Ordering of Hollow Ag-Au Nanospheres with Butterfly Wings as a Bio-template

A biological template strategy is implemented for the fabrication of hollow noble metal composite nanospheres within the ordered array nanostructures by introducing butterfly wings to some convenient technique procedure. Butterfly wings are activated by ethylenediamine to increase the reactive sites on the chitin component, on which Ag nanoparticles are in situ formed and serve as “seeds” to direct further incorporation during the following impregnation procedure. Butterfly wings could function as bio-substrate to provide an ordered array and regulate the synthesis process by providing active reaction sites (e.g. -CONH- and -OH). Thus, hollow Ag-Au nanospheres are loaded on the wings’ surface layer and inside the ordered array nanostructures homogeneously, which would have potential applications in surface enhanced Raman scattering (SERS).

their special 3D periodic sub-microstructures 25 . These 3D sub-microstructures including ridges, ribs and struts, are further divided up to numerous periodic window structures by horizontal and vertical struts between the parallel-aligned ridges. The main components of wings include chitin and a small amount of protein, which can provide some reaction hotspots 26 . Figure 2(a) shows the SEM images of intermediate product: Ag/wing. As can be seen, the intermediate product is covered with a layer of particles distributing on the "ridge" and "strut" uniformly, rougher than that of the original wings in Fig. 1. The mapping image of the intermediate product can be find in Figure S2 in supplementary information. As revealed by the HRTEM images in Fig. 2(b), the particles are nearly round with diameter 5-10 nm. The corresponding SAED patterns ( Fig. 2(b) inset image) match well with the face-centered cubic (fcc) Ag structure reported in JCPDS cards 040783, and the relevant planes are indexed as (1 1 1), (2 0 0), (2 2 0), (3 0 0), and (3 1 1). Besides, HRTEM image in Fig. 2(b) exhibits clear lattice fringes with d spacing 0.23 nm, corresponding to the (1 1 1) plane of fcc Ag structure, which indicates that (111) plane is the most stable crystal plane. The less dark areas among the nanoparticles are wing scales. The structure of wing scale keeps intact and nano-Ag are embedded in wing scales matrix. Hollow Ag-Au nanospheres (Fig. 2(c)) on the wing scales are prepared based on the wing-Ag product. The distribution of hollow Ag-Au nanosphere on butterfly wings is investigated by FESEM and TEM observation. It can be seen that about 60 nm nanospheres distribute on the "ridge" and "strut" uniformly.   Overall, about 60 nm hollow nanospheres with the ordered array nanostructures are synthesized with 5-10 nm fcc Ag in-situ grown on the surface of wings as "seeds". Figure 3(a) presents a typical hollow Ag-Au nanosphere incorporated into butterfly wings structure. Line scanning ( Fig. 3(b)) exhibits a board peak of Au and Ag in the center and two intensive peaks on both sides, further confirming that Au and Ag tend to be distributed at the periphery and form a hollow structure internally. TEM images in Fig. 3(a) show that the thickness of Ag-Au shell is about 10 nm. Atomic percentage of Au and Ag are 44.2% and 55.8%, respectively, according to the semi-quantitatively analysis. The concentration of HAuCl 4 is about 0.2 wt% and one can speculate that the thickness and element content of hollow Ag-Au nanospheres could be regulated by the concentration of HAuCl 4 solution, as can be seen in Figure S3 in supplementary information. Elemental analyses of hollow Ag-Au nanospheres were performed by energy disperse X-ray analysis (EDX) technique. The mapping images ( Fig. 3(c,d,e)) confirm the presence of elemental Ag and Au in the nanocomposite. Besides, both Ag and Au distribute nearly homogeneously across the nanoparticles. Figure 4(a) shows the XRD patterns of the Ag-Au/wing product compared with Ag/wing and original butterfly wing. The result shows that the natural butterfly wing and the processed butterfly wing exhibit the diffractive peaks of crystalline domains around 20°. It indicates that the samples have the same components of chitin, and chitin doesn't change in the process. In addition, the diffraction pattern of Ag/wing has four peaks at 38.1°, 44.2°, 64.4° and 77.5°, corresponding to (1 1 1), (2 0 0), (2 2 0), and (3 1 1) of face-centered cubic (fcc) structure of Ag (JCPDS file No. 04-0783), respectively. The intensity of (1 1 1) plane is twice the value from standard card (JCPDS file No. 04-0783), which proved that (1 1 1) crystal face is the most stable crystal face of nano-Ag seeds. Line C represents the hollow Ag-Au nanospheres with ordered array nanostructures, the peak intensity increases and the peak position moves to the left slightly compared with Line B, which indicates the formation of Ag-Au nanocomposites. To confirm these results, XPS analyses ( Fig. 4(b)) were performed on the Ag-Au/wings systems. Figure 4(c) shows the XPS Au 4 f spectrum of the Ag-Au/wings systems. The result suggests that Au and Au 3+ coexist in the final product, and the relative contents of Au and Au 3+ are 69.3% and 30.7% respectively. The binding energy of the Au are at 84.1 eV and 87.8 eV, and the splitting between the 4f 7/2 and 4f 5/2 lines is equal to 3.7 eV, corresponding to the binding energy of standard bulk Au0 (84.0 eV and 87.7 eV) 27 . Such shift to higher energy is commonly observed for metallic nanoparticles and is attributed to geometry and/or confinement effects 28 . The XPS Ag 3d spectrum is shown in Fig. 4(d). The Ag 3d spectrum exhibits two contributions, between the 3d 5/2 (374.5 eV) and 3d 3/2 (368.5 eV) lines is equal to 6.0 eV, corresponding to Ag0 electronic state 374.2 eV and 368.2 eV reported in the literature 29 . Overall, the XPS analyses confirm the Ag0 valence state, Au0 and Au 3+ of the Ag-Au nanospheres.
FTIR spectra were performed to investigate the mechanism of synthesis of Ag-Au nanoparticles and the participation role of butterfly wing template. ATR-FTIR spectra ( Fig. 5(a)) of the original butterfly wings show peaks around 1626 cm −1 and 1541 cm −1 , assigned to the amide I band of chitin due to C=O bending vibration   The peak at 1058 cm −1 of -OH stretching vibration shifts to a wider peak at 1069 cm −1 , which further indicates the possible existence of coordination reaction between -NH 2 and Ag ions. Butterfly wings acts as a substrate by binding [Ag (NH 3 ) 2 ] + with coordination reaction sites -CONH-and -OH.
According to the ATR-FTIR spectra results, we figure out the schematic procedures for butterfly wings to regulate the fabrication of hollow Ag-Au nano-spheres (Fig. 6). The original butterfly wings include active functional groups (such as -CONH-and -OH). After activation with ethylenediamine, more functional groups (-NH 2 , -OH) generate on the butterfly wing. When activated butterfly wings are immersed in silver-ammonia solution, some [Ag(NH 3 ) 2 ] + are trapped on wings by chelation with -OH, -NH 2 and -CONH-sites provided by chitin and other small nano-Ag are formed in-situ on the surface of wings. Here wings act as both the template and supporting substrate. Then [Ag (NH 3 ) 2 ] + trapped on wings are reduced to nano-Ag by reducing agent (Potassium tartrate). The prepared Ag nanoparticles can serve as seeds of synthesis of Au-Ag nanoparticle. During the immersion of Ag/wing in HAuCl 4 solution, Au III is reduced to nano-Au by nano-Ag because the AuCl 4 − /Au (0.99 V vs NHE) is more positive than that of AgCl/Ag (0.22 V vs NHE).
We hypothesize the mechanism shown in Fig. 7 for hollow Au-Ag nanospheres' formation. The produced Au and Ag are distributed to the surface of nanosphere and interior is AgCl when immersing the Ag-wings sample into HAuCl 4 solution. Hollow Au-Ag nanosphere within the ordered array nanostructures are produced after AgCl was further dissolved. We begin by describing the mechanism for nanosphere's formation. When Ag nanoparticles with (1 1 1) crystal faces (Fig. 2) react with a small amount of HAuCl 4 solution, Ag dissolution occurs at all cube corners for Ag nanoparticles 14 since the (1 1 1) corners are more stable in comparison to the other faces. As the reaction proceeds, Ag is oxidized and electrons are stripped, defects are introduced into the structure due to reaction stoichiometry: three Ag atoms are removed with deposition of one Au atom (3Ag + HAuCl 4 → 3AgC l + Au + HCl) 31 . The released electrons migrate to the nanoparticles faces and are captured by AuCl 4 − , generating Au atoms that grow on the nanoparticles epitaxially and silver chloride precipitates come into being at the same time. Thus, Ag/Au/AgCl nanoparticles are produced given the mutual solubility of Cl, Ag and Au and the high diffusion rates at the reaction temperature. Finally, the AgCl precipitate is removed and hollow Ag-Au nanospheres remained incorporating into the ordered array nanostructures within butterfly wing.
The SERS property of the Ag-Au/wing material was tested at 532 nm wavelength by Senterra R200-L dispersive laser confocal Raman spectrometer using Rhodamine 6 G (R6G) as a Raman signal molecule. We judge the Raman properties by the intensity of the signal peak. Figure 8(a) is comparison of Raman signal from R6G at concentration of 10 −3 mol/L on original butterfly wing, Ag/wing, Ag-Au/wing. One can see that the corresponding curve of the  original butterfly wing is relatively smooth, which indicates that the original butterfly wing has no enhancement of the Raman signal from R6G. Ag/wing and hollow Ag-Au/wing can detect R6G signal, and the signal of hollow Ag-Au/wing is slightly stronger. Figure 8(b) shows that the hollow Ag-Ag/wing substrate can detect the Raman signal from R6G at concentration of 10 −6 mol/L successfully, which is 10 2 times higher than that of Ag/wing. The limit of detection for R6G is calculated to be 8.67 × 10 −10 mol/L using the method reported by C. Zhu et al. 32 and Figure S5 in supplementary information. Here, the advanced SERS performance of hollow Ag-Au/wings probably results from the following factors. First, hollow Ag-Au particles generate more hotspots, resulting in an enhanced SERS effect 13 . Second, the hotspots formed by the combination of Ag-Au nanoparticles with the micro-nanostructure of the butterfly wing also contribute to the improvement of SERS properties. Therefore, the materials with hollow structures and micro-nano structures may have great potential as new SERS substrates.

Conclusions
A facile synthesis strategy is used to fabricate Ag-Au composite with ordered array nanostructures within butterfly wings and control the constituent units to be hollow Au-Ag nanospheres. Butterfly wing scales function as bio-template to stabilize the nanocrystal and provide active functional groups (-CONH-and -OH) on which nanoparticles can in situ grow. The decomposition and reduction route provides a new method for synthesis of hollow metallic spheres. Hollow Ag-Au nanospheres with ordered array nanostructures are produced by the diffusion of Cl, Ag and Au with participation of butterfly wings, which would have significant potential applications such as SERS substrates. The synthetic technique and mechanistic understanding may provide new opportunities to design and fabricate heterogeneous nanostructures with interesting physicochemical properties.

Experimental
In a typical process, butterfly wings were first immersed in diluted nitric acid (13 vol. %) for 1 h and then in ethylenediamine for 4 h (amination) to remove mineralized impurities and activate. Immerse the aminated samples in an aqueous solution of [Ag(NH 3 ) 2 ] OH (0.03 mol/L) for 1 h, then expose in 50 g/L aqueous solution of potassium sodium tartrate for 30 min. Ag-NP seeds were formed on the aminated bio-surface. Immerse the Ag-NP-functionalized butterfly wings into HAuCl4 (0.2 wt%) solutions at 90 °C. Immerse product into ammonia for 2 h at room temperature, rinse them with deionized water, and dry in air. The whole process should be in the dark to avoid the influence of the light. All chemicals were of analytical grade from the Sinopharm Chemical Reagent Co., Ltd. and were used as starting materials without further purification.
X-ray diffraction (XRD) patterns were carried out on Ultima IV instrument operating at a voltage of 40 kV and a current of 30 mA. X-ray photoelectron spectroscopy (XPS) studies were recorded on AXIS Ultra DLD spectrometer using standard Al Kα X-ray source (150 W) and an analyser pass energy of 20 eV. Samples were mounted using double-sided adhesive tape and binding energies were referenced to the C 1 s peak at 284.8 eV of the surface adventitious carbon. FESEM images were recorded on S-4800 field emission scanning electron microscope operated at an acceleration voltage of 10 kV. FETEM were performed on a JEOL JEM-2100F instrument operated at an acceleration voltage of 200 kV. FT-IR spectra were obtained by NICOLET 6700. The Raman signal of the sample was measured by a dispersive laser confocal Raman spectrometer Senterra R200-L.