
-The supernatant (GNRs) was carefully collected with a pipet (Sample B), leaving $50 ml of the solution with the precipitates in the centrifuge tubes.
-The precipitates at the bottom (spheres and cubes) in the 50 ml of the solution were resuspended by the addition of 1.45 ml of Milli-Q water, leading to a final volume of 1.5 ml. -All the resuspended GNRs were then combined (Sample C).
After separation, the colors of the solutions of the three samples were clearly different as shown in Fig. 1. Moreover, the structural and optical properties of the separated NPs (Sample B and C) were characterized by optical extinction spectra and TEM images, compared to Sample A (Figs. 2 and 3).
The absorption spectrum relative to Sample A (Fig. 2) showed a transversal surface plasmon peak with a maximum at 514 nm and a broadening at longer wavelength, indicating the presence of GNRs and spherical and cubical NPs as well. The absorption spectrum of Sample C showed that, after the separation, the maximum of the surface plasmon peak was at 522 nm while the spectrum relative to Sample B showed a transversal surface plasmon peak with a maximum at 510 nm. These differences indicated the loss of spherical and cubical NPs in Sample B while the bulk of the by-products were in Sample C. Moreover, the longitudinal surface plasmon peak with a maximum at 800 nm in the absorption spectra of Sample C indicated that some GNRs were present as well. The size and shape of the reaction products (Sample A) and separated NPs (Sample B and Sample C) are shown in the TEM images (Fig. 3). As a result, Sample B (Fig. 3B) was comprised mainly of GNRs while Sample C (Fig. 3C) contained mostly spherical NPs, confirming the shape separation by the two steps of centrifugation. Step 4: Quantification of the separation The quantification of the separation was estimated as follow:

[ ( F i g . _ 1 ) T D $ F I G ]
-The sizes of 300 NPs (spheres, cubes and GNRs) for each sample were measured with ImageJ software: spherical NPs showed the average diameter of 28.6 AE 4.7 nm, cubes showed the average side length of 14.1 AE 2.9 nm while GNRs showed a width of 7.5 AE 0.8 nm and length of 28.6 AE 3.5 nm. -The volumes of spherical, cubical NPs and GNRs were calculated; to calculate the volume of GNRs, we assumed that GNRs have a shape of a cylinder capped with two half-spheres. -The quantification of the separation was expressed in terms of volume percentage of by-products (spheres and cubes) or GNRs respect to the total volume of all the reaction products (Fig. 4).
Before separation, Sample A was comprised of 41.5% of by-products and 58.5% of GNRs (Fig. 4A). After separation, Sample B contained 79.4% of GNRs and 20.6% of by-products while Sample C consisted 14.2% of GNRs and 85.8% of by-products. As shown in Fig. 1, it is important to notice that the sizes (and the volumes) of most by-products were larger than the size of GNRs, implying that even a small amount of by-products greatly affect the total volume of the sample.

Background
Among the various gold NPs, gold nanorods (GNRs) have attracted particular attention due to their unique properties (structural, optical, and electronic) [5,6]. Moreover, facile and well-established synthetic routes (e.g., seed-mediated growth protocol [7]) permit to produce stable structures of GNRs with high yield and monodispersity.
Rod-shaped NPs exhibit two distinct surface plasmon resonances (SPRs): the transversal mode in the visible region and the longitudinal one in the upper visible or near-infrared part of the spectrum [8]. The longitudinal SPR can be tailored to a particular wavelength [9]. There is a linear relationship between the absorption maximum of the longitudinal SPR band and the mean AR of GNRs, which can be tuned during the synthesis [3]. Due to the SPR, GNRs strongly absorb light, converting absorbed light into localized heat. The strong absorption combined with the facile structural tuning property of GNRs is useful in potential in vivo applications. (e.g., photothermal treatment [10] in the near-infrared window (700-900 nm) where tissue absorption is minimal, along with deeper penetration depth [11]).
The common synthetic method of GNRs, such as the seed-mediated method [7], produces a variety of gold spheres and cubes as by-products. Several methods such as selective precipitation [17,18] or oxidation [19], size exclusion chromatography [20], electrophoresis [21], and centrifugation [2,22], have been reported for the purification of high AR (AR > 4), relatively big sized GNRs (width !10 nm). However, it is difficult to separate small sized GNRs while there is an increasing demand of small GNRs because of their strong absorption properties [23] and their high-payload-to-carrier ratio (e.g., drug delivery) [16]. Here, we optimized the method to allow the separation of small GNRs from reaction byproducts with two cycles of centrifugation. This method does not require the addition of surfactant or salts. Moreover, a large amount of GNRs can be easily obtained from by-products with a high yield.

Acknowledgement
MethodsX thanks the (anonymous) reviewers of this article for taking the time to provide valuable feedback. The authors would like to thank Beneficentia Stiftung (Vaduz, Liechtestein) and the National Institutes of Health (EB014277 and GM077173) for financial support.