Technical Note: Synthesis and Characterization of Anisotropic Gold Nanoparticles ()
1. Introduction
Metal nanoparticles (NPs) in the range of one to a few hundred nanometers are stated to have novel magnetic, electronic and optical properties dependent on their shape, size and composition [1]. This makes them a desirable commodity in the medical, pharmacological and biotechnological industries for use in various applications including drug delivery [2,3], hyperthermal cancer therapies [4] and diagnostic devices [5].
There are a number of advantages to the use of Au-NPs, particularly in biological systems including the following; Au-NPs: 1) bind readily to sulfhydryl groups which allow for simple NP functionalization by the conjugation of various biomolecules and other ligands; 2) are resistant to oxidation; 3) possess surface plasmons (in some cases multiple plasmon peaks depending on their morphology) which allow the Au-NP, and hence the attached ligand/molecule to be monitored by UV-Vis spectroscopy; 4) are biocompatible; 5) are size tunable for variation in Au-NP properties; 6) are electron dense due to the high atomic number of gold, making the Au-NPs easily identifiable by analysis with electron-microscopes and 7) laser illumination of the particles results in an increase in particle temperature due to energy absorption by the surface plasmons [3,4,6-9].
Anisotropic metal NPs of varying size distributions have been synthesized via environmentally-benign approaches using both prokaryotic and eukaryotic organisms including bacteria, fungi, actinomycetes and yeasts [10-12]. The use of biotemplates has also come under the spotlight in an attempt to synthesize nanoparticles by exploiting the structural properties of various proteins/ peptides and viruses to act as scaffolding for the nucleation process [13-15].
Bovine serum albumin (BSA) is one such biological template which has been successfully used by Basu and colleagues (2008) in the production of anisotropic gold nanotriangles [15]. There are a number of advantages to its use in a biological, medical and biotechnological context including the fact that it is an inert, stable protein that is often used to stabilize enzymes and proteins in low concentrations (<1 mg/ml) [16]; and is known to increase the solubility of Au-NPs in aqueous solutions when coated/conjugated to the NPs [17]. This in turn negates the need for inorganic stabilizers such as polyvinylpyrrolidone (PVP).
Although a large amount of literature exists covering the synthesis of a range of nanoparticle compositions and morphologies by various methods, surprisingly little information exists on the characterization of the particles produced.
In this study nanoparticles were produced according to the method of Basu et al. 2008, optimized in our laboratory and were characterized by TEM, SEM, STEM-EDS, UV-Vis spectrophotometry and photoluminescence in an attempt to identify potential applications.
2. Methods and Materials
2.1. Nanoparticle Synthesis
A heterogeneous mixture of anisotropic Au-NPs were synthesized using the BSA protocol described by Basu and colleagues (2008). The method was optimized for BSA template (8.3 µM) and gold chloride (Au3+) (0.25 mM) precursor concentration in our laboratory at 25˚C over 96 hrs [15]. The reproducibility of the particle synthesis was also investigated by performing the method in triplicate.
2.2. Particle Separation
A number of methods were evaluated in an attempt to separate the larger nanoplates from the smaller nanoparticles. These included differential centrifugation; centrifugation in combination with a glycerol density gradient; agarose gel electrophoresis and size exclusion chromatography.
The particles described in the paper were separated using differential centrifugation. After 96 hours incubation in the synthesis medium, a sample (25 ml) was collected, stored and labelled as the “Original” sample. The remaining solution (25 ml) was centrifuged at 1500 rpm for 5 minutes. The supernatant was labelled “Small NPs”, while the pellet was redispersed in dH2O and labelled “Large NPs”.
2.3. UV-Vis Analysis
The UV-Vis spectra of the partially purified colloidal solutions were determined and characterized on a Perkin Elmer LAMDA 750S UV/Vis Spectrometer from 200 - 1200 nm (1 nm intervals).
2.4. Photoluminescence
Photoluminescence spectra of the partially purified colloidal solutions were recorded by a Perkin Elmer, LS 55 Fluorescence Spectrometer covering a wavelength range from 300 - 680 nm with an excitation wavelength of 325 nm.
2.5. Transmission Electron Microscopy (TEM), Scanning Transmission Electron Microscopy (STEM) and Energy Dispersive X-Ray Spectroscopy (EDS)
Immediately upon sampling, 7.5 µl samples were applied to carbon-coated copper grids and allowed to air-dry (in the dark) prior to TEM analysis, to determine particle morphology. Grids were analysed using a JEOL JEM- 2100F transmission electron microscope with an operating voltage of 200 kV. Item analysis image processing software was used to measure between 100 200 particles in each sample to determine the size distribution and average size of the various anisotropic particles present.
The STEM-EDS analysis was performed using a spot size of 0.5 nm (high resolution) with an acquisition time of 220 seconds. The INCAx-sight (Oxford Instruments) EDS analysis software was used to obtain and identify the elemental spectra of various points of interest.
2.6. Scanning Electron Microscopy (SEM)
A high resolution, Zeiss ULTRA plus Field Emission Gun Scanning Electron Microscope (FEG-SEM) operating at low electron voltages of between 0.6 - 1.0 kV was used to image and characterize the surfaces of the AuNPs.
3. Results and Discussion
3.1. Nanoparticle Synthesis and Separation
The method described by Basu et al. (2008) was optimized to produce the highest concentration of compact nanoparticles within the narrowest size distribution. In addition, the effect of alternating two different acids during pH adjustment, namely weak acetic acid and 50% HCl, on particle morphology was investigated. The results showed that acetic acid promoted the synthesis of the smaller more compact particles (Figures 1(a) and 2(a)), while the use of HCl suppressed the formation of the smaller particles while subsequently promoting the growth of very large, well-formed anisotropic nanoplates in the micrometer size range (up to 10 µm) (Figure 2(b)). Unfortunately the size variations of the plate-like particles were extremely large hence the method utilizing acetic acid was chosen as the method of choice (results not shown).
Even with optimization, two types of particles were produced; namely the smaller, more compact particles and the larger, thinner plates. Since the properties of nanoscale materials are largely dependent on the particle size and morphology, a number of separation methods were identified and investigated.
All of the separation methods tested except for centrifugation in combination with a glycerol density gradient provided poor separation efficiencies. Further investtigations into optimizing the most promising method were undertaken but difficulties in extracting the fractionated particles from the glycerol gradient resulted in the method being rejected. In the end, the simplest method of differential centrifugation was chosen even though only partial separation was achieved.
3.2. TEM, SEM and STEM-EDS
Figure 1 illustrates TEM images taken of the Au-NPs