Controlling Yield and Morphology for Gold Nanorods in a Seed-Mediated Synthesis Method for Cell Imaging Hai-Yan Qin, 1,2,3,4 Tao Fu,1,* Zhijun Ning, 3 Hans Ågren, 3 Hjalmar Brismar,4 and Sailing He 1,2 1
Centre for Optical and Electromagnetic Research, Zhejiang University, Zijingang campus, Hangzhou 310058, China Joint Research Center of Photonics of the Royal Institute of Technology (Sweden) and Zhejiang University, Hangzhou 310058, China 3 Department of Theoretical Chemistry, School of Biotechnology, Royal Institute of Technology, SE-10691 Stockholm, Sweden 4 Cell Physics, Department of Applied Physics, Royal Institute of Technology, SE-10691 Stockholm, Sweden
2
(Received , ; CL-; E-mail:
[email protected]
Abstract—Designed experiments are carried out to systematically study the effects of silver ions on morphology and yield of gold nanorods synthesized via a seed-mediated method. Gold nanorods synthesized with different silver ions and CTAB concentrations were characterized by absorption spectroscopy and TEM. The shape and yield of gold nanorods were effectively controlled by adjusting the concentration ratio of CTAB and silver ions for synthesis. Dark field images of a glass slide sample with gold nanorods deposit and a sample with gold nanrods staining cells further confirmed that high quality gold nanorods were obtained. Keywords- gold nanorod; seed-mediated synthesis method; yield and morphology control; cell imaging
I.
INTRODUCTION
Gold nanorods (GNRs) are promising scattering probes for bioimaging studies due to their high biocompatibility and their unique optical properties comparing to organic dyes and quantum dots. One of their favorable properties refers to the formation of the extinction band, which is tunable relative to the scattering color in the visible or near-infrared region by changing the aspect ratio (AR). Furthermore, dark field imaging for the scattering signal from GNRs is free from the photobleaching problem, which exists in most organic dyes and some quantum dots. However, the broad scattering spectral width of GNRs is unfavorable for distinguishing their scattering signal from that of cellular organelles, or for multicolor staining application [1-5]. Therefore, in order to narrow the width of their scattering spectra width as much as possible it is very important to precisely control the yield and morphology of GNRs and to understand the mechanism behind. Recently, the seed-mediated method with cetyltrimethylammonium bromide (CTAB) as surfactant has received considerable attention in the synthesis of gold nanoparticles (GNPs) for its convenience, safety, high efficiency and favorable reproducibility. Furthermore, the
The work is partially supported by Swedish Foundation for Strategic Research (SSF).
morphology and yield of GNPs could be effectively controlled by changing the molar ratio of the reactants or choosing different additives in this method [6-9]. However, the relevant shape formation mechanism is not clearly understood yet [1013]. For example, one important issue is that the size and aspect ratio of the GNR could be tailored by controlling the content of silver ions, but until now the mechanism still remains unclear. In this paper, we systematically studied the yield and shape of the products relying on different concentrations of silver ions in various concentrations of CTAB. GNRs obtained were characterized by absorption spectroscopy and transmission electron microscopy (TEM). Samples of GNRs on a glass slide and GNRs stained COS-7 cells were prepared for dark field imaging. The scattering signals of the GNRs could be easily distinguished from those of impurities or cellular structures, which confirmed that the yield and morphology of GNRs were precisely controlled. This experimental result is consistent with our hypothesis that the yield and shape of GNRs were controlled by the relative concentration of CTAB and silver ions. The excessive silver ions and chloride ions form Ag-Clsurfactants and decrease the aspect ratio of GNRs. II.
EXPERIMENTAL METHODS
A. Materials Cetyltrimethylammonium bromide (CTAB), silver nitrate, chloroauric acid, ascorbic acid, and sodium borohydride were all purchased from Sinopharm Chemical Reagent Co., Ltd. All materials were used as received without any purification. COS7 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin. B. GNR synthesis The GNRs synthesis procedures were based on the work by Perez-Juste et al [14]. Firstly, we synthesized the CTAB-GNP seed solution. To a clean bottle which contains 5 mL 0.2 M CTAB solution, 5 mL water and 50 µL 50 mM chloroauric
acid (about 1 mg), 0.6 mL 0.01 M ice-cold sodium borohydride (NaBH4) was injected all at once while stirring, and the solution turned brown immediately. Keep stirring for 5 min and then place the solution into a 30 ºC water bath for 2 hours. Secondly, we prepared the growth solution of GNRs. The typical synthesis procedures were as follows: 10 mL growth solution containing HAuCl4 (500 μM), AgNO3 (60 μM), and CTAB (0.1 M) was prepared at 25–30 ºC. Then, 55 μL of 0.1 M ascorbic acid was added to the growth solution with gentle stirring. In this step, the Au3+ ions were reduced to Au+ ions by the weak reducing agent ascorbic acid in the presence of Ag+ ions. Finally, 15 μL fresh prepared CTAB-seed solution was added to the growth solution without stirring and initiated further reduction of the Au+ ions on the surfaces of GNP seeds to form GNRs. The solution was stored overnight prior to use. GNRs with different aspect ratios could be obtained by adjusting the concentration of AgNO3 or CTAB used during synthesis procedures. C. Characterizations The absorption spectra were collected using a Shimadzu model UV2550 scanning spectrophotometer over the wavelength range from 450 to 900 nm. Transmission Electron Microscopy (TEM) images were obtained using a JEOL model JEM-1230 microscope with an acceleration voltage of 80 kV. Samples used for TEM observation were prepared by depositing one drop of GNR solution on the carbon-coated copper grid and evaporating spontaneously in the open air. D. Sample preparation for dark field imaging A drop of GNR solution was deposited on a clean glass slide. After dried out in open air, it was covered by a cover glass and ready for imaging. In order to prepare a sample of GNRs stained cells, COS-7 cells were cultured on a 28 mm diameter cover glass with a concentration of ~200 cells/mm2 one night ahead. Before treating with GNRs, we cultured the sample with serum free medium for one hour, in order to empty the binding sites on the cell membrane for nonspecific staining. Cells were incubated with ~100 nM GNR solution for 30 min and washed away the free GNRs with PBS. Lastly, the cover glass was mounted on a glass slide and ready for imaging. Both the GNRs deposit sample and the cell staining sample were imaged using Olympus BX51 imaging system with a ×50 objective and OLYMPUS C-4000 ZOOM digital color camera. III.
RESULTS
It was found that Ag+ ions could effectively adjust the AR and the size distribution of GNRs at high CTAB concentration, similar to previous reports [8, 10, 14]. For example, Fig. 1(a) shows the typical normalized absorption spectra of the asprepared colloidal GNRs grown at five different values of [Ag+] while the CTAB concentration is 0.1 M. On increasing [Ag+], the growth of the GNRs is evident by the shift of
Figure 1. (a) Normalized absorption spectra of GNRs solutions synthesized in 5 different [Ag+]: 20 μM, 40 μM, 60 μM, 80 μM, 100 μM. The concentration of CTAB is 0.1 M. (b) The aspect ratios of the GNRs are plotted against [Ag+]; the solid line is a linear fitting of the data points. (c) TEM image of GNRs with longitudinal band absorptions at 805 nm, corresponding to 60 µM [Ag+].
longitudinal band to a longer wavelength. The transverse band absorption of the GNRs, corresponding to the first peak of the absorption spectra, is located around 520 nm; while the longitudinal band absorption, corresponding to the second peak, falls into near infrared region (NIR) close to 850 nm. The relative intensities of the two absorption peaks indicate the high yields of the GNRs indirectly [15] and the narrow full widths at half-maximum (FWHMs) reveal the narrow size distribution of the prepared GNRs [17]. Fig. 1(c) shows the transmission electron microscopy (TEM) image of the sample with the [Ag+] of 60 µM. The longitudinal band absorption peak is 805 nm, corresponding to an AR of 4.This image directly confirms the monodisperse, narrow size distribution and high yield (> 97%) of the as-obtained GNRs. Since the longitudinal absorption of GNRs is AR-dependent, we can find an approximate linear relationship between the [Ag+] and the AR of NRs, which can be well fitted by the following equation (Fig. 1(b)): AR = 2.73124 + 0.01809 × [Ag+]
(1)
+
where the [Ag ] is in the unit of µM. However, when [Ag+] is continuously increased, a critical point appeared around [Ag+] of 120 µM. The yield of the GNRs decreased and the longitudinal band peaks of the solution displayed a slightly blue-shift when the [Ag+] exceeded the critical point. Meanwhile, some irregular particles were found in TEM images, which probably were caused by the precipitation of AgCl salt. We further studied the influence of [Ag+] with various CTAB concentrations. Fig. 2 shows the absorption spectra of the GNPs synthesized with four different concentrations of CTAB (0.02 M, 0.04 M, 0.06 M and 0.08 M), and in each case four different [Ag+] (20 μM, 40 μM, 60 μM and 80 μM) were used. Notably, the [Ag+] solution maintained the ability of controlling the AR and the yield of the GNRs when the concentration of CTAB was decreased from 0.1 M to 0.04 M. However, large changes in shape and yield occurred when the CTAB concentration decrease to 0.02 M. From the spectra in
Figure 2. Normalized absorption spectra of GNRs solutions synthesized in 4 different concentrations of CTAB: (a) 0.02 M, (b) 0.04 M, (c) 0.06 M, and (d) 0.08 M.
Fig. 2(a), we note that the intensity of the transverse band absorption peak was gradually enhanced with the increment of [Ag+], which indicates that the yield of the GNRs decreased. Furthermore, when the [Ag+] was introduced at 80 μM, the longitudinal band peak disappeared and only one peak near 560 nm could be detected, which demonstrated that no GNR was formed and bigger GNPs were synthesized in this reaction condition. Fig. 3 shows the corresponding TEM images of the GNPs of Fig. 2(a). Clearly, the yields of GNRs are gradually reduced with the increasing of [Ag+] (Fig. 3 (a) ~(c)). At the [Ag+] of 80 μM, few GNRs were formed and particles with two different sizes were obtained (Fig. 3(d)). This result implies that the microcapsules formed by CTAB and water are very sensitive to the concentration of [Ag+] at low concentration of CTAB. Under dark field illumination, it is hard to avoid the white scattering background causing by impurities or cellular structures. GNRs with red, orange, green or cyan scattering color are popular in dark field bioimaging applications. One of the main reasons is that these colors are easier to distinguish from the white background than some other colors. Precisely control the yield and morphology of GNRs can provide high quality samples with narrow FWHM and bright scattering signals. Therefore a series of high quality GNR samples with all sorts of colors of the visible spectrum are possible to obtain for multi-color staining imaging applications. By controlling the yield and morphology of GNRs, we obtained high quality GNRs with yellow scattering color. In order to further confirm this, we prepared two samples for dark field scattering imaging. The first one is a glass slide with yellow GNRs deposit. From Fig. 4 (a), one can easily distinguish the GNRs with yellow scattering color from white impurities. The second one is a sample of COS-7 cells staining with yellow GNRs. The amino groups on the surface of GNRs carry strong positive charges, while the heparan sulfate (HS) proteoglycans at the cell surface are negatively charged. Therefore, the cell surface has a strong electrostatic interaction
Figure 3. TEM images of GNRs synthesized with the concentration of CTAB 0.02 M and 4 different [Ag+]: (a) 20 μM, (b) 40 μM, (c) 60 μM, and (d) 80 μM.
with GNRs and nonspecific staining occurs. Fig. 4 (b) shows the dark field image of COS-7 cells stained by GNRs unspecifically. The GNRs scattering yellow signals are easy to distinguish from the cellular structures which give scattering white signals. IV.
DISCUSSION AND CONCLUSIONS
The notion of a “Soft template mechanism” postulated by El-Sayed’s group is so far one of the most popular explanations for the formation of GNRs. In this mechanism, the template is formed by the CTAB micelle with a size depending on the CTAB concentration and the ionic strength of the solution. The surfactant-capped seed links to the micelle surface, and it starts growing accompanied by new atoms joining the nanocrystal lattice [10]. It is found that an Ag-Br-surfactant complex is formed in the growth solution, similar to the Au-Br-surfactant complex [16], and the Ag-Br-surfactant complex would promote the attachment between seed GNPs and micelles [13]. Moreover, the formation of AgBr precipitates reduced the ionic strength of the solution, resulting in a CTAB template elongation [10].
Figure 4. Dark field images of (a) GNRs deposited on glass slide and (b) COS-7 cells staining with GNRs.
However, in our experiments, the occurrence of critical point indicates that the Ag-Br-surfactant complex was saturated. Increasing the silver content would cause the formation of an Ag-Cl-surfactant complex, which makes the shape of the product uncontrollable [17]. Hence, we supposed that the CTAB controlled the shape of the GNRs based on the following three observations: (1) most CTAB molecules formed micelles as templates for restricting the growth of GNRs; (2) parts of the CTAB molecules strayed in the solution freely and formed Ag-Br-surfactant complexes with silver ions; (3) the rest in the status of hydrolysis, which could release free and CTA+, enabled the formation of Brcetyltrimethylammonium chloride (CTAC) molecules and then Ag-Cl-surfactant complexes. Since the amount of free CTAB molecules is related to the concentration of CTAB in the solution, solutions with high concentration of CTAB have more free CTAB molecules than those with low concentrations. Moreover, at the coexistence of both CTAB and CTAC, silver ions prefer to combine with CTAB first because the affinity between silver ions and bromide ions is stronger than that between silver ions and chloride ions, which could be deduced from the Ksp values of AgBr (3.3 × 10-13) and AgCl (1.6×10-10). When the molar ratio of [Cl-]/CTAB was fixed, increasing the concentration of silver ions could result in the formation of, first, Ag-Br-surfactant and then Ag-Cl-surfactant complexes. Thus, at high CTAB concentration, AR would increase until the free CTAB was used up. Later on, excessive silver ions and free Cl- formed Ag-Cl-surfactant complexes, which led to a decline in the quality of GNRs. At low concentration of CTAB, a small quantity of silver ions could exhaust all free Br- (in our experiment reaction, [Ag+] was 20 μM corresponding to 0.02 M CTAB). As a result, the increment of silver content could only lead to the formation of Ag-Cl-surfactant and the decrease of the GNRs. This mechanism could also be extended to explain the similar results reported in Refs. [8, 10]. In conclusion, the effects of silver ions on synthesizing GNRs by a seed-mediated method were experimentally studied and qualitatively discussed. Our current results provide evidence that the shape and yield of GNRs can be effectively controlled by adjusting the concentrations ratio of CTAB and silver ions. Excessive silver ions and chloride ions form Ag-Clsurfactants and decrease the aspect ratio of GNRs. REFERENCES [1]
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