Core-Shell Structure of Gold Nanoparticles with Inositol Hexaphosphate Nanohybrids for Label-Free and Rapid Detection by SERS Nanotechnology

1Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan 2Department of Materials Engineering, Ming Chi University of Technology, New Taipei City 24301, Taiwan 3Center for Condensed Matter Sciences, National Taiwan University, Taipei 10617, Taiwan 4Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan 5Department of Physics, National Taiwan University, Taipei 10617, Taiwan


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][3][4], because they produce localized surface plasma resonance (LSPR), which can increase the intensity of the Raman signal by at least 10 9 .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][6][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][3][4].Therefore these materials showed promising potential in the application of SERS [8,9] and biosensing [10,11].
Inositol hexaphosphate (IP 6 ) 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 IP 6 contains six phosphate acid groups (negatively charged) which are able to link with metal particles and have good absorption capability.Therefore we will employ IP 6 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 HAuCl 4 to produce monodispersed AuNPs.In this present work, IP 6 was added during the reduction procedure and its adsorption onto the AuNPs led to the final product AuNPs/ IP 6 .The final product will be further tested in the application of SERS for detecting microorganism Staphylococcus aureus.

TEM of the AuNPS of the Gold
Colloids.An aliquot of 5 L 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.

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/IP 6 .Raman microscope (HR800, Horiba, Japan) with He-Ne laser (632.8 nm) was used to detect the presence of S. aureus (ATCC 6538P).50 L of the varied AuNPs/IP 6 and 50 L of S. aureus (1 × 10 5 cfu/mL grown for 18 h at 37 ∘ C) were placed in 1.5 mL microcentrifuge tubes and mixed well.Then 5 L 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.

Characterization Analysis of
AuNPs/IP 6 .The interaction between AuNPs and IP 6 samples were analyzed by X-ray photoelectron spectroscope (XPS, VG ESCA Scientific, Theta Probe) and surface electric properties of AuNPs/IP 6 samples were analyzed by zeta potential analyzer (Nano S90, Malvern Instruments) as described below.without IP 6 .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.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 M of IP 6 showed more uniform morphology and diameter as compared to the traditional process of thermal citrate reduction method without addition of IP 6 .

TEM Analysis of the Gold Colloids.
The IP 6 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/IP 6 formed irregular structures [14][15][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 IP 6 increased from 190 M to 260 M.The morphology of AuNPs changed to spherical structures again when the concentrations of IP 6 reached 320 M, which displays a small second peak at 655 nm and just little irregular shaped AuNPs were found.4 and Table 3, the exhausted time increased greatly with the addition of 190 and 260 M of IP 6 , as compared to the addition of the 26∼130 M of IP 6 .The calculation method for temporal evolution curve of HAuCl 4 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 IP 6 was not reactive enough to produce AuNPs and might go through nucleation twice to cause aggregation; (2) IP 6 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.

Exhausted Time and
Figure 5 shows that with the addition of 26 and 65 M of IP 6 , the initial reaction rate was much faster as compared to 0 M of IP 6 ; therefore HAuCl 4 can be exhausted immediately to develop spherical shaped AuNPs.With the addition of 190 and 260 M of IP 6 , the initial reaction rate was slower than those of 0∼130 M of IP 6 , because the reactivity of auric salt decreased with the increasing of pH of the solution [18,19] due to increase of IP 6 concentration.With the addition of 320 M of IP 6 , 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 IP 6 concentration on the morphology of AuNPs.26 and 65 M of IP 6 caused the reaction to develop faster as compared to formation of AuNPs without the addition of IP 6 .With the addition of 190 and 260 M of IP 6 , the formation of AuNPs is slow along with formation of irregular shaped AuNPs as mentioned in Section 3.4.When the concentrations of IP 6 were increased to 320 M, 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/IP 6 .Figure 7 shows the SERS spectra of S. aureus using AuNPs/IP 6 .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 280040 ± 74600 and was about 40 times that of A0 (7705 ± 3295).Therefore the AuNPs reduced with the presence of 65 M of IP 6 would enhance the SERS signal more than that without the addition of IP 6 .This is also confirmed with the TEM images in Figure 3, where the addition of 65 M of IP 6 produced most visible IP 6 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/IP 6 .The interaction between AuNPs and IP 6 was further investigated by zeta potential and XPS analysis, as shown in Figure 10. Figure 9 shows that the zeta potential decreased from −42.9 ± 2.15 to −54.83 ± 1.22 mV when the concentrations of IP 6 increased from 0 to 65 M.Thus the negative charge on the surface of AuNPs increased with increasing IP 6 concentrations and higher IP 6 will induce more IP 6 molecules absorbed on the surface of AuNPs.XPS analysis showed 0.4 eV and 0.3 eV binding energy shifting of Au by electrostatic force.Therefore, IP 6 definitely formed a layer on the surface of AuNPs with 65 M of IP 6 .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 M of IP 6 was an ideal concentration leading to formation of IP 6 layer on the surface of AuNPs.The IP 6 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 M and 260 M of IP 6 .Thus in addition to improve the monodispersity of these anisotropic structures, irregular AuNPs can also be produced by adjusting the IP 6 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 welldispersed AuNPs, which exhibited interparticle distance of ∼1-2 nm, while providing excellent biocompatibility.Therefore, 65 M of IP 6 bound to AuNPs has great potential for further industrial application of SERS biosensing of bacteria or cancer cells.

Conclusion
The core-shell structure of AuNPs/IP 6 nanohybrids was successfully in situ synthesized by modified Frens method, which was applied in the rapid SERS detection of bacteria.In particular, by reducing HAuCl 4 in 65 M of IP 6 , the morphology and distribution of AuNPs were greatly improved as compared to the AuNPs without IP 6 .Furthermore, AuNPs formed in 65 M of IP 6 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.

Figure 4 :
Figure 4: Temporal evolution of HAuCl 4 concentration for the reaction with different IP 6 concentrations as labeled.

Figure 5 :
Figure 5: Effect of IP 6 concentration on the initial reaction rates of HAuCl 4 .

Figure 9 :Figure 10 :
Figure 9: Effect of IP 6 concentration on the zeta potential of AuNPs/IP 6 .
Inositol hexaphosphate (IP 6 ), sodium citrate dihydrate (Na 3 Ct⋅2H 2 O), hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4 ⋅3H 2 O), and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich.Nitric acid (HNO 3 ) 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.Table1lists the solution for preparing AuNPs/IP 6 by mixing 0.01% HAuCl 4 with 1.0 mM IP 6 stock solution.When the solutions were boiling for 10 min, 3.5 mL of 1% 2.1.Materials.

Table 1 :
The solution compositions for the preparation of HAuCl 4 /IP 6 .

Table 2 :
Wavelength and absorbance of AuNPs/IP 6 in different concentrations of IP 6 .

Table 3 :
Figure 2: Effect of concentrations of IP 6 on the size distributions of AuNPs/IP 6 .Dependence of exhausted time of HAuCl 4 /IP 6 on the concentration of IP 6 .
Initial Reaction Rate of Gold Colloids.The reason why the morphology of AuNPs became irregularly shaped when the concentration of IP 6 reaches 190 and 260 M was because IP 6 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 IP 6 caused the pH of the solution to increase and [AuCl 4 ] − is converted to a less reactive [AuCl  (OH) 4− ] − .Therefore at higher pH, [OH − ] will increase and react with [AuCl 4 ] − , forming a less reactive [AuCl  (OH) 4− ] − substance.Therefore higher concentration IP 6 will cause the exhausted time of HAuCl 4 /IP 6 to increase.As shown in Figure 7/2 (84.5 to 84.9) and Au 5/2 (88.1 to 88.4) from 0 M to 65 M of IP 6 addition, respectively.This showed that IP 6 interacted with Au + NPs The mechanism of AuNPs formation with different IP 6 concentration.