Gold Micro-Flowers: One-Step Fabrication of Efficient, Highly Reproducible Surface-Enhanced Raman Spectroscopy Platform
We present a new method enabling simultaneous synthesis and deposition of gold micro-flowers (AuMFs) on solid substrates in a one-pot process that uses two reagents, auric acid and hydroxylamine hydrochloride, in aqueous reaction mixture. The AuMFs deposited onto the substrate form mechanically stable gold layer of expanded nanostructured surface. The morphology of the AuMFs depends on and can be controlled by the composition of the reaction solution as well as by the reaction time. The nanostructured metallic layers obtained with our method are employed as efficient platforms for chemical and biological sensing based on surface-enhanced Raman spectroscopy (SERS). SERS spectra recorded by such platforms for p-mercaptobenzoic acid and phage lambda exhibit enhancement factors above 106 and excellent reproducibility.
KeywordsSurface-enhanced Raman spectroscopy Surface modification Micro-flowers Microstructures and nanostructures
Noble metal nano- and microstructures of nonspherical shapes have received much attention in recent years [1, 2, 3, 4, 5]. Their morphological forms—including flower-, rod-, wire-, and plate-like structures—exhibit unusual optical , electronic , and catalytic [8, 9] properties. Very promising area of applications of gold nano- and microflowers is surface-enhanced Raman spectroscopy (SERS). Surfaces modified with such flower-like structures have proved [10, 11, 12, 13, 14] to provide large SERS enhancement factors. Known methods [1, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22] of synthesis of gold microflowers (AuMFs) require fairly elaborate techniques. With these methods, AuMFs are typically synthesized in reaction solutions containing gold salts, reducing agents, buffers, and additional reagents that strongly affect both size and the morphology of the resulting structures. For instance, reagents like polianiline , chitosan [15, 16], surfactants [17, 18], or DNA  have been utilized to obtain gold particles of ragged, flower-like shapes. Similar structures have also recently been fabricated mechanically by a centrifuging process . Synthesis of rough flower-like complexes directly on surfaces is usually carried out electrochemically via electrodeposition  or seed-mediated growth approach . In this study, we present a novel facile method enabling simultaneous synthesis and deposition of the AuMFs on hydrophilic solid substrates. The method developed is a one-pot process using only two simple reagents in aqueous reaction mixture. We demonstrate that substrates covered with a layer of the AuMFs using our technique can be employed as efficient, highly reproducible SERS platforms, providing the enhancement factor of the order of 106.
Auric acid (HAuCl4) and hydroxylamine hydrochloride (NH2OH·HCl) were purchased from Sigma-Aldrich. The reagents were all analytical grade and used without additional purification. Concentrated sulfuric acid, hydrochloride, hydrogen peroxide, and nitric acid were purchased from Chempur. We employed as solvents acetone and methanol from Chempur and deionized water (15 MΩ). The glass microscope slides were obtained from Roth. The silicon-polished wafers were obtained from Cemat Silicon.
Preparation of the Reaction Mixture
To prepare the reaction mixture, solution of auric acid was added dropwise while stirring to an aqueous solution of hydroxylamine hydrochloride. The resulting reaction mixture was fully characterized by the HAuCl4/NH2OH molar ratio and the concentration of hydroxylamine. In this paper, the mixture concentration is given relative to the reference solution, c1, containing 0.5 mM of NH2OH. For example, to obtain the c1 (3:8) reaction mixture containing 3:8 HAuCl4/NH2OH molar ratio, one volume of 0.8 mM NH2OH with 0.6 volume of 0.5 mM HAuCl4 were mixed. Mixtures diluted ten times are marked—irrespectively of their HAuCl4/NH2OH molar ratio—as c0.1. Similarly, mixtures with ten times higher concentration of hydroxylamine are marked as c10, etc.
Covering Substrates with AuMFs
Before use, the substrate plate was cleaned by sonication first in water and then in acetone. Next, the plate was immersed in a freshly prepared piranha solution (3:1 H2SO4:30% H2O2) for 3 h. Finally, the plate was richly rinsed with deionized water and then with methanol. The reaction mixture was prepared according to the procedure described previously in this section. As soon as the reagents were stirred, the dried slides were placed horizontally in a vial containing this mixture for 1 h. Then, the plate was carefully washed subsequently with water and with methanol, and dried.
Fabrication of SERS Platform
To obtain the SERS platform, we followed the general covering procedure outlined in Fig. 1. Silicon plates were applied as substrates. Before used, the plate was roughened by scratching it with sandpaper (grit size 500). The roughened plate, cleaned as described above, was placed horizontally at the bottom of a vial containing a c50 (3:8) fresh reaction mixture [c50 (3:8) mixture consists one volume of 40 mM NH2OH with 0.6 volume of 25 mM HAuCl4]. The height of the solution column above the plate was about 10 mm. After 24 h, the plate with deposited AuMFs porous film was carefully washed subsequently with deionized water and with methanol and then dried in air. Finally, the plate was—without additional cleaning—immersed in the analyte solution for 6–48 h and dried in air before the SERS measurements.
Surfaces covered with AuMFs were analyzed using field emission scanning electron microscopy (SEM) with Neon 40-Auriga Carl Zeiss apparatus. The absorption spectra were recorded on Ocean Optics USB 2000+ spectrophotometer in the spectral range 300–800 nm in quartz micro cuvette (10 mm of path length). The sample was illuminated with UV-VIS-NIR LIGHTSOURCE DH-2000, Ocean Optics. The absorption spectra were recorded starting from 35th second of the reaction. SERS measurements were performed using the Renishaw InVia Raman system equipped with a He-Ne laser emitting a 632.5 nm line used as the excitation source. The light from the laser was passed through a line filter, and focused on a sample mounted on an X-Y-Z translation stage with a ×50 microscope objective. The Raman-scattered light was collected by the same objective through a holographic notch filter to block out Rayleigh scattering. A 1,800 groove/mm grating was used to provide spectral resolution of 1 cm−1. The Raman scattering signal was detected by the 1,024 × 256 pixel RenCam CCD detector. The SERS signal was collected for a dried sample on the SERS substrate. The SERS spectra were acquired using 150 s integration time and processed with the Renishaw software WiRE 3.2.
Results and Discussion
Effect of Composition of the Reaction Mixture on the Morphology of AuMFs
Our experimental results suggest that the flower-like particles are not observed for the concentrations lower than c0.4 because of insufficient amount of gold ions in the reaction mixture. As we found from the analysis of the SEM images, the average number of particles created during the reaction was similar for the concentrations from the range c0.05–c1. However, the average size of the particles observed for the low concentrations was significantly smaller than that observed for the concentrations greater than c0.4. This observation indicates that in the low concentration reaction mixtures there is not enough matter for the nuclei to grow and develop properly shaped “petals”.
Effect of Reaction Time on the Morphology of AuMFs
Kinetics of the AuMF Formation
We employed a minimalistic reaction model that accounts for the kinetic data obtained. According to this model, the reaction proceeds in two stages, which are nucleation and autocatalytic growth of the metallic clusters. From the plot, it follows that the first step of the reaction occurs in the first 5 min. The second stage takes place roughly between fifth and twelfth minute of reaction. In general, the reaction time of the chemical reduction of gold ions observed in our method is similar to that reported by other researchers for the seedless methods [8, 14, 15, 16, 17, 18].
This two-step model follows from the fact that hydroxylamine is thermodynamically capable to reduce Au3+ ions directly to metallic gold , but this reaction is dramatically accelerated by the presence of gold surfaces [24, 25]. For this reason, production of new nuclei is suppressed in a solution that contains colloidal gold nanoclusters . One therefore expects that the formation of the AuMFs occurs in the following two stages. (1) Initially, the Au3+ ions are reduced in the bulk solution to form nuclei. Although the nuclei created grow autocatalytically, at this stage, the consumption of the gold ions is due mainly to the nucleation processes. (2) As the concentration of the nuclei (seeds) increases, the nucleation process is gradually suppressed and replaced by the autocatalytic (surface catalyzed) reduction of Au3+. The stages are described below.
Here [A]1 and [B]1 denote, respectively, the concentration of the Au3+ ions and surface atoms of the growing clusters (seeds) present at the beginning of the autocatalytic growth stage. The quantities [A]0 and [A]1 are linked by the relation [A]1 = [A]0exp(−k 1 t n), where t n is the duration of the nucleation stage.
We fitted the functions given by Eqs. 2 and 4 to the absorbance data using t n, [A]0, k 1, α, β, and k 2 as the fitting parameters. The fitting procedure yielded t n = 5.0 ± 0.5 min, [A]0 = 1.040 ± 0.002, and k 1 = 0.0550 ± 0.0006 for the nucleation reaction and α = 0.853 ± 0.008, β = 0.0024 ± 0.0004, and k 2 = 0.85 ± 0.02 for the autocatalytic reaction. The resulting kinetic curve is plotted in Fig. 4a. In our attempts to model the formation of the AuMFs, we considered also two-step growth reaction that comprises both nucleation and the autocatalytic growth (Eqs. 1 and 3) that is referred to as the Finke–Watzky (FW) kinetic model [26, 27]. The FW model predicts, however, an S-shaped sigmoidal kinetic curve of the concentration of the unconsumed metal ions that did not fit our experimental data.
For the reaction times less than ~12 min, corresponding to late stages of the nucleation regime and the autocatalytic growth stage, we observed the sharp-edged morphology of the AuMFs. When the reagents were consumed, in late phases of the autocatalytic growth stage, the round-edge morphology was observed. The three stages of the AuMF formation—nucleation followed by the autocatalytic growth, and the edge smooting—are illustrated schematically in Fig. 4b.
In order to estimate the detection limit for PMBA molecules, the SERS measurements were performed for substrates that were immersed in solutions containing different concentrations of PMBA. The substrates were immersed in each solution for the same period of time (2 h). Three analyte concentrations were applied: 10−3, 10−9, and 10−9 M. The SERS spectra recorded for these concentrations are shown in Fig. 6c. As the amount of the analyte in solution was reduced, the SERS intensity decreased but the Raman band at 1,080 cm−1 was observed even for the lowest PMBA concentration used (marked as A in Fig. 6c).
To demonstrate the applicability of the platforms for biomolecule sensing, we carried out SERS-based detection of the bacteriophage λ. This phage contains double-strainded linear DNA as its genetic material, and infects Escherichia coli. To prepare the SERS platform, 10 μM probe of bacteriophage λ in phosphate-buffered saline (PBS; 1 M, pH = 6.7) was immobilized on the surface of the AuMF-functionalized plate. The plate was kept in the solution at room temperature for 6 h. Then, excessive bacteriophage was washed with sodium dodecyl sulfate solution in PBS (0.1%, v/v) for 5 min. Figure 6d shows the SERS spectrum of DNA of bacteriophage λ, which clearly displays Raman bands at 883, 1,276, 1,496, 1,558, and 1,670 cm−1 assigned to, respectively, O-P-O backbone, cytosine, guanine, adenosine, and thymine vibrational modes of DNA . This result suggests that SERS platforms fabricated with our method can be successfully applied for identification of biomolecules.
In summary, we developed a novel facile one-pot method for covering solid substrates with stable, thick layer of micrometer-sized AuMF. By changing the composition of the reaction mixture and/or the reaction time, we can easily control the morphology of the AuMFs, that is, the average number and shape of petals of which the AuMFs are composed. Our method is suitable to fabricate platforms for SERS-based chemical and biological sensing. The SERS spectra recorded by platforms for PMBA and bacteriophage λ provided enhancement factors above 106 and exhibited excellent reproducibility both within single substrates and between different substrates. The fact that our platforms allowed detection of Raman bands of the bacteriophage’s DNA is remarkable. It proves that the AuMF-functionalized substrates can be potentially employed for bioanalytical applications. To end, note that due to hazardous substrate minimization, mild reaction conditions, and lack of power consuming processes, our method meets the goals of green chemistry.
This work was supported by Polish Ministry of Science and Higher Education as scientific projects (2007–2010) and "Iuventus Plus" (IP2010025970). The research was partially supported by the European Union within European Regional Development Fund, through grant Innovative Economy (POIG.01.01.02-00-008/ 08).
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