Evaluation of Antimicrobial Activity of Ionic Liquid-Assisted Synthesis of Monometallic Silver and Bimetallic Copper-silver Nanoparticles

The formation of monometallic silver and bimetallic copper-silver nanoparticles in 1-butyl-3methylimidazolium methanesulfonate ionic liquid, through chemical reduction is reported. The synthesized particles were characterized using SEM/EDX, UV-vis, and FTIR spectroscopy. UV-vis and FTIR revealed the formation of nanoparticles with active components being adsorbed on the surface of the particles, as stabilizers. SEM revealed uniformed microspheres and microcubes for AgNPs and AgCuNPs, respectively. On the bactericidal and fungicidal activity of AgNPs and AgCuNPs against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebseilla pneumonia (bacteria) and Candidaalbicans (fungus), we observed that AgNPs inhibited Pseudomonas aeruginosa (23 mm) and Candida albicans (29 mm) higher than the bimetallic particles and the antibiotics used as control. It is interesting to note that AgCuNPs inhibited Staphylococcus aureus (21 mm) better than AgNPs (15 mm) indicative of the synergistic effect of two metals. Introduction The interest in metal / or bimetallic nanoparticles stems from their applications in diverse areas such as catalysis, chemical sensors, biocatalysts and nanomaterial technology (Yu et al., 2012; Esteban et al., 2015; Marcos et al., 2015; Hatakeyama et al., 2016; Janiak , 2014). In particular, silver nanoparticles (Ag-NPs) are potential building blocks for the creation of new materials with tailored properties for optical and medical applications (Graf et al., 2009; Mantion et al., 2008; 2011). Silver nanoparticles have proven useful in sports wears due to their anti-bacterial tendency and their compatibility with the human system at low concentration which have made them useful as disinfectants. Also, their unique optical property has made them useful in decorative arts and human make up (Ayi et al., 2014; Pacioni et al., 2015). It has been demonstrated by different research groups that ionic liquids (ILs) offer outstanding possibilities as a green solvent in the synthesis of metal nanoparticles (Wegner and Janiak 2017; Zhu and Hou, 2012, Ayi et al., 2011; 2015). This is not only as a result of achievable high reaction and nucleation rates with the formation of small particles, but also for their electronic and steric stabilization that prevents particle aggregation [Richter et al., 2013]. Imidazolium-based ILs have been extensively utilized in the formation and stabilization of M-NPs (Khare et al., 2010; Ayi et al., 2010; Ayi et al., 2015; Zhang et al., 2015 ). Imidazolium ILs are air, water and electrochemically stable with a wide liquidus range. 1-Alkyl-3-alkyl’-imidazolium ILs simultaneously act as reaction media, hydrogen sources, catalysts, templating agents and stabilizers for the synthesis of metal nanoparticles. According to Werner and Janiak (2017), there are ILs which have a strong influence on particle formation, good nucleation aids, but poor stabilizers, good nucleation aids and good stabilizers, and those which are none of this. ILs with the 1-n-butyl-3-methylimidazolium [BMIM] cation and the relatively weakly coordinating anions such as tetrafluoroborate, hexafluorophosphate and trifluoromethanesulfonate, are liquids over a large range of temperatures, possess high thermal and chemical stability, a large electrochemical window, high ion density, relatively low viscosity, and negligible vapor pressure [ Dupont et al., 2002]. The present study reports on the antimicrobial activities of silver monometallic and copper-silver bimetallic nanoparticles synthesized in 1-n-butyl-3methylimidazolium methane sulfonate [BMIM][MS] ionic liquid. Evaluation of Antimicrobial Activity of Ionic Liquid-Assisted Synthesis of Monometallic Silver and Bimetallic Copper-silver Nanoparticles http://www.ijSciences.com Volume 7 – May 2018 (05) 26 Materials and Methods Materials All chemicals (1-Butyl-3-methyl-imidazolium methane sulfonate (C9H10N2O3S) ionic liquid, silver nitrate (AgNO3), sodium borohydride (NaBH4)) were of analytical grade and were used as purchased from Sigma-Aldrich. Synthesis of AgNPs (monometallic) The procedure adopted for the synthesis of silver nanoparticles (AgNP) used 1-butyl-3-methylimidazolium methane sulfonate ionic liquid as a solvent and sodium borohydride as reducing agent. In a typical synthesis, AgNO3 (1.0g, 5.83 ×10 -06 mM)was dispersed in 1-butyl-3-methyl-imidazolium methane sulfonate ionic liquid (1.4g, 5.97× 10 mM)followed by the addition ofNaBH4(0.74g, 1.938×10 mM) under continuous stirring. The resultant black mixture was heated at 120 °C under reflux. Six portions of 1cm of the mixture was taken out from the reaction vessel after every 1 h interval, the reaction was stopped after 6 hours, a colour change from black to ash with a dirty white supernatant was observed. The products were centrifuged at 3000 rpm for 15 min, filtered and wash with distilled water, dried at room temperature and stored in airtight container for further analysis. Synthesis of CuAgNPs bimetallic nanoparticles The procedure adopted for the synthesis of Cu-Ag bimetallic nanoparticles (Cu-AgNP) used Copper (II) chloride, silver nitrate, 1-butyl-3-methyl-imidazolium methane sulfonate, sodium borohydride. In a typical synthesis, AgNO3 (2.0g, 1.17×10 -05 mM) was dispersed in BMIMMS ionic liquid (4.0g, 1.74×10 -05 mM) followed by CuCl2 (2.0g, 2.02 × 10 -05 mM). The reducing agent, NaBH4 (0.3g, 7.930 ×10 mM) was then added and the reaction mixture was stirredcontinuously for 30 mins. The resultant homogenous black colloidalmixture was heated under reflux at 120°C for 6 h. Six portions of 1cm of the mixture were taken out from the reaction vessel after every 1h for UV-vis measurements. A colour change from black to light green was observed at the end of the reaction time. The products were centrifuged at 3000 rpm for 15 min, washed with distilled water, dried at room temperature and stored in an airtight container for further analysis. Characterization The reductions were monitored using UV-visible spectrophotometer (Evolution 201 spectrophotometer) at regular interval with samples dissolved in ethanol using quartz cuvette operated with a resolution of 1cm. The FTIR Spectrophotometer (Shimadzu IR Affinity1S) in the spectral range 4000–500 cm KBr pellets (sample: KBr = 20 : 1). The scanning electron microscopy (SEM) was performed on a Hitachi S4800 microscope attached with EDX at a voltage of


Introduction
The interest in metal / or bimetallic nanoparticles stems from their applications in diverse areas such as catalysis, chemical sensors, biocatalysts and nanomaterial technology (Yu et al., 2012;Marcos et al., 2015;Hatakeyama et al., 2016;Janiak , 2014). In particular, silver nanoparticles (Ag-NPs) are potential building blocks for the creation of new materials with tailored properties for optical and medical applications (Graf et al., 2009;Mantion et al., 2008;2011). Silver nanoparticles have proven useful in sports wears due to their anti-bacterial tendency and their compatibility with the human system at low concentration which have made them useful as disinfectants. Also, their unique optical property has made them useful in decorative arts and human make up (Ayi et al., 2014;Pacioni et al., 2015). It has been demonstrated by different research groups that ionic liquids (ILs) offer outstanding possibilities as a green solvent in the synthesis of metal nanoparticles (Wegner and Janiak 2017; Zhu and Hou, 2012, Ayi et al., 2011;. This is not only as a result of achievable high reaction and nucleation rates with the formation of small particles, but also for their electronic and steric stabilization that prevents particle aggregation [Richter et al., 2013]. Imidazolium-based ILs have been extensively utilized in the formation and stabilization of M-NPs Ayi et al., 2010;Ayi et al., 2015;Zhang et al., 2015 ). Imidazolium ILs are air, water and electrochemically stable with a wide liquidus range. 1-Alkyl-3-alkyl'-imidazolium ILs simultaneously act as reaction media, hydrogen sources, catalysts, templating agents and stabilizers for the synthesis of metal nanoparticles. According to Werner and Janiak (2017), there are ILs which have a strong influence on particle formation, good nucleation aids, but poor stabilizers, good nucleation aids and good stabilizers, and those which are none of this. ILs with the 1-n-butyl-3-methylimidazolium [BMIM] cation and the relatively weakly coordinating anions such as tetrafluoroborate, hexafluorophosphate and trifluoromethanesulfonate, are liquids over a large range of temperatures, possess high thermal and chemical stability, a large electrochemical window, high ion density, relatively low viscosity, and negligible vapor pressure [ Dupont et al., 2002].
The present study reports on the antimicrobial activities of silver monometallic and copper-silver bimetallic nanoparticles synthesized in 1-n-butyl-3methylimidazolium methane sulfonate [BMIM][MS] ionic liquid.

Synthesis of AgNPs (monometallic)
The procedure adopted for the synthesis of silver nanoparticles (AgNP) used 1-butyl-3-methylimidazolium methane sulfonate ionic liquid as a solvent and sodium borohydride as reducing agent. In a typical synthesis, AgNO 3 (1.0g, 5.83 ×10 -06 mM)was dispersed in 1-butyl-3-methyl-imidazolium methane sulfonate ionic liquid (1.4g, 5.97× 10 -06 mM)followed by the addition ofNaBH 4 (0.74g, 1.938×10 -05 mM) under continuous stirring. The resultant black mixture was heated at 120 °C under reflux. Six portions of 1cm 3 of the mixture was taken out from the reaction vessel after every 1 h interval, the reaction was stopped after 6 hours, a colour change from black to ash with a dirty white supernatant was observed. The products were centrifuged at 3000 rpm for 15 min, filtered and wash with distilled water, dried at room temperature and stored in airtight container for further analysis.

Synthesis of CuAgNPs bimetallic nanoparticles
The procedure adopted for the synthesis of Cu-Ag bimetallic nanoparticles (Cu-AgNP) used Copper (II) chloride, silver nitrate, 1-butyl-3-methyl-imidazolium methane sulfonate, sodium borohydride. In a typical synthesis, AgNO 3 (2.0g, 1.17×10 -05 mM) was dispersed in BMIMMS ionic liquid (4.0g, 1.74×10 -05 mM) followed by CuCl 2 (2.0g, 2.02 × 10 -05 mM). The reducing agent, NaBH 4 (0.3g, 7.930 ×10 -06 mM) was then added and the reaction mixture was stirredcontinuously for 30 mins. The resultant homogenous black colloidalmixture was heated under reflux at 120°C for 6 h. Six portions of 1cm 3 of the mixture were taken out from the reaction vessel after every 1h for UV-vis measurements. A colour change from black to light green was observed at the end of the reaction time. The products were centrifuged at 3000 rpm for 15 min, washed with distilled water, dried at room temperature and stored in an airtight container for further analysis.

Characterization
The reductions were monitored using UV-visible spectrophotometer (Evolution 201 spectrophotometer) at regular interval with samples dissolved in ethanol using quartz cuvette operated with a resolution of 1cm 3 . The FTIR Spectrophotometer (Shimadzu IR Affinity-1S) in the spectral range 4000-500 cm -1 KBr pellets (sample: KBr = 20 : 1). The scanning electron microscopy (SEM) was performed on a Hitachi S-4800 microscope attached with EDX at a voltage of 15Kv.

Antimicrobial studies
Antimicrobial susceptibility test was done in the bacteriology lab of the General Hospital Calabar, Cross River State, Nigeria. The agar diffusion test (disc diffusion method) was adopted [Prescott et al., 2005]. Muller-Hilton agar was prepared from a dehydrated base according to the manufacturer's instruction. The medium was allowed to cool to 47 °C and poured into petri-dishes that were arranged and labeled according to their microbial isolates, and allowed to set on a level surface to a depth of approximately 4 mm. When the agar had solidified, the plates were dried for 20 minutes at 35°C by placing them in an upright position in hot air oven with the lids tilted. A discrete colony of each of the isolate was picked with a sterile wire loop and streaked on the Muller-Hilton agar according to their names as labeled: staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebseilla pneumonia (bacteria) and Candidaalbicans (fungus).

Results and Discussions
Nanoparticles of silver and copper-silver alloy were synthesized in 1-butyl-3-methylimidazolium methanesulfonate ionic liquid via chemical reduction byNaBH 4 . The reduction reactions were performed under reflux at a temperature of 120 o C. The colour of AgNPs changed from dark to grey, while that of AgCuNPs changed from dark to green, indicating the possible oxidation of metallic copper to copper chloride. The effect of heating time on the reaction was monitored with UV-vis spectroscopic analysis. Figure 1 shows representative UV/vis spectra of AgNPs (Fig. 1a) and AgCuNPs (Fig. 1b  According to Derjaguin-Landau-Verwey-Overbeek theory (DLVO theory), the anion is the primary source of stabilization for the metal nanoparticles (Verwey and Overbeek 1999;Finke, 2002). The metal nanoparticles and their anion layer on the surface form an overall negatively charged particle and are subject to Coulomb repulsion within the DLVO theory [Vollmer and Janiak, 2011]. Thus the ionic liquid cations are attracted to the surface of a negatively charged nanoparticle to form a positive ion layer, and then counter anions form a second layer on the nanoparticle surface by electrostatic attraction [Obliosca et al., 2010;Rubim et al., 2008;Schrekker et al., 2007].To determine the possible functional groups involved in the stabilization of both AgNPs and the bimetallic AgCuNPs, FTIR spectroscopy was employed (Fig. 4). The spectra showed broad absorption band at 3458 cm -1 for AgNPs, which can be assigned to stretching vibration  Antimicrobial activities of the synthesized AgNPs and AgCuNPs were investigated using standard agar diffusion assay. The nanoparticles showed inhibition zone against all the studied bacteria and fungus (Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas and Candida albicans sp) as presented in Figure 5 and Figure 6. The zone of inhibition of AgNPs against Pseudomonas and Candida albicans are 23 and 29 mm, respectively which are higher than the inhibition zones of the bimetallic particles (AgCuNPs) and also the antibiotics used as control. Monometallic silver nanoparticles inhibited other organisms except staphylococcus aureus better than the bimetallic copper-silver nanoparticles. While AgCuNPs inhibited Staphylococcus aureus (21 mm) better than AgNPs (15 mm)  . On a general scale, it is worthy of note that; nanobiotics (nanoparticles) synthesized from 1-butyl-3-methyl imidazolium methane sulfonate showed prominent inhibition against the microbes under study than antibiotics most especially, racinef.

Conclusion
Using ionic liquid as a solvent and stabilizer, silver monometallic and silver-copper bimetallic nanoparticles have been successfully synthesized via chemical reduction with NaBH 4 . The UV-vis and IR measurements showed that nanoparticles were formed and that molecules of the ionic liquids were adsorbed on the surface of the particles. An EDAX measurement also lends credence to this fact. The silver monometallic nanoparticles were found to be spherical while the bimetallic particles are cubic in shape. The larger zone of inhibition obtained against microbes proves that the synthesized nanoparticles in ionic liquids have good potentials in drug delivery applications.