Abstract
Gold nanoparticles bound with inositol hexaphosphate (IP6) (AuNPs/IP6) were prepared by in situ reduction of various concentrations of IP6 (0~320 µM) through modified Frens method for surface-enhanced Raman scattering (SERS) detection. The resultant AuNPs/IP6 were subject to characterization including UV/Vis spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, and X-ray photoelectron spectroscopy (XPS). The results showed that AuNPs with 65 µM of IP6 would result in a core AuNPs-shell (IP6 layer) structure, which exhibited the strongest SERS signal, due to the “hot spot effect” generated from the 1-2 nm interparticle gaps of AuNPs/IP6 nanohybrids (ionic interaction of IP6 and Au+). Furthermore, the reaction kinetics of Au and IP6 were also investigated in this work. Higher concentration of IP6 (190 and 260 µM) will make AuNPs become irregularly shaped, because IP6 is a basic salt and served as a pH mediator. The morphology and distribution of AuNPs were greatly improved by addition of 65 µM of IP6. This novel AuNPs/IP6 nanohybrid showed great stability and Raman enhancement. It is promising in the application of rapid and label-free biological detection of bacteria or tumor cells.
1. Introduction
Raman scattering was discovered by C. V. Raman in 1928 and SERS technology was developed by Fleischman and others in 1974 [1]. In recent years, SERS has been employed for label-free sensing of bacteria such as Escherichia coli (E. coli) or various molecules, exploiting its tremendous enhancement of the Raman signal. Gold and silver nanoparticles are widely used in this field [2–4], because they produce localized surface plasma resonance (LSPR), which can increase the intensity of the Raman signal by at least 109. Gold and silver nanoparticles have unique optical, electrical, and magnetic properties because of their particle size and morphology. Therefore controlling the size and morphology is important when synthesizing nanoparticles [5–7]. Gold and silver nanoparticles (NPs) increase Raman signal under specific frequency because LSPR produces electromagnetic field, which will increase the Raman signal of the absorbed molecule. If we further limit the space between these metal nanoparticles at 1-2 nm, it will produce “hot spot” effect, which will further increase the intensity of the SERS signals [2–4]. Therefore these materials showed promising potential in the application of SERS [8, 9] and biosensing [10, 11].
Inositol hexaphosphate (IP6) is known as phytic acid sodium salt, a naturally derived material. It could be used to prepare oral cleansing agent, water treatment agent, food additive, and so on because of its nontoxic and natural properties [12]. The structure of IP6 contains six phosphate acid groups (negatively charged) which are able to link with metal particles and have good absorption capability. Therefore we will employ IP6 as a tunable cross linker (or spacer) to obtain AuNPs with a distance between each other at 1-2 nm.
In this work, AuNPs were produced by a procedure developed by Frens et al. in 1973 using sodium citrate to reduce HAuCl4 to produce monodispersed AuNPs. In this present work, IP6 was added during the reduction procedure and its adsorption onto the AuNPs led to the final product AuNPs/IP6. The final product will be further tested in the application of SERS for detecting microorganism Staphylococcus aureus.
2. Experimental Procedure
2.1. Materials
Inositol hexaphosphate (IP6), sodium citrate dihydrate (Na3Ct·2H2O), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O), and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich. Nitric acid (HNO3) was purchased from Scharlau, Spain. Silicon oil was purchased from Choneye Pure Chemical. Luria-Bertani (LB) broth was purchased from Difco. Bacteriological agar was obtained from Oxoid Ltd., UK. All glassware was cleaned with aqua-regia and rinsed with deionized water prior to the experiment. Staphylococcus aureus was obtained from Super Laboratory Co., Taiwan.
2.2. Synthesis of Gold Nanoparticles
The gold nanoparticles were synthesized on the basis of the method developed by Frens et al. Table 1 lists the solution for preparing AuNPs/IP6 by mixing 0.01% HAuCl4 with 1.0 mM IP6 stock solution. When the solutions were boiling for 10 min, 3.5 mL of 1% sodium citrate was then added drop wisely. During the reaction, the color of the solution turned from light yellow color to brick red color. The reaction completed when the color no longer changed and the final solution result was referred to here as gold colloids. The samples of HAuCl4/IP6 solutions were analyzed using TEM and DLS as described in the following section. Scheme 1 depicts the core-shell structure of AuNPs/IP6 nanohybrids.
2.3. Reaction Time for HAuCl4/IP6 Solutions
The reaction time of the gold colloids is the time required for the reaction of HAuCl4 and IP6 to be completed when the color no longer changes. The exhausted time for HAuCl4 was monitored according to the way developed by Ji et al. [13]. Temporal evolution of HAuCl4 was obtained by measuring the pH 10 times before the exhaustion of HAuCl4 with IP6 and initial reaction rate was also obtained.
2.4. TEM of the AuNPS of the Gold Colloids
An aliquot of 5 μL of the gold colloids described in Section 2.2 was placed on the copper grid and dried in the autoclave. Afterwards, they were placed in a copper grid box and analyzed using TEM (H7650, Hitachi, Japan) for the size distribution (diameter) and morphology of AuNPs.
2.5. Dynamic Light Scattering (DLS) of the Gold Colloids
The gold colloids (1 mL) were placed in a DLS cuvette followed by sonicating for 5 s before DLS (Nano ZS, Malvern Instruments, UK) analysis. Each sample was analyzed 3 times.
2.6. SERS Measurements of AuNPs/IP6
Raman microscope (HR800, Horiba, Japan) with He-Ne laser (632.8 nm) was used to detect the presence of S. aureus (ATCC 6538P). 50 μL of the varied AuNPs/IP6 and 50 μL of S. aureus (1 × 105 cfu/mL grown for 18 h at 37°C) were placed in 1.5 mL microcentrifuge tubes and mixed well. Then 5 μL of each sample was dropped on the aluminum sheet. Raman spectra in the range of 600 and 900 cm−1 were evaluated for these 6 samples. Intensity of the Raman signal at 733 cm−1 (SERS signal from the cell wall of S. aureus) for the samples was further investigated, as shown in Figure 8.
2.7. Characterization Analysis of AuNPs/IP6
The interaction between AuNPs and IP6 samples were analyzed by X-ray photoelectron spectroscope (XPS, VG ESCA Scientific, Theta Probe) and surface electric properties of AuNPs/IP6 samples were analyzed by zeta potential analyzer (Nano S90, Malvern Instruments) as described below.
3. Results and Discussions
3.1. Characterization of AuNPs
LSPR wavelength and colors of solutions displayed different pattern of adsorption as shown in Figure 1. Gold colloids of various IP6 concentrations displayed various colors (Figures 1(a)–1(g)). Solutions A0 to A2 displayed brick red color or high concentration of AuNPs, A3 exhibited purple red color, A4 and A5 displayed blue color, and A6 showed purple color. UV/Vis spectroscopy showed a strong single absorption peak of the AuNPs/IP6 and absorbed wavelength of the major peak gradually increases (red-shift) from 525 nm (A0) to 534 nm (A3) (Table 2; Figures 1(a) to 1(c)), implying that the diameter of the spherical AuNPs was about 30 nm and the color of Au colloids is brick red. Moreover, the peaks of A1 and A3 were sharper than the peak of A0; thereby the morphology and diameter of AuNPs are more homogeneous than AuNPs without IP6. Figures 1(e) and 1(f) showed 2 peaks and implied that AuNPs display anisotropic structures. The first peak with lower wavelength was fraction of transverse absorption and the second peak with higher wavelength was longitudinal absorption. Further analysis by TEM also confirmed that both of them are irregular shaped AuNPs.
3.2. DLS Analysis of the Gold Colloids
DLS measurements of the particle diameter from A0, A1, and A2 were , , and nm, respectively (Figure 2). This result agreed with the results of UV/Vis spectra for being spherical shaped. Since the smallest standard deviation of the particle diameter was from A2, the AuNPs with 65 μM of IP6 showed more uniform morphology and diameter as compared to the traditional process of thermal citrate reduction method without addition of IP6.
3.3. TEM Analysis of the Gold Colloids
The IP6 layer can clearly be observable in Figures 3(b) and 3(c), especially in Figure 3(c) for A2. There are two absorption peaks shown in samples A4 and A5 (Figures 1(e) and 1(f)); the second absorption peak was observed at 648 and 725 nm, respectively. This indicates that AuNPs/IP6 formed irregular structures [14–16], which was also observable in the TEM images (Figures 3(e) and 3(f)). There are many oval and prism shaped AuNPs. TEM and DLS results showed that the irregular nanoparticles were observed as the concentrations of IP6 increased from 190 μM to 260 μM. The morphology of AuNPs changed to spherical structures again when the concentrations of IP6 reached 320 μM, which displays a small second peak at 655 nm and just little irregular shaped AuNPs were found.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
3.4. Exhausted Time and Initial Reaction Rate of Gold Colloids
The reason why the morphology of AuNPs became irregularly shaped when the concentration of IP6 reaches 190 and 260 μM was because IP6 is a basic salt and served as a “pH mediator.” It can change the pH of auric acid solutions, leading to the growth of irregular shaped AuNPs. Higher concentration of IP6 caused the pH of the solution to increase and [AuCl4]− is converted to a less reactive []−. Therefore at higher pH, [OH−] will increase and react with [AuCl4]−, forming a less reactive []− substance. Therefore higher concentration IP6 will cause the exhausted time of HAuCl4/IP6 to increase. As shown in Figure 4 and Table 3, the exhausted time increased greatly with the addition of 190 and 260 μM of IP6, as compared to the addition of the 26~130 μM of IP6. The calculation method for temporal evolution curve of HAuCl4 concentration used at least eight concentration points for each reaction into a Taylor expansion polynomial (average -squared value for all reactions in Figure 4 is 98%) [17]. The formation of irregular shaped AuNPs is probably caused by the following: (1) AuCl under high concentration of IP6 was not reactive enough to produce AuNPs and might go through nucleation twice to cause aggregation; (2) IP6 attached to specific surface of Au seeds and induced AuCl to grow on the specific surface of Au seeds leading to the formation of irregular AuNPs.
Figure 5 shows that with the addition of 26 and 65 μM of IP6, the initial reaction rate was much faster as compared to 0 μM of IP6; therefore HAuCl4 can be exhausted immediately to develop spherical shaped AuNPs. With the addition of 190 and 260 μM of IP6, the initial reaction rate was slower than those of 0~130 μM of IP6, because the reactivity of auric salt decreased with the increasing of pH of the solution [18, 19] due to increase of IP6 concentration. With the addition of 320 μM of IP6, the initial reaction rate was very low and the extremely slow reaction rate caused the formation of some spherical shaped AuNPs once again.
Figure 6 illustrates our hypotheses explaining the effect of IP6 concentration on the morphology of AuNPs. 26 and 65 μM of IP6 caused the reaction to develop faster as compared to formation of AuNPs without the addition of IP6. With the addition of 190 and 260 μM of IP6, the formation of AuNPs is slow along with formation of irregular shaped AuNPs as mentioned in Section 3.4. When the concentrations of IP6 were increased to 320 μM, some AuNPs spherical structures were formed (Figure 3(g)). This is because of extreme slow reaction rate causing Au seeds to form slowly and therefore there is less formation of second nucleation or aggregation of AuNPs.
3.5. SERS Application of AuNPs/IP6
Figure 7 shows the SERS spectra of S. aureus using AuNPs/IP6. The SERS peak at 733 cm−1 was from the cell wall of S. aureus and the strongest enhancement was observed for A2 as shown in Figure 7. Figure 7 also shows that A2 exhibits the strongest SERS intensity, which was and was about 40 times that of A0 (). Therefore the AuNPs reduced with the presence of 65 μM of IP6 would enhance the SERS signal more than that without the addition of IP6. This is also confirmed with the TEM images in Figure 3, where the addition of 65 μM of IP6 produced most visible IP6 layer formed on the surface of AuNPs. This core-shell structure not only made AuNPs disperse well but also led to a specific distance of 1-2 nm between AuNPs. This is the reason why A2 greatly increased the intensity of the Raman signal by detecting S. aureus.
3.5.1. Zeta Potential and XPS Analysis of AuNPs/IP6
The interaction between AuNPs and IP6 was further investigated by zeta potential and XPS analysis, as shown in Figure 10. Figure 9 shows that the zeta potential decreased from to mV when the concentrations of IP6 increased from 0 to 65 μM. Thus the negative charge on the surface of AuNPs increased with increasing IP6 concentrations and higher IP6 will induce more IP6 molecules absorbed on the surface of AuNPs. XPS analysis showed 0.4 eV and 0.3 eV binding energy shifting of Au7/2 (84.5 to 84.9) and Au5/2 (88.1 to 88.4) from 0 μM to 65 μM of IP6 addition, respectively. This showed that IP6 interacted with Au+ NPs by electrostatic force. Therefore, IP6 definitely formed a layer on the surface of AuNPs with 65 μM of IP6. This layer not only controlled the interparticle gaps of AuNPs within 1-2 nm from each other but also produced huge “hot spots” effect which greatly increased the Raman intensity of the sample molecules.
Zeta potential and XPS analysis indicated that 65 μM of IP6 was an ideal concentration leading to formation of IP6 layer on the surface of AuNPs. The IP6 layer not only kept AuNPs within a specific interparticle distance (1-2 nm) by ionic force, it also increased dispersion of AuNPs. On the other hand, irregular AuNPs or prismatic AuNPs formed with 190 μM and 260 μM of IP6. Thus in addition to improve the monodispersity of these anisotropic structures, irregular AuNPs can also be produced by adjusting the IP6 concentration.
In summary, A2 showed more uniform morphology and diameter as compared to A0. A2 exhibited nanoscale interparticle gaps (1-2 nm) between AuNPs, thereby producing very huge “hot spots” effect, leading to greater enhancement of SERS signal. This is a convenient way to fabricate well-dispersed AuNPs, which exhibited interparticle distance of ~1-2 nm, while providing excellent biocompatibility. Therefore, 65 μM of IP6 bound to AuNPs has great potential for further industrial application of SERS biosensing of bacteria or cancer cells.
4. Conclusion
The core-shell structure of AuNPs/IP6 nanohybrids was successfully in situ synthesized by modified Frens method, which was applied in the rapid SERS detection of bacteria. In particular, by reducing HAuCl4 in 65 μM of IP6, the morphology and distribution of AuNPs were greatly improved as compared to the AuNPs without IP6. Furthermore, AuNPs formed in 65 μM of IP6 exhibited enormous “hot spots” effect, leading to greater enhancement of SERS signal. Thus, our works demonstrated a convenient way to fabricate well-dispersed AuNPs that can induce outstanding SERS enhancement that is applicable for label-free detection and biodetection of microbes and cancerous cells.
Conflict of Interests
The authors declare no competing financial interests.
Authors’ Contribution
The paper was written by contributions of all authors. All authors have given approval for the final version of the paper.
Acknowledgments
This work was financially supported by Ministry of Science and Technology of Taiwan (MOST 103-2628-M-001-002 and MOST 103-2221-E-131-019). The authors also thank National Taiwan University for allowing them to use their TEM Lab in order to obtain TEM images for this experiment.