On demand release of ionic silver from gold-silver alloy nanoparticles: fundamental antibacterial mechanisms study

Materials Today Chemistry 16 (2020) 100237

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On demand release of ionic silver from gold-silver alloy nanoparticles: fundamental antibacterial mechanisms study S. Panicker a, I.M. Ahmady b, C. Han c, M. Chehimi d, A.A. Mohamed a, * a

Center for Advanced Materials Research, Research Institute of Sciences and Engineering, University of Sharjah, Sharjah, 27272, United Arab Emirates Department of Applied Biology, University of Sharjah, Sharjah, 27272, United Arab Emirates c Department of Environmental Engineering, 100 Inha-ro, Michuhol-gu, INHA University, Incheon, Republic of Korea d Institut de Chimie et des Mat
eriaux Paris Est (ICMPE)-SPC-UMR 7182 CNRS-Universit
e Paris Est Cr
eteil, 2-8 Rue Henri Dunant, 94320, Thiais, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 October 2019 Received in revised form 7 December 2019 Accepted 10 December 2019 Available online xxx

Synthesis and biomedical research of bimetallic gold-silver nanoparticles (AueAg NPs) have gained much attention due to their unique properties. Antibacterial mechanism of gold-silver nanoparticles is a current topic of interest in nanomedicine engineering. We used three routes in the synthesis of AueAg NPs alloy: i) Co-reduction of [HOOC-4-C6H4N^N]AuCl4/AgNO3, ii) Seeding of AuNPs-COOH/AgNO3 and iii) immobilization of AgNPs over the parent AuNPs-COOH. Two mild reducing agents, NaBH4 and 9-BBN (9-borabicyclo(3.3.1)nonane), were used. Colloidal alloy nanoparticles structure was confirmed using transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The particles reduced using NaBH4 were larger (~20 nm) than those synthesized using 9-BBN (<10 nm). The synthesized nanoparticles showed high stability under notoriously leaching conditions of chloridecontaining electrolytes. Moreover, we studied the AueAg NPs antibacterial activity against the growth of Gram-negative Escherichia coli ATCC strain 25922 and Gram-positive Staphylococcus aureus ATCC strain 29213. The antibacterial mechanisms were evaluated by studying the time-dependent generation of reactive oxygen species (ROS). A major destruction of the bacterial cell wall and leakage of cell components were observed by scanning electron microscopy (SEM), which is clearly visible towards E. coli more than S. aureus bacterial strain. The destruction of the bacterial cell wall was further confirmed by detecting the DNA leakage using gel electrophoresis. The synergistic effect of gold enhanced the antibacterial properties, however, with low cytotoxicity to human dermal fibroblast cells. This study deals with the important aspects of time-dependent mechanisms of the antibacterial action of AueAg NPs since the leaching out of Ag ion is slow compared to AgNPs. The AueAg NPs alloy efficiently tackles microbial activity that can be controlled to minimize cytotoxicity and thus opens their future applications as antibacterial agents. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Gold-silver nanoparticles Diazonium Silver oxidative dissolution Antibacterial mechanism Bacterial morphology

1. Introduction Bimetallic gold-silver nanoparticles (AueAg NPs) have been of a continuous interest because of their unique optical properties [1,2]. They display surface plasmon resonance (SPR) bands [3,4] that can be utilized in many applications as biosensors [5e8], phototherapy treatment of cancer [9,10], surface-enhanced Raman scattering [11,12] and catalysts [13e16]. Their exceptional properties and wide applications are due to synergistic effects of the individual metals

* Corresponding author: E-mail address: [email protected] (A.A. Mohamed). https://doi.org/10.1016/j.mtchem.2019.100237 2468-5194/© 2019 Elsevier Ltd. All rights reserved.

[17,18]. Silver nanoparticles have the highest SPR among all metals while gold nanoparticles have high chemical stability [19]. Gold-silver nanoparticles can form alloys [20e27], core-shell entities [22,26,28,29], or Janus particles, depending upon the synthesis method [30e34]. El-Sayed synthesized gold-silver alloys by a simple co-reduction of the two metal ions with sodium citrate in an aqueous medium [3]. While alloys of AueAg were prepared by Murphy in an aqueous medium using sodium borohydride as a reducing agent and sodium citrate as a capping agent [20]. Pal reported the alloy formation using stabilizing agents like SDS [26] and polyacrylamide solution [27]. Calagua formed the core-shell arrangement with sodium citrate as a reducing agent and ascorbic acid as capping agent using a green approach [28]. In a study by

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Bakshi, phospholipids were used to synthesize pearl-necklace AueAg NPs as bioconjugate materials [29]. There are also a few reports on the use of molecular linkers/impeded linkers such as 4aminothiophenol in the synthesis of gold-silver Janus nanostructures [5,30]. Synthesis [31e33] and phase stability [34] of Janus gold-silver nanoparticles have also been reported. Chen prepared bimetallic Janus structures by an interfacial galvanic exchange of Ag nanoparticles with Au complex [32]. Janus nanoparticles of gold core-semi shell arrangement were prepared by the same group using an etching approach of the core-shell gold-silver nanoparticles in ammonia/hydrogen peroxide mixture [33]. The key properties of silver nanoparticles are their antibacterial effects with limited resistance [35]. However, their major drawback is the toxicity towards mammalian cells, which makes it difficult to use under physiological conditions [36e40]. Many studies have shown that the combination of silver with gold enhances the antibacterial properties of silver. Moreover, the presence of Au facilitates the controllable release of Agþ ion and improves the biocompatibility of AgNPs [41e43]. Gold nanoparticles are easy to prepare and form bioconjugates with proteins, antigens, antibodies and DNA compared to AgNPs which are not biocompatible and unstable under physiological conditions [44e46]. For example, Banerjee fabricated positively charged AueAg nanoparticles, which exhibited antibacterial activity against both Gram-negative and Gram-positive bacteria [47]. Yang synthesized AueAg NPs with a fixed diameter of gold core. Silver concentration varied to control the thickness of Ag on Au core and poly(vinyl pyrrolidine) was used as a stabilizing agent [48]. They showed enhanced antibacterial property of AueAg NPs. As anticipated, the dual nature of the AueAg NPs can be utilized in a wide area of applications, as they are easy to prepare and are biocompatible. Last few years, our group fabricated gold-carbon nanoparticles by reducing aryldiazonium gold(III) salts with mild reducing agents [49e52]. The aim of this research is to synthesize bimetallic AueAg nanoparticles using aryldiazonium salts, which contain diazotized 4-aminobenzoic acid as the linker ligand. It is predicted to connect gold through aryl carbon (AueC) and silver through carboxyl oxygen (AgeO). The synthesis was conducted in three ways: (i) coreduction of aryldiazonium gold(III) and silver nitrate salts, (ii) seeding by the reduction of silver nitrate onto the surface of AuNPs, and finally (iii) immobilization of AgNPs onto AuNPs obtained by the reduction of the aryldiazonium [AuCl4] salt. The study is divided into three main sections: (1) synthesis and characterization of AueAg NPs to confirm their structures; core-shell, alloy or Janus arrangement, (2) determination of ratios of Au and Ag in the six different solid AueAg NPs prepared and (3) evaluation of the biocompatibility and mechanism of the antibacterial properties of the bimetallic nanoparticles. 2. Experimental 2.1. Materials Sodium nitrite, 0.5 M solution of 9-borabicyclo(3.3.1)nonane (9BBN) in tetrahydrofuran, hydrochloric acid (36.5e38.0%), 4aminobenzoic acid, calcium chloride, resazurin, glutaraldehyde, 20 ,70 -dichlorofluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), ethanol, agarose and ethidium bromide from SigmaAldrich. Sodium borohydride from Fisher Chemicals. 5X TAE buffer-1L (tris-acetate-EDTA) from NORGEN, BIOTEK CORP. Silver nitrate from PanReac AppliChem. Muller-Hinton agar and broth from HIMEDIA, India. Sodium chloride LR from WARDLE chemicals. Phosphate buffer saline (pH 7.4) and Penicillin Streptomycin from Gibco by life technology. Primary Dermal Fibroblast Normal; Human, Neonatal (HDFn) (ATCC PCS201010) cell line, Dulbecco's

Modified Eagle's Medium High glucose DMEM, EDTA, phenol red and fetal bovine serum (FBS) from Sigma-Aldrich. Gram-negative Escherichia coli ATCC strain 25922 and Gram-positive Staphylococcus aureus ATCC strain 29213 from ATCC. 2.2. Instruments UVeVis absorption spectra were measured using a scanning spectrophotometer (Spectro UV-2510TS, Labomed Inc.) in the 200e800 nm range with 2 nm resolution. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were recorded on a Bruker (Platinum ATR) Tensor II FT-IR spectrophotometer. The hydrodynamic diameters were determined at room temperature using dynamic light scattering (Nanotrac Wave II, model MN42x, USA). X-ray powder diffraction (XRD) data were collected using Bruker D8 ADVANCE diffractometer with Cu-Ka X-ray source at l ¼ 1.5406 Å operating at 40 kV tube voltage and 40 mA current. Energy dispersive X-ray spectroscopy (EDS) analyses were performed with map and point modes at 50 kV and 20 mA. Raman spectra were obtained using Renishaw In Via Raman Spectrophotometer, UK. The laser used was at wavelength 514 nm. A JEOL JEM2100 transmission electron microscope (TEM) was used to investigate the morphology of the nanoparticles. Dispersed nanoparticles in acetonitrile were immobilized on 400 mesh copper grids coated by FORMVAR carbon film (FCF 400-Cu, Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). Thermo Scientific K Alpha X-ray photoelectron spectrometer (XPS) system fitted with monochromatic Al-Ka X-ray source of 400 mm spot sizes and a flood gun for static charge compensation was used to record XPS spectra. The pass energy was set to 200 eV for the survey spectra and 80 eV for the narrow regions. The elemental composition of the samples was determined using the manufacturer sensitivity factors. Gold and silver concentrations in solution were determined with simultaneous ICP-OES (inductively coupled plasma-optical emission spectroscopy-ICP-Varian, Vista MPX CCD model). Reactive oxygen species (ROS) concentration was determined using SHEMATZU RF-6000 fluorophotometer model RF-6000. TESCAN VEGA XM variable pressure SEM, accelerating voltage: max. 30 kV, was used to study the morphological changes in bacteria. NanoDrop 2000 UVeVis spectrophotometer (Thermo Scientific, USA) was used to quantify the nucleic acids. Agarose gel apparatus and gel documentation system from Bio-Rad, USA. DensiCHEK Plus turbidity meter from BioMerieux. 2.3. Preparation of AuNPs seeds The water-soluble aryldiazonium gold(III) salt [HOOC-4C6H4N^N]AuCl4 was synthesized following our published procedure [49]. The gold-aryl nanoparticles were synthesized by reducing the salt using 0.1 M NaBH4. Briefly, 0.48 g (0.234 mM) of aryldiazonium gold(III) salt was dissolved in 50 mL of distilled water, and 0.1 M NaBH4 was added dropwise to the solution under stirring. The nanoparticles were formed as purple colored solution, purified and stored in the refrigerator for further use. 2.4. Preparation of AgNPs AgNPs were synthesized using the procedure described by Creighton [53]. Briefly, 30 mL of 0.002 M NaBH4 was taken in an Erlenmeyer flask and placed in ice bath under stirring. The solution was kept under stirring for 30 min, and then 10 mL of 0.001 M AgNO3 solution was added dropwise into the stirring NaBH4 solution. The stirring was stopped as soon as all AgNO3 was added and a clear yellow solution of AgNPs was formed.

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2.5. Preparation of AueAg NPs alloy

2.8. Reactive oxygen species concentration determination

The solid AueAg NPs were synthesized using three methods and using two mild reducing agents 0.5 M 9-BBN and 0.1 M NaBH4. The concentration of aryldiazonium gold(III) salt and AgNO3 was constant in all preparations. The three routes were as follows: (1) Coreduction of diaz-Au þ AgNO3. In two beakers, 0.001 M aryldiazonium gold(III) salt was dissolved in 10 mL water and 10 mL of 0.001 M AgNO3 was added. 0.5 M 9-BBN was added to one of the beakers while 0.1 M NaBH4 was added to the other. (2) Seeding of AuNPs þ AgNO3. In the seeding method, AuNPs were first synthesized as described above and then 0.001 M AgNO3 was added and reduced using either 0.5 M 9-BBN or 0.1 M NaBH4. (3) Immobilization of AgNPs over AuNPs. In the immobilization method, AuNPs and AgNPs were pre-synthesized by reducing with 0.5 M 9-BBN or 0.1 M NaBH4, and then mixed together and stirred for 24 h. All AueAg NPs thus synthesized were washed with deionized water and purified. The solutions were then centrifuged at 12,000 rpm. Pellets were collected, dried under vacuum and stored in the desiccator.

Reactive oxygen species generation was determined by the fluorogenic dye 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) as previously described [45]. Briefly, 2 mM of DCFH-DA was prepared in ethanol, and 1 mL each of the diluted suspensions of E. coli and S. aureus of concentration 1  108 CFU/mL was taken into different vials and 2.5 mL of DCFH-DA solution was added. The cells were kept in the shaker and allowed to incubate in the dark for 20 min at 37  C. Nanoparticles were then added to each vial and again incubated in the dark for 2 h. The fluorescence intensity was measured at excitation/emission of 488/525 nm.

2.6. Stability of nanoparticles in chloride electrolytes Stability of AgNPs and AueAg NPs were tested by incubation of 2 mL of each with 500 mL of 0.1 M NaCl, CaCl2 and HCl solutions at room temperature for 1 h. UVeVis spectra were recorded to monitor the change in the plasmon peaks. 2.7. Assessment of antibacterial activities Gram-negative bacteria E. coli ATCC strain 25922 and Grampositive bacteria S. aureus ATCC strain 29213 were used as organism models for antibacterial studies. Minimum inhibitory concentrations (MICs) that completely inhibited visible bacterial growth were measured according to Clinical and Laboratory Standards Institute (CLSI) guidelines [54]. First, solutions sterility was tested by spreading 100 mL on nutrient agar and incubated for 24 h. Then the testing solutions were diluted in Muller Hinton broth in microtitration plates ranging from 1:2 to 1:128. Samples were tested in triplicate. Standard inoculums of both bacteria were prepared in a saline solution from 24 h agar plate using a direct colony suspension method following CLSI guidelines [54]. Initial inoculums density was adjusted to 0.5e0.63 using DensiCHEK Plus turbidity meter, this density is equal to 0.5 McFarland solution that gives bacterial concentration of 1  108 CFU/mL. Then the inoculums were further diluted to give final bacterial concentration of 5  105 CFU/mL in 100 mL of diluted testing solution. Control wells of broth with bacteria and sterile wells containing broth only were prepared. Plates were sealed and incubated overnight in a shaking incubator at 37  C. MIC was detected visually by the unaided eyes and confirmed with Resazurin sodium reagent [55]. The growth in the wells containing nanoparticles was compared with the control wells to determine the growth end points. Control of 2 mm button or definite turbidity in the growth-control well is considered as acceptable growth. For MIC values confirmation, 30 mL/well of 0.015% Resazurin sodium reagent was added and plates were incubated for 2 h to observe color change, then MICs were recorded based on color change visually such that blue-purple indicates no bacterial growth, however, red, pink or colorless indicates bacterial growth [55]. Furthermore, the minimum concentrations that kill 99.9% of the initial inoculum were detected by sub-culturing 10 mL of the samples from all wells that showed inhibition after 24 h onto nutrient agar and incubated for 24 h at 37  C. Number of colonies was counted to calculate the concentrations that inhibit 99.9% of the bacteria.

2.9. Silver ion oxidative dissolution Silver ion oxidative dissolution was measured using ICP-OES. Briefly, 1 mg/mL nanoparticles solution was added to 1  108 CFU/mL of E. coli and S. aureus and incubated at 37  C. After time intervals of 2, 4 and 24 h, solutions were centrifuged at 11,000 rpm for 10 min at 4  C to remove the bacterial cells and nanoparticles. The concentration of Agþ released in the supernatant was determined. 2.10. Probing bacterial morphology change Bacterial morphology change was probed using SEM. Bacteria at 1  108 CFU/mL concentration were incubated with the different preparations of nanoparticles for 2 h, and then 10 mL of the treated bacterial suspensions were gently placed onto SEM sample holders, air dried and fixed with 2.5% glutaraldehyde. The samples were then dehydrated with ethanol for 15 min and allowed to air dry, gold coated and mounted for SEM analysis. 2.11. Nucleic acids leakage measurement The amount of released nucleic acids (DNA and RNA) was determined from the maximum absorbance at 260 nm using NanoDrop spectrophotometer. Briefly, the nanoparticles were incubated with E. coli and S. aureus for 2 h. The bacterial suspensions were then centrifuged at 12,000 rpm for 5 min, and the supernatants were collected. The optical density (OD) was then measured at 260 nm [56]. The pellets were dissolved in PBS, and the suspension was electrophoresed to analyze the fragmentation of DNA. 20 mL of AueAg NPs and AgNPs treated bacterial suspensions were taken into vials and 2 mL of DNA loading buffer was added to each. DNA samples were electrophoresed in 1.5% agarose gel and visualized by ethidium bromide staining. The gel was photographed under ultraviolet light. 2.12. AueAg nanoparticles cytotoxicity assessment Cytotoxicity of AueAg NPs was evaluated using MTT tetrazolium colorimetric assay on human dermal fibroblast cell line PCS-201010 at density of 35  103 cell/well. Cells were cultured according to ATCC recommendations in Dulbecco's Modified Eagle's Medium High glucose-DMEM, supplemented with 10% fetal bovine serum and 2  penicillin/streptomycin and incubated at 37  C in humidified atmosphere incubator with 5% CO2. 100 mL of the cell solutions was added into 96-well plate and incubated for 24 h at 37  C. The cells were treated with different concentrations of the nanoparticles for another 24 h 10 mL of 0.5 mg/mL MTT tetrazolium dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added into each well followed by incubation for 3 h to allow for the formation of the dark purple formazan crystals, which were dissolved in 100 mL DMSO. Finally, the absorbance was measured at

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570 nm using UV spectrophotometer. All samples were tested in triplicate. Positive control was prepared using cells in DMEM medium, while empty wells with MTT reagent and DMSO only were used as a negative control. The cell viability percentage of the metabolically active cells was measured using Equation (1)

Cell viability% ¼

Sample abseNegative control abs Positive control abseNegative control abs

 100 (1) Statistical significance was calculated using Student's-t tests. A difference of p < 0.05 was considered statistically significant.

3. Results and discussion 3.1. Synthesis and characterization of AueAg alloy nanoparticles The AueAg alloy nanoparticles were synthesized by means of three routes using two mild reducing agents, 0.1 M NaBH4 and 0.5 M 9-BBN (Fig. 1a). The nanoparticles thus obtained were subjected to different characterization techniques to establish their structures and to estimate the gold/silver percentage. The three routes are: (i) [HOOC-4-C6H4N^N]AuCl4/AgNO3: co-reduction of gold(III) in the form of aryldiazonium-AuCl4 salt in the presence of silver nitrate. The reduction of the diazonium cation cleaves the molecular nitrogen and the aryl shell binds to the gold nanoparticles [50]. Thus, the co-reduction facilitates the continuous growth of silver nanoparticles over the carboxylate shell. (ii) AuNPs-COOH/AgNO3: seed-mediated growth of silver nitrate, by in situ reduction, over the preformed AuNPs-COOH. (iii) Ag NPs/ AuNPs-COOH: immobilization of AgNPs over AuNPs-COOH. In the immobilization route, silver nanoparticles propagate over the gold nanoparticles. In addition, the formation of AueAg NPs can occur without the need for a reducing agent. Since this route involves preformed nanoparticles, it avoids the influence of chloride ion from the AuCl4 anion.

UVeVis spectra of monometallic, NaBH4 reduced AuNPs and AgNPs prepared separately, showed typical bands at 560 nm (purple solution) and 390 nm (yellow solution) (Fig. 1b). The bimetallic AueAg NPs showed a blue shifted plasmon peak appeared at 410 nm (dark brown solution) due to the plasmon resonance of the AueAg NPs alloy pattern [47]. To further confirm the formation of the alloy structure, the AueAg NPs solutions were treated with different volumes of 0.1 M HNO3 and the change in the plasmon peaks was observed by UV. The dark brown solution became lighter after the addition of increasing volumes of HNO3. The plasmon peak at 410 nm started to disappear, and the peak for the benzoic acid ligand became clear at 290 nm (Fig. S1). Dynamic light scattering (DLS) was used to determine the hydrodynamic diameter of AueAg NPs. The results are shown in the Supporting Information (Fig. S2 a-b). The average size distribution range is 300e600 nm for all the AueAg NPs samples. The crystalline nature of the AueAg NPs was further confirmed from X-ray diffraction (XRD) analysis. The results depicted in Figs. S3aeb show the XRD pattern of AueAg NPs obtained after reduction using both 9-BBN and NaBH4. All the nanoparticles showed Bragg reflection peaks in the 2q range between 30 and 80 . The hkl values corresponding to (111), (200), (220) and (311) planes of face centered cubic (fcc) for both gold (PDF Card #4e784) and silver (PDF Card #4e783) can be seen. We observed the presence of low intensity peaks in the diffractograms at ~32.35, 46.38, 54.03 and 57.66 due to AgNO3 impurities. The impurities can be seen in the co-reduction of the diazonium salt in the presence of AgNO3 using 9-BBN. Meanwhile, this is not observed in the case of NaBH4 reduction where no AgNO3 impurities were seen in XRD diffractograms. Hence, it can be concluded that NaBH4 is a stronger reducing agent than 9-BBN. Spherical AueAg alloy nanoparticles were formed by the reduction using 0.5 M 9-BBN and 0.1 M NaBH4, confirmed from TEM images, Figs. 2e3. They are closely packed and it seems that the particles are interconnected with each other in a worm-like arrangement by formation of junction between particles during the synthesis (seen in Fig. 2 (c), (f) and (i), and Fig. 3 (c), (f) and (i) of high resolution TEM images). In both cases, AueAg alloy

Fig. 1. (a) Scheme showing the different routes to synthesize gold-silver nanoparticles, (b) UVevisible spectra of pure AuNPs and AgNPs and bimetallic AueAg NPs alloy. Conditions: AuNPs formed by reducing aryldiazonium gold(III) salt with 0.1 M NaBH4; AgNPs synthesized by adding AgNO3 in concentrated NaBH4 solution; and AgNO3 reduced in the solution of pre-synthesized AuNPs using 0.1 M NaBH4 to form alloy structure of AueAg NPs.

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Fig. 2. TEM images of AueAg NPs (9-BBN). (aec) Diaz-Au þ AgNO3 (co-reduction), (def) AuNPs þ AgNO3 (seeding), (gei) AuNPs þ AgNPs (immobilization).

nanoparticles were fully crystallized by observing their lattices. The measured lattice spacings using an ImageJ software provided by United States National Institutes of Health were about 0.235 nm, corresponding to (111) planes of Ag or Au, and thus confirmed the fully crystallized AueAg NPs [57,58]. This result is in a good agreement with a previous study by Sun [23]. They reported that the measured interplanar distance of AueAg alloy nanoparticles was 0.24 nm, corresponding to (111) planes of Au or Ag. The particles reduced using NaBH4 were larger (~20 nm) than those synthesized using 9-BBN (<10 nm). The nanoparticles were characterized using EDS (Figs. S4aeb) and XPS (Fig. 4, Figs. S5aeb and Figs. S6aeb) in order to determine the stoichiometric ratio of gold and silver. Figs. S4aeb shows the weight % of gold and silver in all alloys determined using EDS. The AueAg NPs reduced with NaBH4 showed higher weight % of both gold and silver compared to those reduced with 9-BBN. The weight % of gold is 13.9, 28.9 and 16.8% whereas the weight % of silver is 6.8, 10.8 and 7.6% in the three AueAg NPs reduced with 9-BBN. While the AueAg NPs reduced with NaBH4 displays the weight % of gold to be 56.4, 55 and 64.3%, for silver is 33.6, 36.6 and 29.5%. Surface elemental composition (in at. %) of AueAg NPs was estimated by using XPS. Fig. 4aec displays the survey regions from the three AueAg NPs reduced with 9-BBN. From the spectra one can note the main peaks C1s, Au4f doublet, Ag3d doublet, and O1s centered at ~284, 84, 368 and 532 eV, respectively. High resolution Au4f and Ag3d doublets are shown in insert with Au4f7/2 and Ag3d5/2 peaks centered at 84 and 368.2 eV in line with metallic

state for both elements. The C1s are much more intense compared to Au4f and Ag3d doublets, indicating an organic-rich surface composition for the reduction route utilizing 9-BBN. This is illustrated quantitatively in Fig. 4d which shows quasi 0 at.% for both precious metals. Fig. 4eeg displays survey spectra for the same type of nanoparticles but prepared with NaBH4. A completely different picture is offered; indeed, sharp Au4f and Au3d doublets are noted and at binding energy positions characteristic of metallic states as judged from the high-resolution peaks shown in inset. One can note the very low relative peak intensity of C1s which suggests a rather metal rich surface. Again, this is well illustrated by the respective at.% for the various elements (Fig. 4h). Interestingly, EDS shows higher at.% for the precious metals in both series of materials; this is due to the sensitivity of EDS to the bulk of materials compared to XPS which is more surface specific. However, whilst for the reduction route using NaBH4, EDS suggests almost 2-fold gold to silver, XPS indicates a reverse situation, that is two times more silver than gold. It is thus likely that silver is segregated to the surface. This could be due to two possible pathways: (i) by design, that is by synthesis of silver on top of gold particles, or (ii) by segregation after synthesis of the bimetallic particles which means partial de-alloying resulting in particles with silver-rich surfaces. For the reduction with 9-BBN, the Au4f and Ag3d peaks are too noisy to tempt interpret the results. The presence of the carboxylate group linker and its connectivity is supported by ATR-FTIR (Fig. S7) and Raman measurements

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Fig. 3. TEM images of AueAg NPs (NaBH4). (aec) Diaz-Au þ AgNO3 (co-reduction), (def) AuNPs þ AgNO3 (seeding), (gei) AuNPs þ AgNPs (immobilization).

(Figs. S8 and S9). Earlier, we studied the gold-carbon bonding in several gold nanoparticles using DFT calculations and Raman spectroscopy. DFT calculations of the optimized model system Au38eC6H4eCOOH showed an AueC (aryl) distance of 2.04 Å, which is related to a binding energy of 59.2 kcal/mol [52]. The most important band in the Raman modes is the AueC stretch, which can be assigned to a weak experimental band at 516 cm1. Evidence of the aryl shell presence is provided by observation of n(C]O) at 1685 cm1, n(C]C) at 1566 cm1 and n(CeO) at 1085 cm1 in the infrared. The absence of the diazonium band at 2270 cm1 is also notable. The peaks at 1390 cm1, 850 cm1, and 224 cm1 are assigned to the n(OeCeO), n(AgeO) and n(Ag-OCO) bands in Raman clearly support the binding of Ag nanoparticles with the carboxylic group through the AgeO interaction. 3.2. Stability of AueAg alloys in chloride electrolytes Chloride ion is abundant in bacterial growth media and is expected to deposit the leached silver ion. The AgNPs and AueAg NPs solutions were treated with three chloride electrolytes to monitor the silver oxidative dissolution after 1 h of incubation at room temperature. Figs. S10aeb shows the UVeVis spectra after adding 0.1 M NaCl, CaCl2 and HCl to the nanoparticles in aqueous solution. It is observed that the oxidative dissolution from AgNPs was quite high as expected; the high molar extinction coefficient plasmon peak disappeared completely. This is in accordance with previously reported studies, where the presence of Cl resulted in the loss of

particle dispersity and AgNPs were completely oxidized and leached into the electrolyte solution to form AgCl [59]. The AueAg NPs solutions are more robust compared to AgNPs as the plasmon peak still is observed after 1 h of incubation. This clearly indicates that in the case of AueAg NPs, the oxidative dissolution of silver in aerobic water is attenuated after alloying with gold. It is possible that silver nanoparticles stabilization originates from bonding with the carboxylate oxygen. As a result, this minimizes the formation of silver oxides and hydroxides by hydrolysis at a neutral pH. Previous studies reported similar results with AueAg NPs solutions, which showed enhanced chemical stability compared to AgNPs [17]. In another study, it was suggested that carboxylic groups in glutamic acid and phosphate groups in teichoic acid bind with silver and enhance its stability [38]. We hypothesize the stability of our AueAg alloy to originate from the alloy structure, difficulty of oxygen diffusibility to silver and silver-oxygen bonding. 3.4. Antibacterial activity of AueAg nanoparticles The antibacterial activity of AueAg NPs was evaluated against Gram-negative E. coli and Gram-positive S. aureus bacteria. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined. The gold and silver concentrations were determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES). From the results given in Tables S11 and S12, AueAg NPs demonstrated greater antibacterial activities than pure AgNPs, while pure AuNPs did not

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Fig. 4. XPS survey spectra of AueAg NPs reduced with 9-BBN (aec) and corresponding apparent composition in atomic % (d); and the same types of particles prepared with NaBH4 reducing agent (eeg) and the corresponding composition (h). Survey spectra correspond to diaz-Au þ AgNO3 (a,e); AuNP þ AgNO3 (b,f); and AuNP þ AgNP (ceg). High resolution spectra are shown in insert for Au4f and Ag3d.

exhibit any antibacterial activities. We observed that diazAu þ AgNO3 prepared by co-reduction with 9-BBN showed the highest antibacterial activity which is indicated by the low total MICs of 0.012 mg/mL for E. coli and 0.024 mg/mL for S. aureus with least total concentrations and MBCs of 0.096 mg/mL for E. coli and 0.76 mg/mL for S. aureus. AuNPs þ AgNO3 prepared using NaBH4 was on the second position with total MIC of 0.031 mg/mL for both strains and MBC value of 0.213 mg/mL and 0.980 for E. coli and S. aureus respectively. AgNPs showed the least activity and the highest MIC value of 0.095 mg/mL and no bactericidal activity was recorded against both bacterial strains. This result indicates that AueAg NPs prepared by reducing with 9-BBN has significantly much higher antibacterial activity than all other AueAg NPs as well as the pure AuNPs and AgNPs.

3.5. Quantification of reactive oxygen species One of the most widely accepted mechanisms of the antibacterial effect of silver is explained by the release of active species or free radicals, which can induce further damage to the bacterial cell wall [35]. ROS are oxygen species such as hydroxyl radical, reactive oxygen and superoxide anion that are highly reactive and are produced during basic metabolic pathways [35]. However, the concentrations of ROS are prone to increase significantly under high levels of stress conditions. Studies have shown that one of the primary mechanisms of action displayed by the nanoparticles in inhibiting the growth of the bacterial cells is by generating high amounts of ROS by breaking the bacterial membrane [35]. To validate this mechanism, the ROS concentration was evaluated using the fluorogenic dye 20 ,70 -dichlorofluorescein diacetate (DFCH-DA). Results show (Fig. 5) the fluorescence intensity increase with

incubation time of bacteria with the nanoparticles. The concentration of ROS in AgNPs treated E. coli and S. aureus increased after 2 h of incubation, which was significantly higher than the AueAg NPs treated bacterial solutions. Whereas the fluorescence intensity increased in two of the AueAg NPs to higher levels than the AgNPs treated bacteria after 24 h of incubation, indicating that AueAg NPs can cause ROS production in water and this effect is time dependent. Thus, demonstrating that one of the mechanisms of action of AueAg NPs is similar to AgNPs, which is caused by a high level of oxidative stress in bacteria.

3.6. Measurement of silver ion From the ROS concentration measurements, we found that the two bimetallic AueAg nanoparticles which produced the highest levels of ROS more than AgNPs were diaz-Au þ AgNO3 mixture reduced using both 9-BBN and NaBH4. So, we used these two AueAg NPs along with AgNPs to study the release of Agþ in bacterial solutions after certain time intervals. E. coli and S. aureus were incubated with the nanoparticles for 2, 4 and 24 h at 37  C and the concentration of silver was determined using ICP-OES. Results showed (Fig. 6) that the concentration of silver ion released in both bacterial solutions after 2 h was higher in AgNPs treated cells than the two AueAg NPs. Moreover, the amount of Agþ released was gradually increased in all the nanoparticles treated solutions after 4 h. After 24 h of incubation, the amount of Agþ was higher in cells treated with both types of AueAg NPs than AgNPs, reaching a maximum concentration of 18.60 mg/mL and 16.50 mg/mL for the two AueAg NPs and 12.76 mg/mL in AgNPs treated E. coli. In S. aureus solution, the amount of Agþ released from the two AueAg NPs were 27.20 mg/mL and 26.70 mg/mL, respectively, whereas for

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Fig. 5. (a) ROS concentration in E. coli cells tested with DFCH-DA. E1, E2 and E3 are the AueAg NPs (9-BBN); E4, E5 and E6 are the AueAg NPs (NaBH4) and incubated for 2 and 24 h at 37  C, (b) ROS concentration in S. aureus cells tested with DFCH-DA. S1, S2 and S3 are the AueAg NPs (9-BBN); S4, S5 and S6 are the AueAg NPs (NaBH4) and incubated for 2 and 24 h at 37  C.

Fig. 6. (a) Concentration of Agþ released in E. coli cells treated with AgNPs and the two types of AueAg NPs over 24 h determined using ICP-OES, (b) Concentration of Agþ released in S. aureus cells treated with AgNPs and the two types of AueAg NPs over 24 h determined using ICP-OES.

Fig. 7. SEM images of E. coli. (a, b) Untreated control cells, showing normal rods. (c, d) treated with diaz-Au þ AgNO3, (9-BBN) and (e, f) diaz-Au þ AgNO3 (NaBH4) showing cellular damage and nanoparticles adsorbed to cell wall.

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Fig. 8. SEM images of S. aureus (a, b) untreated control cells, showing normal round cells. (c, d) treated with diaz-Au þ AgNO3 (9-BBN), (e, f) diaz-Au þ AgNO3 (NaBH4) showing cellular damage and nanoparticles adsorbed to the cell wall. AueAg NPs are accumulated on the surface of bacteria.

Fig. 9. (a) Concentration of nucleic acids released from bacteria after 2 h of incubation with AueAg NPs (9-BBN), (b) concentration of nucleic acids released from bacteria after 2 h of incubation with AueAg NPs (NaBH4). 1.5% of agarose gel for DNA analysis in E. coli after 2 h of treatment with nanoparticles. (c) L1: DNA ladder; L2: Control; L3, L4, L5: AueAg NPs (9-BBN); L6, L7, L8: AueAg NPs (NaBH4); L9: AgNPs. 1.5% of agarose gel for DNA analysis in S. aureus after 2 h of treatment with nanoparticles. (d) L1: DNA ladder; L2: Control; L3, L4, L5: AueAg NPs (9-BBN); L6, L7, L8: AueAg NPs (NaBH4); L9: AgNPs.

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Fig. 10. Cell viability assay of human dermal fibroblast cell line PCS-201-010, (a) Cells treated with AueAg NPs (9-BBN) at three different concentrations (dilutions %) and incubated for 24 h (1) diaz-Au þ AgNO3, (2) AuNPs þ AgNO3, (3) AuNPs þ AgNPs, (4) AuNPs and (5) AgNPs. (b) Cells treated with AueAg NPs (NaBH4) at three different concentrations (dilutions %) and incubated for 24 h (1) diaz-Au þ AgNO3, (2) AuNPs þ AgNO3, (3) AuNPs þ AgNPs, (4) AuNPs and (5) AgNPs.

AgNPs it was 18.83 mg/mL. Comparing the release of Agþ with ROS concentrations, both showed similarity as they gave maximum antibacterial activity after 24 h of incubation with the AueAg NPs. 3.7. Destruction of bacterial morphology SEM was used to visualize the direct structural change in the bacterial cell morphology following the treatment with the nanoparticles. After 2 h of incubation, the Gram-negative E. coli control group in Fig. 7 showed the unique cell envelope of the normal cell structure and typical rod-shaped morphology. Moreover, the cells treated with AueAg NPs synthesized using both 9-BBN and NaBH4 showed extensive damage to the cellular structure. The visible rupture of cell wall which has swollen into a bigger shape probably is caused by ROS generation. The formation of fragments is more visible in the AueAg NPs prepared using NaBH4. This result is novel in the sense that other AueAg nanoparticles showed cellular morphology change only due to increased cell permeability but no visible fragmentation. Likewise, Gram-positive S. aureus control group in Fig. 8 showed the typical grape-shaped arrangement

morphology after 2 h of incubation. Pits or holes can be seen after AueAg NPs treatment, but significantly less flawed compared to Gram-negative E. coli. The difference in the AueAg NPs effect on both bacteria can be explained based on the cellular wall components. Gram-positive bacteria S. aureus are more protected by a thicker cell wall than Gram-negative bacteria E. coli. Our conclusion is supported by the minor damage to the cell wall of S. aureus by the effect of nanoparticles synthesized using both reducing agents. In addition, SEM images showed that dead bacteria are not aggregated around the nanoparticles and thus continue to behave as antibacterial and remain active by releasing silver ions over long time. It has been reported that AgNPs could form electrostatic interactions with the bacterial membrane, which leads to the disruption of the membrane integrity and cell death [60]. SEM images prove a clear support to the association of nanoparticles with the bacteria wall. 3.8. Nucleic acids release Earlier studies on the AgNPs antibacterial activity have shown them to interfere with the DNA replication process either directly or

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indirectly [44]. Reports showed that the released silver ions bind to DNA and cause its damage. Also the high levels of ROS produced by the nanoparticles result in the degradation of the genetic material. To verify the extent of DNA damage and prove the leakage of nucleic acids into the cytoplasmic solutions, we incubated the bacterial cells of 1  108 CFU/mL with AgNPs and all the different types of AueAg NPs for 2 h at 37  C. Solutions were centrifuged, and the supernatants were analyzed for nucleic acids concentration by NanoDrop spectrophotometer. The exposure of both E. coli and S. aureus to AueAg NPs and AgNPs for 2 h leaked a very high amount of nucleic acids into the bacterial solutions, Fig. 9a and b. This result indicates the damage caused to the bacterial genome in the presence of nanoparticles. We further investigated the change in the DNA integrity as a result of the nanoparticles action on the genetic material. For this, 20 mL of the supernatant from each sample was electrophoresed in 1.5% agarose gel, Fig. 9c and d. As shown in the gel image, a high amount of DNA fragments exists in almost all the bacterial cells treated with the nanoparticles. These observations thus further confirm the mechanisms involved in the bacterial cell death.

3.9. Cytotoxicity of AueAg nanoparticles towards human dermal fibroblast cells Other than exhibiting high antibacterial activities, the biocompatibility of AueAg NPs is also a key factor in determining their applications in the medical field. To check the extent of biocompatibility of AueAg NPs compared to AgNPs, human dermal fibroblast cell line PCS-201-010 was treated with the nanoparticles at different concentrations including the MICs for well evaluation of the cytotoxicity (Fig. 10a and b). Fibroblast cells exhibited low viability between 13% and 50% at high AueAg nanoparticles concentrations, while the viability increased at the low concentrations and reached up to 100%. It was observed that AueAg NPs synthesized using 9-BBN showed less cytotoxicity than those prepared using NaBH4, and is concentration dependent, Fig. 10 a-b. The highest cell viability percentage recorded at the lowest MICs values that were obtained with diaz-Au þ AgNO3 (9-BBN) and AuNPs þ AgNO3 (NaBH4) in which 81%e97% of the cells were viable, meanwhile, AgNPs gave the least cell viability of 13% at their MIC of 0.095 mg/mL, but it increased as the concentrations decreased. In contrast, AuNPs did not show cytotoxic effect against fibroblast cells at the investigated concentrations. Thus, the results showed the high biocompatibility of AueAg NPs towards human cells when used at low concentrations. 4. Conclusions In summary, we hereby present a novel bimetallic AueAg NPs alloy synthesized from aryldiazonium gold(III) salts in a combination with silver nitrate. These nanoparticles are easily synthesized and stable for long duration. The alloys showed high level of robustness as proved from the characterization studies. The incorporation of Au and Ag in the same nanoparticles further makes it a strong antibacterial agent while amenable to human cells. Moreover, we propose the potential antimicrobial mechanism of AueAg NPs. These results will help in future studies with drug resistant bacteria. Furthermore, the presence of gold along with silver makes it more potent biocompatible bimetallic nanoparticles. Authors contribution All authors contributed equally.

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Declaration of competing interest There are no conflicts to declare. Acknowledgement AAM acknowledges the University of Sharjah support of SEED grant VC-GRC-SR-83-2015, competitive grants 160-2142-029-P and 150-2142-017-P, Organometallic Research Group grant RISE-0462016 and Functionalized Nanomaterials Synthesis Lab grant (1510039). CH acknowledges the support by INHA University research grant INHA-60121-1. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtchem.2019.100237. References [1] L. Lu, G. Burkey, I. Halaciuga, D.V. Goia, Core-shell gold/silver nanoparticles: synthesis and optical properties, J. Colloid Interface Sci. 392 (2013) 90e95. [2] M.B. Cortie, A.M. McDonagh, Synthesis and optical properties of hybrid and alloy plasmonic nanoparticles, Chem. Rev. 111 (2011) 3713e3735. [3] S. Link, Z.L. Wang, M.A. El-Sayed, Alloy formation of gold-silver nanoparticles and the dependence of the plasmon absorption on their composition, J. Phys. Chem. B 103 (1999) 3529e3533. [4] K.B. Mogensen, K. Kneipp, Size-dependent shifts of plasmon resonance in silver nanoparticle films using controlled dissolution: monitoring the onset of surface screening effects. J. Phys. Chem. C 118 (2014) 28075e28083, https:// doi.org/10.1021/jp505632n. [5] Z. Luo, K. Chen, D. Lu, H. Han, M. Zou, Synthesis of p-aminothiophenolembedded gold/silver core-shell nanostructures as novel SERS tags for biosensing applications, Microchim. Acta 173 (2011) 149e156. [6] A. Steinbruck, O. Stranik, A. Csaki, W. Fritzsche, Sensoric potential of goldsilver core-shell nanoparticles, Anal. Bioanal. Chem. 401 (2011) 1241e1249. [7] H. Tu, T. Sun, K.T. Grattan, SPR-based optical fiber sensors using goldesilver alloy particles as the active sensing material, IEEE Sens. J. 13 (2013) 2192e2199. [8] K.D. Gilroy, A. Ruditskiy, H.C. Peng, D. Qin, Y. Xia, Bimetallic nanocrystals: syntheses, properties, and applications, Chem. Rev. 116 (2016) 10414e10472. [9] T. Jiang, J. Song, W. Zhang, H. Wang, X. Li, R. Xia, L. Zhu, X. Xu, AueAg@Au hollow nanostructure with enhanced chemical stability and improved photothermal transduction efficiency for cancer treatment, ACS Appl. Mater. Interfaces 7 (2015) 21985e219894. [10] P. Wu, Y. Gao, H. Zhang, C. Cai, Aptamer-guided silveregold bimetallic nanostructures with highly active surface-enhanced Raman scattering for specific detection and near-infrared photothermal therapy of human breast cancer cells, Anal. Chem. 84 (2012) 7692e7699. [11] T. Bai, J. Sun, R. Che, L. Xu, C. Yin, Z. Guo, N. Gu, Controllable preparation of core-shell Au-Ag nanoshuttles with improved refractive index sensitivity and SERS activity, ACS Appl. Mater. Interfaces 6 (2014) 3331e3340. [12] M.F. Cardinal, B.R. Gonzaìlez, R.A. Alvarez-Puebla, J. Peìrez-Juste, L.M. LizMarzaìn, Modulation of localized surface plasmons and SERS response in gold dumbbells through silver coating, J. Phys. Chem. C 114 (2010) 10417e10423. [13] H. Zhang, J. Okuni, N. Toshima, One-pot synthesis of AgeAu bimetallic nanoparticles with Au shell and their high catalytic activity for aerobic glucose oxidation, J. Colloid Interface Sci. 354 (2010) 131e138. [14] X. Li, Y. Yang, G. Zhou, S. Han, W. Wang, L. Zhang, W. Chen, C. Zou, S. Huang, The unusual effect of AgNO3 on the growth of Au nanostructures and their catalytic performance, Nanoscale 5 (2013) 4976e4985. [15] J. Gu, Y.W. Zhang, F.F. Tao, Shape control of bimetallic nanocatalysts through well-designed colloidal chemistry approaches, Chem. Soc. Rev. 41 (2012) 8050e8065. [16] T. Sinha, M. Ahmaruzzaman, High-value utilization of egg shell to synthesize silver and goldesilver core shell nanoparticles and their application for the degradation of hazardous dyes from aqueous phase-a green approach, J. Colloid Interface Sci. 453 (2015) 115e131. [17] D.M. Mott, D.T. Anh, P. Singh, C. Shankar, S. Maenosono, Electronic transfer as a route to increase the chemical stability in gold and silver core-shell nanoparticles, Adv. Colloid Interface Sci. 185-186 (2012) 14e33. [18] I.J. Godfrey, A.J. Dent, I.P. Parkin, S. Maenosono, G. Sankar, Structure of goldesilver nanoparticles, J. Phys. Chem. C 121 (2017) 1957e1963. [19] C. Gao, Y. Hu, M. Wang, M. Chi, Y. Yin, Fully alloyed Ag/Au nanospheres: combining the plasmonic property of Ag with the stability of Au, J. Am. Chem. Soc. 136 (2014) 7474e7479. [20] M.P. Mallin, C.J. Murphy, Solution-phase synthesis of sub-10 nm Au-Ag alloy nanoparticles, Nano Lett. 2 (2002) 1235e1237.

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