In situ formation of gold/silver bi-metal nanodots on silica spheres and evaluation of their microbicidal properties

Abstract

An environmentally benign synthetic method of seedless and one-step growth of 2–4 nm sized gold/silver (Au/Ag) bi-metal nanodots (BNDs) on preformed amine functionalized silica (SiO₂) spheres via co-reduction of metal precursors at room temperature and their antimicrobial activity are reported. The resulting BNDs on SiO₂ spheres (SiO₂@/Au/Ag) exhibited a distinctive surface plasmon resonance (SPR) band between the Ag and Au nanoparticle (NP) SPR absorption regions. The deposition of BNDs on SiO₂ spheres was confirmed from the high resolution transmission electron microscopy (HRTEM) and X-ray photoelectron spectroscopy (XPS) analyses. The SiO₂@Au/Ag BNDs with different concentrations of Au and Ag (SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅ BNDs) exhibited a broad-spectrum of antimicrobial activities involving significant growth inhibition of Gram negative bacterial strains (Escherichia coli, Pseudomonas aeruginosa), a Gram positive bacterial strain (Bacillus thuringiensis) and a fungal strain (Trichoderma longibrachiyatum MKU-1). The mechanism of interaction between the BNDs and microbial strains was analyzed through scanning electron microscopy (SEM). The results confirmed that the treated microbial cells were damaged and have pits on the cell surface. The results evidently demonstrated the antimicrobial activity of the SiO₂@Au/Ag BNDs even at lower concentrations of Ag (SiO₂@Au₇₅/Ag₂₅), which may be suitable for the formation of potential microbicidal materials.

---

Introduction

Metal nanostructured materials with controlled size and shape have received great scientific and technological interest in the fields of catalysis and sensing over the years due to their unique optical, electronic and catalytic properties.^1–3 Recently, bi-metal nanoparticles (NPs), in particular gold/silver (Au/Ag) were found to show numerous potential applications due to their distinctive catalytic properties compared to their mono-metal counterparts.^4,5 A number of synthetic procedures have been explored for the synthesis of Au/Ag bi-metal NPs by various research groups.^6–9 Huang et al.¹⁰ reported the preparation of luminescent Au/Ag bi-metal nanodots (BNDs) protected by 11-mercaptoundecanoic acid. Similarly, the syntheses of hybrid bi-metal Au/Pt¹¹ and bi-metallic Au/Ag NPs supported on silica (SiO₂)^4,12 and their catalytic applications were discussed. The SiO₂ supported metal NPs are gaining significant interest due to their stability and enhanced optical and catalytic properties.^13,14 However, only limited reports have been published about the synthesis of Au/Ag BNDs and their applications. Generally, seed-mediated growth method is preferred for the synthesis of Au/Ag bi-metallic NPs.¹⁵ In this, Au seeds are produced first by reducing the corresponding metal ions with suitable reducing agent like NaBH₄ and in the subsequent step AgNO₃ is introduced along with suitable surfactants which lead to the formation of Au/Ag nanomaterials via a two-step process. The resulting nanomaterials are surrounded by surfactants that can hinder the direct interaction of analytes especially for biological applications. In addition, the use of reagents like NaBH₄ and surfactants are not in accordance with the principles of green chemistry. Hence, in the present work a single step facile route was employed to prepare sub-4 nm sized Au/Ag BNDs on amine functionalized SiO₂ spheres (SiO₂@Au/Ag BNDs). A modified Stober method¹⁶ was employed for the preparation of amine functionalized SiO₂ spheres and on this SiO₂, Au/Ag BNDs were deposited by in situ method in the presence of amine functionalized silicate sol–gel (SSG) matrix. Herein, N-[3-(trimethoxysilyl) propyl] diethylenetriamine (TPDT) acts as both reducing as well as stabilizing agent for the in situ growth of Au/Ag BNDs.

Interestingly, the bi-metallic core–shell nanostructures and other classes of core–shell NPs have been explored for widespread applications in bio-imaging, drug delivery, cell labelling and catalysis.¹⁷ In addition, bi-metallic Au/Ag NPs have been known as potential antimicrobial agents.¹⁸ At the same time, the antimicrobial properties of BNDs containing Ag supported on SiO₂ need to be explored, because the infection caused by microbes, emergence of new pathogens, increasing number of multiple antibiotic/drug resistant (MDR) strains and the continuing emphasis on health care cost led the scientists to develop new effective antimicrobial agents. Based on these view points, the SiO₂@Au/Ag BNDs were prepared and the studies showed excellent antimicrobial effects against Gram negative bacterial strains (Escherichia coli, Pseudomonas aeruginosa), Gram positive bacterial strain (Bacillus thuringiensis) and fungal strain (Trichoderma longibrachiyatum MKU-1). The selected organisms have great ecological importance and the risk associated with them is highly relevant to practical applications.

Materials and methods

Chemicals, microorganisms and cultivation conditions

Gold(III) chloride hydrate (HAuCl₄), silver nitrate (AgNO₃), tetraethylorthosilicate (TEOS) and TPDT were received from Sigma-Aldrich. Bacterial strains Escherichia coli DH5α, Pseudomonas aeruginosa MTCC no. 7925, Bacillus thuringeinsis MTCC no. 6905 and fungus Trichoderma longibrachiyatum MKU-1 from Department of Genetics, School of Biological Sciences, Madurai Kamaraj University were used as model organisms in antimicrobial studies. Cells were cultivated at 37 °C in 2 L Erlenmeyer flasks containing 100 mL of Lysogeny Broth (LB) (which is composed of 10 g L⁻¹ of peptone and NaCl and 5 g L⁻¹ of yeast extract) supplemented with 2% (w/v) bacto agar as solidifying agent. Fungal organism Trichoderma longibrachiyatum MKU-1 was grown on Potato Dextrose Agar (PDA) at 28 °C for 72 h. The Milli-Q water was used for preparation of solutions (all glassware used for the preparation of SiO₂@Au/Ag BNDs was thoroughly cleaned with aqua regia and rinsed extensively with doubly distilled water prior to use).

Synthesis of SiO₂@Au/Ag BNDs

The TPDT functionalized SiO₂ spheres were prepared by modified Stober method.¹⁶ Briefly, ammonium hydroxide (1.7 mL; 25–28%) was added to ethanol (50 mL) along with TEOS (1.5 mL) and water (1 mL). The solution was vigorously stirred and after 3 h an additional 1 mL of TEOS was added. The SiO₂ spheres were obtained after 12 h of stirring and then TPDT (0.4 mL) was added to the solution and the stirring was continued for another 6 h. The TPDT functionalized SiO₂ particles were purified twice by centrifugation and re-dispersion in water (40 mL). For the preparation of SiO₂@Au/Ag BNDs, about 5 mg of preformed amine functionalized SiO₂ was added to 5 mM TPDT silane present in 5 mL of water and the solution was sonicated for 5 min. To this solution, HAuCl₄ and AgNO₃ (each 0.5 mM final concentration for Au₅₀/Ag₅₀ BNDs) were added and the solution was stirred for 12 h. The observed of brownish red color indicated the formation of Au/Ag BNDs on SiO₂ spheres (SiO₂@Au₅₀/Ag₅₀ BNDs). The same synthetic procedure was followed for the preparation of SiO₂@Au₂₅/Ag₇₅ and SiO₂@Au₇₅/Ag₂₅ BNDs.

Characterization of the samples

Absorption spectra of the SiO₂@Au/Ag BNDs were recorded on an Agilent Technologies 8453 spectrophotometer. The HRTEM analysis was performed on a JEOL JEM 2100 instrument operating at 200 kV. Specimen for HRTEM analysis was prepared by placing a drop of fresh BNDs solution on carbon coated copper grid and then evaporating the solvent under vacuum. The Au and Ag contents present in the Au/Ag BNDs were measured using inductively coupled plasma spectrometry (ICP-MS, PerkinElmer Optima 5300DV). X-ray photoelectron spectroscopy (XPS) analysis was performed using a Kratos AXIS Ultra DLD X-ray photoelectron spectrometer with a monochromatic Al Kα X-ray source (hν = 1486.69 eV) and a hemispherical analyzer. Survey spectrum was obtained with 160 eV pass energy and 0.7 eV step size. High resolution spectra were collected with 40 eV pass energy and 0.05 eV step size. The analysis chamber was maintained at pressure in the 1 × 10⁻⁸ Torr range throughout the data collection. The spectra were collected at a photoelectron take-off angle of 90°. The binding energies were aligned with reference to the C 1s hydrocarbon carbon peak at 285 eV. The SEM analysis of microorganisms treated with the BNDs was performed on Quanta250 FEG and VEGA3 TESCAN instruments. Microorganisms treated with 100 μL (1 mM Au/Ag BNDs) of as prepared BNDs were harvested by centrifugation at 8000 rpm for 10 min at 4 °C after overnight incubation. The pellet was washed twice with 0.5 M NaCl and sterile doubly distilled water. The washed cells were subjected to 30, 50, 70 and 90% absolute ethanol treatment. Finally, the pellet was suspended in 95% absolute ethanol. Then the samples were drop-casted on indium titanium oxide (ITO) coated glass plate and dried at room temperature prior to SEM analysis.

Antimicrobial activity by well diffusion method

Antimicrobial activity of SiO₂@Au/Ag BNDs (SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅) was experimentally verified by disc diffusion method. The sterile filter paper discs of 10 mm were dripped with 100 μL (1 mM Au/Ag BNDs) of SiO₂@Au/Ag BNDs solution and allowed to dry completely. The pure sub-cultured Gram negative (E.coli, P.aeruginosa), Gram positive (B.thuringeinsis) and fungal strain (T.longibrachiyatum MKU-1) were swabbed uniformly on the individual LB and PDA plates using sterile cotton swab and then the disks with BNDs were placed, opposing the side with BNDs to the microorganism. Sterile water (3 μL) was added above the disk in order to stick them to the agar. Petri plates swabbed with microbes and the filter disk with sterile water alone was used as negative control. The respective bacterial and fungal plates were incubated at 37 °C/28 °C for 24 h after which the different levels of zone of inhibition surrounding the disk were determined. Each experiment was performed at least three times to confirm the reproducibility.

Preservation of microbial cultures

The bacterial cultures to be preserved were streaked on nutrient agar slant and incubated at 37 °C overnight and then refrigerated at 4 °C with routine sub-culturing for every 1 or 2 months for further use. The purity of the strain was ascertained by standard microbiological methods.¹⁹ The fungal culture storage was done at −80 °C.

Results and discussion

TEM, absorption spectra and XPS analyses of SiO₂@Au/Ag BNDs

The morphology of SiO₂@Au/Ag BNDs was analyzed by HRTEM. The HRTEM images recorded for SiO₂@Au₅₀/Ag₅₀ BNDs at different magnifications (Fig. 1A–C) revealed that the Au₅₀/Ag₅₀ BNDs are effectively attached and uniformly distributed on the SiO₂ spheres. The approximate size of SiO₂ spheres was found as 250 nm. The average particle size of Au₅₀/Ag₅₀ BNDs was estimated to be 3.6 nm and they were comfortably accommodated on the TPDT functionalized SiO₂ surface due to their interaction with the –NH₂ groups present in TPDT. The SiO₂ provides stable accommodation sites for the Au₅₀/Ag₅₀ BNDs and prevents the aggregation among them and leads to the formation of uniformly distributed Au₅₀/Ag₅₀ BNDs. Furthermore, the EDX analysis (Fig. 1D) showed the signatures of the elemental Au and Ag. The well identified peaks observed in the EDX spectrum confirm the presence of Au and Ag in Au₅₀/Ag₅₀ BNDs on SiO₂. From the ICP-MS analysis, the ratio of Au-to-Ag present in the Au₅₀/Ag₅₀ BNDs was found to be 48.79 : 51.2. The HRTEM images help us to analyse whether the BNDs are in alloy or core–shell form. Usually, TEM images of bimetallic Au/Ag core–shell NPs show a banding in electron density with the dark and bright regions for gold and silver, respectively.²⁰ The HRTEM images of BNDs (Fig. 1C) prepared in this work did not show any banding and showed uniform contrast on BNDs. This feature points out that the scattering was distributed evenly throughout the particle volume, which is only possible if the BNDs are alloy. The HRTEM images for SiO₂@Au₂₅/Ag₇₅ and SiO₂@Au₇₅/Ag₂₅ BNDs were obtained at different magnifications and are shown in Fig. S1.† From the images, it is observed that the Au₂₅/Ag₇₅ and Au₇₅/Ag₂₅ also formed as BNDs on SiO₂ spheres and the average particle sizes were found to be 3.3 and 3.2 nm for Au₂₅/Ag₇₅ and Au₇₅/Ag₂₅ BNDs, respectively.

Fig. 1 HRTEM images of SiO₂@Au₅₀/Ag₅₀ BNDs at different magnifications (A, B and C) and the corresponding EDX spectrum (D).

The absorption spectra for mono-metallic SiO₂@Au NPs, SiO₂@Ag NPs and bi-metallic SiO₂@Au/Ag BNDs with different molar ratios of Au and Ag are shown in Fig. 2A. The SPR absorption bands observed at 533, 420 and 475 nm correspond to SiO₂@Au NPs, SiO₂@Ag NPs and SiO₂@Au₅₀/Ag₅₀ BNDs, respectively. The position of the SPR band for SiO₂@Au₅₀/Ag₅₀ BNDs was observed in between the SPR band positions of SiO₂@Au NPs and SiO₂@Ag NPs. The distinctive SPR band and noticeable color change of the solution (inset of Fig. 2) suggest that the Au₅₀/Ag₅₀ BNDs are actually formed and they are not the physical mixtures of the mono-metallic Au and Ag NPs. Furthermore, a single SPR band observed for SiO₂@Au₅₀/Ag₅₀ BNDs indicates that the formed Au₅₀/Ag₅₀ BNDs may be in alloy form.²¹ From the previous reports,^22,23 it is well understood that the observation of two peaks in the absorption spectrum is due to the formation of either mixed, single metal or core–shell metal NPs. Moreover, a single absorption peak is the indication for the formation of either single metal NPs or bimetallic alloy NPs. Moreover, the position of the SPR band of SiO₂@Au₅₀/Ag₅₀ BNDs (475 nm) demonstrated that the obtained Au₅₀/Ag₅₀ BNDs are in alloy form. In this report, both Au and Ag precursors are co-reduced by the –NH₂ groups present in the TPDT and the SSG matrix provides a good stabilization matrix for the BNDs and no other external reducing or stabilizing agents were used for the growth of BNDs. The prepared BNDs were stable for more than a month without any noticeable change in the intensity of SPR band. For the first time, we report a facile and environmentally benign synthetic method to decorate SiO₂ with Au/Ag BNDs without adding any external reducing and protecting reagents.

Fig. 2 Surface plasmon absorption spectra of SiO₂@Au NPs, SiO₂@Ag NPs and SiO₂@Au/Ag BNDs with different% molar compositions of Au and Ag (A) and the plot of SPR band against different SiO₂@Au/Ag BNDs (B).

The formation mechanism of Au/Ag BNDs on SiO₂ via seedless one-step process may be explained as follows: the Au³⁺ ions are first reduced in the mixture of AuCl₄⁻ and Ag⁺ when compared to Ag⁺ ions due to the lower reduction potential of AuCl₄⁻/Au (0.99 vs. SCE for AuCl₄⁻/Au, 0.80 vs. SCE for Ag⁺/Ag). In a mixture, Ag⁺ also gets reduced by the competitive reduction and the formed Ag NPs may favor the reduction of AuCl₄⁻ by the following galvanic replacement reaction.²⁴

3Ag(s) + HAuCl₄(aq) → Au(s) + 3AgCl(s) + HCl(aq)

The formed AgCl may cause the morphological transformation to produce irregular shaped NPs. In other way, the formation of AgCl is inhibited by the formation of ammine chloride complex between AuCl₄⁻ and amine groups present in the TPDT SSG matrix.²⁵ By this way, the growth of BNDs is controlled in the present system. The reduction of both Au and Ag ions occurs in a competitive fashion and the formed BNDs may be in alloy form. When Au and Ag ions are reduced simultaneously in the mixture solution, Au/Ag alloy particles formation occurs.²⁶ The alloy nature of the BNDs is further supported by the SPR band shift with respect to the concentrations of Au and Ag.^21,26,27 The SPR band of SiO₂@Au NPs was blue shifted by the influence of Ag (Fig. 2B). This blue shift confirmed the formation of Au/Ag alloy BNDs on the SiO₂ spheres. The schematic representation of Au/Ag alloy BNDs formation on SiO₂ spheres is shown in Scheme S1.†

The existence and the chemical states of elements present in the SiO₂@Au₅₀/Ag₅₀ BNDs were further analysed by XPS and the results are shown in Fig. 3. The XPS peaks observed at 84.6 and 88.3 eV (Fig. 3A) are assigned to the Au 4f_7/2 and Au 4f_5/2 peaks, respectively for metallic Au²⁸ and the peaks at 368.7 and 372.3 eV (Fig. 3B) are assigned to the Ag 3d_5/2 and Ag 3d_3/2 peaks, respectively for metallic Ag.²⁸ These results demonstrated that Au and Ag are present in the form of zero-valent Au and Ag in SiO₂@Au₅₀/Ag₅₀ BNDs. The Si 2p spectrum (Fig. 3C) showed two peaks at 99 and 102.5 eV corresponding to Si 2p and particularly, the peak observed at 102.5 eV is due to presence of Si in SiO₂.²⁹ The peak observed at 402 eV in N 1s spectrum (Fig. 3D) is assigned to the ‘N’ present in the TPDT SSG matrix. The XPS analysis showed that the signals of Au 4f and Ag 3d peaks are stronger than that of N 1s and Si 2p signals. From this observation, it is understood that the Au₅₀/Ag₅₀ BNDs are present at the top of the SiO₂ surface. Since the N 1s spectrum showed a relatively stronger signal than Si 2p spectrum it confirmed the functionalization of TPDT SSG on the SiO₂ surface. The –NH₂ groups present in the TPDT served as anchoring sites for BNDs and through which the BNDs bind with SiO₂ surface. The survey of XPS analysis of SiO₂@Au₅₀/Ag₅₀ BNDs is shown in Fig. S2.†

Fig. 3 XPS analysis of Au 4f (A), Ag 3d (B), Si 2p (C) and N 1s (D) recorded for SiO₂@Au₅₀/Ag₅₀ BNDs.

Antimicrobial activity of SiO₂@Au/Ag BNDs

Antibacterial activity. The antibacterial activity of SiO₂@Au/Ag BNDs (SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅) was evaluated against Gram negative (E.coli and P.aeruginosa) and Gram positive (B.thuringiensis) bacterial strains, and their effect on bacterial growth is shown in Fig. 4. Indeed as presented in the Fig. 4, clearing zone around Gram negative bacterial growth was more prominent when compared to Gram positive strain upon treatment with SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅ BNDs. The SiO₂@Au₂₅/Ag₇₅ BNDs exhibited maximum antibacterial activity against E.coli, P.aeruginosa and B.thuringiensis with the clearing zone values of 17, 20 and 16 mm, respectively due to the presence of higher concentration of Ag.^30,31 The antibacterial activity of SiO₂ spheres, SiO₂@Ag and SiO₂@Au NPs against both Gram negative and Gram positive bacterial strains was also evaluated and are shown in Fig. S3.† As the Ag NPs are known to possess antibacterial activity^32,33 the SiO₂@Ag NPs showed clearing zones around Gram negative and Gram positive bacterial growth. The SiO₂@Au NPs showed a minimum activity against E.coli only. Interestingly, the SiO₂@Au/Ag BNDs showed the antibacterial activity against both the bacterial strains even at lower concentration of Ag (SiO₂@Au₇₅/Ag₂₅ BNDs). The clearing zone values of SiO₂@Au₇₅/Ag₂₅ BNDs against E.coli, P.aeruginosa and B.thuringiensis bacterial strains growth were measured as 10, 13 and 10 mm, respectively.

Fig. 4 Images of untreated (control) and treated E.coli, P.aeruginosa and B.thuringeinsis with SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅ BNDs (arrows indicate the growth inhibition zone).

To further confirm the antibacterial activity and the morphological changes of bacterial cells, the SEM analysis was performed using bacterial strains treated with SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅ BNDs (Fig. 5). The SEM micrographs demonstrated that the control cells (bacterial cells not treated with the BNDs) were rod-shaped and cell surface was intact. On the other hand, the cells treated with SiO₂@Au/Ag BNDs exhibited irregular shape (flattened and pits on the cell surface) instead of rod shaped cells. Also, SEM studies demonstrated that the BNDs attached to the bacterial surface which caused membrane damage leading to cell death. Previous reports^33,34 reveal the mode of action of Ag NPs on bacterial strains is that the Ag NPs get attached to the sulfur-containing proteins on the cell wall, leading to increased permeability of the membrane, finally causing cell death. Further, the action of BNDs against Gram negative cells was more prominent than Gram positive cells. This can be explained as the Gram negative cell envelope consists of outer membrane, thin peptidoglycan layer and cell membrane whereas Gram positive cell consists of lipoteichoic acid containing thick peptidoglycan layer and cell membrane.³⁵ Therefore, the BNDs can more easily damage Gram negative cells having a few nanometers thick peptidoglycan layer than Gram positive cells having 30 to 100 nm thickness.

Fig. 5 SEM micrographs of untreated (control) and treated E.coli, P.aeruginosa and B.thuringeinsis with SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅ BNDs (arrows indicate the adherence of BNDs on the cell surface).

Antifungal activity. The antifungal activity of SiO₂@Au/Ag BNDs (SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅) was also evaluated against T.longibrachiyatum, and their effect on fungal growth is shown in Fig. 6. The highest antifungal activity was observed for SiO₂@Au₇₅/Ag₂₅ BNDs with the clearing zone of 10 mm around fungal growth when compared to SiO₂@Au₂₅/Ag₇₅ and SiO₂@Au₅₀/Ag₅₀ BNDs with clearing zone values of 6 and 7 mm, respectively. The SiO₂@Ag NPs (Fig. S4b†) exhibited a minimum antifungal activity while SiO₂ spheres (Fig. S4a†) and SiO₂@Au NPs (Fig. S4c†) did not show any activity. The observed antifungal activity in SiO₂@Au/Ag BNDs can be ascribed to the combination of Au and Ag metals and more specifically to the formation of Au/Ag BNDs. The SEM analysis of fungal strain treated with SiO₂@Au/Ag BNDs showed the presence of BNDs on the cells, the cell membrane was well fragmented with many pits and gaps (Fig. 7). All the three different compositions of Au and Ag (SiO₂@Au₂₅/Ag₇₅, SiO₂@Au₅₀/Ag₅₀ and SiO₂@Au₇₅/Ag₂₅) exhibited good antifungal activity. On the other hand, the control cells were smooth and intact and some filaments around the cells were obvious and clear (Fig. 7(control)). The control plates (SiO₂@Ag and SiO₂@Au NPs) demonstrated the absence of clearing zone because of the strong cell wall composition of fungus (Fig. S4†). The cell wall of the fungus consists of structural components of chitin microfibrils, chitosan in Zygomycota, β-linked glucans and Mannoproteins.³⁶ This strong cell wall provides a very few anchoring sites for NPs thus leading to difficulty in penetration. But in the case of SiO₂@Au/Ag BNDs, the sub-4 nm sized Au/Ag BNDs formed on SiO₂ might anchor the fungus walls and caused the cell damage. Moreover, due to the presence of SiO₂ support the release time of Au/Ag BNDs can be delayed for a long time so that SiO₂@Au/Ag BNDs have great effects for antimicrobial activity.³⁷

Fig. 6 Images of untreated (control) and treated T.longibrachiyatum MKU-1 with SiO₂@Au₂₅/Ag₇₅ (a), SiO₂@Au₅₀/Ag₅₀ (b) and SiO₂@Au₇₅/Ag₂₅ (c) BNDs (arrows indicate the growth inhibition zone).

Fig. 7 SEM micrographs of untreated (control) and treated T.longibrachiyatum with SiO₂@Au₂₅/Ag₇₅ (a), SiO₂@Au₅₀/Ag₅₀ (b) and SiO₂@Au₇₅/Ag₂₅ (c) BNDs.

In the present work, an eco-friendly SiO₂ sphere was used as support material for the accommodation of Au/Ag BNDs on its surface and also it helps to avoid the aggregation of metal nanoparticles. The amine functionalized TPDT matrix provided anchoring sites for BNDs and the TPDT acted as reducing and stabilizing agent for the formation of BNDs. Interestingly, the Au/Ag bimetal was formed as BNDs on SiO₂ sphere in a single step without employing any external reagents or multi-step synthetic protocols. The so formed SiO₂@Au/Ag BNDs showed microbicidal property not only against Gram negative and Gram positive bacterial strains but also against fungal strain.

Conclusion

In conclusion, a facile and environmentally benign synthetic method for the formation of 2–4 nm sized Au/Ag BNDs on amine functionalized SiO₂ spheres was presented. The amine functionalized silicate served as both reducing and stabilizing agent for the in situ formation of BNDs. The presence of Au/Ag BNDs on the SiO₂ spheres was confirmed by the HRTEM images, EDX and XPS analyses. The formed BNDs were confirmed as alloy of Au and Ag by absorption spectral studies. The SiO₂@Au/Ag BNDs were tested for the antimicrobial activity against Gram negative, Gram positive bacterial strains and fungal strain. The BNDs showed excellent antimicrobial effect on the growth of bacterial and fungal strains. The SEM micrographs evidently demonstrated the antimicrobial action of the present BNDs. Also, the Au/Ag BNDs supported on SiO₂ spheres were able to inhibit the growth of microorganisms even at lower concentration of Ag (Au₇₅/Ag₂₅). Therefore, the newly synthesized SiO₂@Au/Ag BNDs of the present study are an excellent biocide to eliminate Gram negative, positive and fungal strains which can endanger human beings at short time.

Acknowledgements

RR acknowledges the financial support from the Science and Engineering Research Board (SERB) (Grant no. SB/S1/IC-03/2013), New Delhi. The authors thank the Co-ordinator UGC-NRCBS, School of Biological Sciences, Madurai Kamaraj University, Madurai for providing infra-structural facility to carry out antimicrobial studies and Dr P. Manisankar, Alagappa University, Karaikudi for SEM analysis.

References

1. Y. Xia, Y. Xiong, B. Lim and S. E. Skrabalak, Angew. Chem., Int. Ed., 2009, 48, 60.
2. D. V. Talapin and C. B. Murray, Science, 2005, 310, 86.
3. M. C. Daniel and D. Astruc, Chem. Rev., 2004, 104, 293.
4. X. Y. Liu, A. Q. Wang, X. F. Yang, T. Zhang, C. Y. Mou, D. S. Su and J. Li, Chem. Mater., 2009, 21, 410.
5. S. Manivannan and R. Ramaraj, J. Chem. Sci., 2009, 121, 735.
6. C. Gao, Y. Hu, M. Wang, M. Chi and Y. Yin, J. Am. Chem. Soc., 2014, 136, 7474.
7. H. Zhang, M. Haba, M. Okumura, T. Akita, S. Hashimoto and N. Toshima, Langmuir, 2013, 29, 10330.
8. Q. Zhang, J. Lee, J. Yang, C. Boothroyd and J. Zhang, Nanotechnology, 2007, 18, 245605.
9. S. Pande, S. K. Ghosh, S. Praharaj, S. Panigrahi, S. Basu, S. Jana, A. Pal, T. Tsukuda and T. Pal, J. Phys. Chem. C, 2007, 111, 10806.
10. C. C. Huang, H. Y. Liao, Y. C. Shiang, Z. H. Lin, Z. Yang and H. T. Chang, J. Mater. Chem., 2009, 19, 755.
11. S. Guo, J. Zhai, Y. Fang, S. Dong and E. Wang, Chem.–Asian J., 2008, 3, 1156.
12. C. W. Yen, M. L. Lin, A. Wang, S. A. Chen, J. M. Chen and C. Y. Mou, J. Phys. Chem. C, 2009, 113, 17831.
13. R. D. Jean, K. C. Chiu, T. H. Chen, C. H. Chen and D. M. Liu, J. Phys. Chem. C, 2010, 114, 15633.
14. D. Gajan, K. Guillois, P. Delichere, J. M. Basset, J. P. Candy, V. Caps, C. Coperet, A. Lesage and L. Emsley, J. Am. Chem. Soc., 2009, 131, 14667.
15. Y. Ma, W. Li, E. C. Cho, Z. Li, T. Yu, J. Zeng, Z. Xie and Y. Xia, ACS Nano, 2010, 4, 6725.
16. P. Rameshkumar, S. Manivannan and R. Ramaraj, J. Nanopart. Res., 2013, 15, 1639.
17. N. Sounderya and Y. Zhang, Recent Pat. Biomed. Eng., 2010, 1, 34.
18. M. Banerjee, S. Sharma, A. Chattopadhyay and S. S. Ghosh, Nanoscale, 2011, 3, 5120.
19. N. J. Palleroni, Introduction to the family Pseudomonadaceae, 1992, p. 3071.
20. I. Srnova-Sloufova, F. Lednicky, A. Gemperle and J. Gemperlova, Langmuir, 2000, 16, 9928.
21. Z. Y. Li, J. P. Wilcoxon, F. Yin, Y. Chen, R. E. Palmer and R. L. Johnston, Faraday Discuss., 2008, 138, 363.
22. T. Yan, X. Zhong, A. E. Rider, Y. Lu, S. A. Furmanb and K. Ostrikov, Chem. Commun., 2014, 50, 3144.
23. M. P. Mallin and C. J. Murphy, Nano Lett., 2002, 2, 1235.
24. X. Liu, A. Wang, L. Li, T. Zhang, C. Y. Mou and J. F. Lee, Progress in Natural Science: Materials International, 2013, 23, 317.
25. S. Manivannan and R. Ramaraj, Chem. Eng. J., 2012, 210, 195.
26. S. Link, Z. L. Wang and M. A. El-Sayed, J. Phys. Chem. B, 1999, 103, 3529.
27. K. S. Lee and M. A. El-Sayed, J. Phys. Chem. B, 2006, 110, 19220.
28. L. Shang, L. Jin, S. Guo, J. Zhai and S. Dong, Langmuir, 2010, 26, 6713.
29. S. W. Lin and D. H. Chen, Small, 2009, 5, 72.
30. J. R. Morones, J. L. Elechiguerra, A. Camacho, K. Holt, J. B. Kouri, J. T. Ramírez and M. J. Yacaman, Nanotechnology, 2005, 16, 2346.
31. L. Liu, J. Yang, J. Xie, Z. Luo, J. Jiang, Y. Y. Yang and S. Liu, Nanoscale, 2013, 5, 3834.
32. P. Sanpui, A. Murugadoss, P. V. D. Prasad, S. S. Ghosh and A. Chattopadhyay, Int. J. Food Microbiol., 2008, 124, 142.
33. M. Banerjee, S. Mallick, A. Paul, A. Chattopadhyay and S. S. Ghosh, Langmuir, 2010, 26, 5901.
34. P. Gopinath, S. K. Gogoi, A. Chattopadhyay and S. S. Ghosh, Nanotechnology, 2008, 19, 075104.
35. T. J. Silhavy, D. Kahne and S. Walker, Cold Spring Harbor Perspect. Biol., 2010, 2, 000414.
36. J. M. Willey, L. M. Sherwood and C. J. Woolverton, Prescott, Harley and Klein’s Microbiology, McGraw Hill Higher Education, 7th edn, 2008.
37. Y. H. Kim, D. K. Lee, H. G. Cha, C. W. Kim and Y. S. Kang, J. Phys. Chem. C, 2007, 111, 3629.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra12042f

---