Nano silver copper (Ag / Cu bimetallic) increases antibacterial activity
Silver and copper nanoparticles are produced by chemically reducing their respective nitrate salts with ascorbic acid in the presence of chitosan using a microwave oven. Particle size increased with increasing nitrate concentration and decreasing chitosan concentration. The zeta potential on the surface was positive for all the nanoparticles produced and they varied from 27.8 to 33.8 mV. The antibacterial activity of Ag, Cu, Ag and Cu mixture, and copper silver nanoparticles (Ag / Cu bimetallic) were tested using Bacillus subtilis and Escherichia coli. Out of the two, B. subtilis proved more susceptible under all investigated conditions.
The silver nanoparticles had higher activity than the copper nanoparticles and the mixture of nanoparticles with the same average particle size. However, when compared on the basis of equal concentrations, Cu nanoparticles were more lethal due to their higher surface area. The highest antibacterial activity was obtained with Ag / Cu bimetallic nanoparticles with minimum inhibitory concentrations (MIC) of 0.054 and 0.076 mg / L for B. subtilis and E. coli, respectively.
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1. INTRODUCTION
Interest in metal nanoparticles as antimicrobial agents has been around for more than a decade (Dai & Bruening, 2002). The large number of particles can be produced and the high surface area to volume ratio allows efficient nanoparticles in very small quantities (Sundaresan, Sivakumar, Vigneswaran, & Ramachandran, 2012).
Ag nanoparticles have been widely used for biomedical applications (Marambio-Jones & Hoek, 2010). Cu is relatively non-toxic to mammals (Flemming & Trevors, 1989) but is toxic to many microorganisms and this offers new prospects for antibiotic treatments (Hsiao, Chen). , Shieh, & Yeh, 2006).
Several methods of producing Ag and Cu nanoparticles have been developed using both physical and chemical approaches. A commonly used method to prepare Ag nanoparticles is to chemically degrade a silver salt solution in water or an organic solvent to create a colloidal suspension. The most common approach to synthesis of Cu nanoparticles is to create micro-emulsions (Solanki, Sengupta, & Murthy, 2010).
However, micro-emulsion techniques requiring large amounts of surfactants and organic solvents increase production costs (El-Nour, Eftaiha, Al-Warthan, & Ammar, 2010). Physical methods using laser ablation, radiolysis, or aerosol, although effective, require expensive equipment and large amounts of energy (Thakkar, Mhatre, & Parikh). , 2010). These methods often use agents that are both toxic and polluting (Lim & Hudson, 2004).
Chemical-based methods offer the best opportunity at both low cost and environmental friendliness. Ag nanoparticles have been synthesized using water as solvents and starch as capping agents and they have been shown to have advantages over conventional methods involving chemical agents. related to environmental toxicity (Sharma, Yngard, & Lin, 2009).
The synthesis of Ag nanoparticles using chitosan as a reducing and acting agent has also been developed (Sanpui, Murugadoss, Prasad, Ghosh, & Chattopadhyay, 2008). In addition, Cu nanoparticles have been fabricated using alginate as stabilizer (Díaz-Visurraga et al., 2012).
More recently, nanoparticles related to alloys of two metals have been produced. Valodkar, Modi, Pal, & Thakore (2011) synthesized Ag / Cu copper silver bimetallic nanoparticles using starch and Said-Galiev et al. (2011) synthesized Ag and Cu nanoparticles using chitosan.
They were treated with supercritical carbon dioxide, then reduced Ag and Cu metal-mechanical complexes with hydrogen to form metal-chitosan nanoparticles. Ag / Cu copper silver bimetallic nanoparticles are also produced from a solution of silver nitrate and copper acetate with hydrazine hydrate as a reducing agent (Taner, Sayar, Yulug, & Suzer, 2011).
This work is an attempt to further develop the green synthesis of Ag and Cu nanoparticles, a mixture of Ag and Cu nanoparticles (denoted “Ag + Cu”) and alloyed nanoparticles. of Ag and Cu (nano silver copper denoted “Ag / Cu”) with chitosan as a stabilizer and using microwave heating.
The attraction of using chitosan for this function compared to starch is that it has antibacterial properties (No, Park, Lee, & Meyers, 2002), and can be easily dissolved using organic acids. (Muzzarelli et al., 1984; Muzzarelli, 1985). Chitosan can form various chemical bonds with metal components thereby enhancing the stability of the nanoparticles (Muzzarelli, 2011).
It has low toxicity and is therefore safe for human applications (Muzzarelli, 2010), although it is recognized that the metal nanoparticles produced may have some environmental toxicity ( Li et al., 2010).
In this study, the nanoparticles were synthesized at different concentrations of chitosan. The synthesized nanoparticles are characterized by spectroscopic measurements and by using a zetasizer. Their antibacterial properties were tested using Bacillus subtilis and Escherichia coli.
2. MATERIALS AND METHODS
2.1 Reagents
Copper (II) nitrate (Cu (NO3) 2. XH2O) (Sigma Aldrich Chemie GmbH, Steinheim, Germany), silver nitrate (AgNO3) (BDH Ltd, Poole, UK), L-ascorbic acid (Sigma Aldrich, Poole , UK), acetic acid (Fisher Scientific, Loughborough, UK) and chitosan (Sigma Aldrich, Poole, UK) have been used for the synthesis of nanoparticles.
The molecular weight of chitosan ranges from 50 000 to 190 000 Da and has been reduced from 75-85%. All reagents were used without further refining.
2.2 Preparation of chemical solution
All of the following solutions were prepared using distilled water: silver nitrate (10, 30 and 50 mM), copper nitrate (10, 30 and 50 mM), ascorbic acid (10% w / v). A solution of chitosan (1, 2 and 3% w / v) was prepared by dissolving chitosan in 1% (v / v) acetic acid solution. They were then left for 3 days to allow the chitosan to completely dissolve (Wei, Sun, Qian, Ye, & Ma, 2009).
2.3 Preparation of nanoparticles
2.3.1 Ag and Cu nanoparticles
To prepare Ag or Cu nanoparticle solution, 40 mL silver nitrate solution or copper nitrate (10, 30 or 50 mM) mixed with 40 mL chitosan solution (1, 2 or 3% w / v) and 4 mL 10% ascorbic acid solution (w / v). The reduction reaction was performed by heating in a microwave oven (EM-SI067 UK, Sanyo, China) at a maximum power of 800 W for 4 minutes (Valodkar, Modi, Pal, & Thakore, 2011).
2.3.2 The bimetallic Ag / Cu nanoparticles
To prepare the following nano silver copper (Ag/Cu bimetallic) called “Ag / Cu” nanoparticles, 20 mL of silver nitrate solution and 20 mL of copper nitrate solution were mixed with 40 mL 3% chitosan (w / v) and 4 mL ten%. (w / v) ascorbic acid solution. The reaction was then performed in a microwave oven at a maximum power of 800 W for 4 minutes (Valodkar et al., 2011).
2.3.3 Mixture of Ag and Cu nanoparticles
To prepare a simple mixture of Ag and Cu nanoparticles, then called “Ag + Cu” nanoparticles, 40 mL of Ag nanoparticles and 40 mL of Cu nanoparticles were separately synthesized in 3% (w / v) chitosan as described in section 2.3.1 and then mixed together (Valodkar et al., 2011).
2.4 Properties of nanoparticles
2.4.1 Spectral measurement
UV-vis absorption spectra of Ag / Cu nanoparticle solutions, Ag / Cu copper silver bimetallic Ag / Cu and Ag + Cu were performed over the wavelength range 200 to 800 nm by UV-vis spectrophotometer model UV mini-1240 (Shimadzu Corporation, Kyoto, Japan).
2.4.2 Particle size and zeta potential analysis
The particle size of Ag, Cu, nano silver copper (Ag/Cu bimetallic) and Ag + Cu nano solutions was measured with a Zetasizer device (Model ZEM5002, Malvern Instruments Ltd, Malvern, UK) using UV Grade cuvettes after treatment in a super water bath. negative (Model FB11012, Fisherbrand, Loughborough, UK) for 30 minutes to subdivide whatever population is present (Ribeiro, Hussain, & Florence, 2005). The zeta potential of each nanoparticle was measured using the Zetasizer 3000HS model (Malvern Instrument Ltd, Malvern, UK). All measurements were taken in triplicate.
2.5 Microbiological method
2.5.1 Bacteria
Gram-positive bacteria, Bacillus subtilis ATCC 6633 were obtained from the National Collection of Industrial, Food and Marine Bacteria (NCIMB), Aberdeen, Scotland, and Gram-negative bacteria, E. coli K12 by Dr. Jon Hobman in Nottingham is pleased to donate to University, Nottingham, UK.
2.5.2 Bacterial culture
Bacteria were collected from frozen storage (-80 oC) and spread over Tryptone Soy Agar (TSA) and incubated overnight at 37 oC. A single colony was then used to inoculate 100 mL of Tryptone Soy Broth (TSB) in a 500 mL Erlen flask, then placed in an incubator shaken at 37 ° C at 140 rpm for 12 hours.
This 100µL inoculum was then used to inoculate 100 mL of fresh TSB incubated under the same conditions as described above until a mid logarithmic phase was reached. The culture at this time is appropriately diluted in phosphate buffered saline (PBS) to produce a suspension containing colony-forming units (CFU) per mL. Identical procedures were followed for both B. subtilis and E. coli.
2.5.3 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
A series of dilutions of the nanoparticles in sterile distilled water were prepared and 4 mL of the dilutions were added to 20 mL of TSB medium with 20 mL of 108 CFU / mL bacteria and incubated in a shaker. incubators (CERTOMAT® BS-1 type, Sartorius, Göttingen, Germany) at 37 ° C overnight (Cao et al., 2010).
MIC was determined by visual observation and confirmed by turbidity measurements with a UV-vis spectrophotometer (Jenway 6300, Bibby Scientific Ltd, Essex, UK) at a wavelength of 600 nm before and after incubation. Diluted portions (100µL) of non-observed bacterial growth were coated using TSB to determine the minimum lethality concentration (MBC).
Samples were incubated at 37 ° C overnight and colonies formed were observed. MIC was determined to be the lowest concentration inhibiting visible bacterial growth tested (Wei et al., 2009).
2.6 Statistical analysis
Data from the three-fold experiments are presented as mean and standard error values of mean. Statistical analysis of results was performed using one-way ANOVA with the Bonferroni comparative multi-post hoc test. All statistical analyzes were performed using IBM SPSS Statistics 21.0 (SPSS UK Ltd., Surrey, United Kingdom).
3. RESULTS AND DISCUSSION
3.1 Physical Characteristics
Pictures of the nanoparticle suspension prepared according to the methods described above are shown in Figure 1. The formation of a colloidal suspension of the nanoparticles is evident when visually inspecting the reagent mixture after processing. microwave heat.
The light yellow Ag nanoparticles (Figure 1a) became darker with increasing chitosan concentration. The formation of metal nanoparticles was confirmed by UV-vis spectroscopy. Figure 2a shows an absorption spectrum with a maximum at about 420 nm, indicating the formation of Ag nanoparticles (Ahmad et al., 2003).
Ag nanoparticles have previously been reported to be yellowish in aqueous solution (Perera et al., 2013). Color and absorption spectra at 420 nm are due to excitation of the surface plasmon oscillation of Ag atoms (Twu, Chen, & Shih, 2008).
The increase in chitosan concentration was found to lead to a small change in peak absorbance along with an overall increase in absorption. These results are consistent with those obtained by Huang, Yuan, & Yang (2004), who synthesized Ag nanoparticles in chitosan by reduction with sodium borohydride.
Cu nanoparticles (Figure 1b), whose color changes from light pink to dark red with increasing chitosan concentration. UV spectra (Figure 2b) showed the maximum at 550 nm, confirming the presence of Cu nanoparticles. They have been reported to range from 500 to 600 nm (Mallick, Witcomb, & Scurrell, 2006).
Similarly, it has been reported that the color of the newly synthesized Cu nanoparticles stabilized using a water-soluble aminoclay matrix suspension is the red color, characteristic of Cu nanoparticles (Datta, Kulkarni, & Eswaramoorthy, 2010).
Figure 1c shows the mixture of Ag and Cu nanoparticles that are orange in color (as expected from the blend) but the Ag and Cu alloy nanoparticles are dark brown. Taner et al. (2011) formed bimetallic Ag / Cu nanoparticles by desalting metal in aqueous solution with hydrazine hydrate and also reported that the color of the suspension was dark brown. UV-vis spectra of Ag + Cu and nano silver copper (Ag/Cu bimetallic) are shown in Figure 2c.
Ag + Cu spectra showed two distinct peaks at 420 and 550 nm, showing the physical mixture of Ag and Cu nanoparticles. On the other hand, the nano silver copper (Ag/Cu bimetallic) showed a single absorption peak at an intermediate ratio confirming that a bimetallic alloy was formed (Valodkar et al., 2011).
In this study, chitosan was dissolved with dilute acetic acid solution. Chitosan reacts with H + from acid solution to produce proton chitosan with –NH3 + function group. The inclusion of these functional groups in the frame of the chitosan improves its solubility in water. When silver and copper nitrate are added to chitosan solution, Ag + and Cu2 + ions will attach to the chitosan macromolecules by electrostatic interaction, since the electron-rich oxygen atoms of polar hydroxyl groups and ethers of chitosan have ability to interact with electrified metal cations.
A reducing agent is required to provide the free electrons needed for deionization and the formation of nanoparticles (Tolaymat et al., 2010). In this study, ascorbic acid was used as a reducing agent. Therefore, Ag and Cu nanoparticles formed by reducing Ag + and Cu2 + ions correspond to residual ascorbic acid (for 100% conversion).
The Ag and Cu nanoparticles were stabilized with protonated chitosan to prevent agglutination and control the size of the final generated nanoparticles. The same mechanism applies to the synthesis of nano silver copper (Ag/Cu bimetallic).
3.2 Average particle size
The changes in UV-vis absorption mentioned above show that the size of the nanoparticles formed has changed with the concentration of chitosan, acting as a nucleation controller as well as a stabilizer. (Huang et al., 2004).
Figure 3 shows the average particle size of Ag and Cu nanoparticles synthesized with 10 mM saline solution at different chitosan concentrations. It was observed that the mean particle size of both Ag and Cu nanoparticles decreased with increasing chitosan concentration and for the same chitosan concentration Ag nanoparticles were slightly larger than Cu nanoparticles as shown. This reduction is due to the protective effect of chitosan whereby chitosan can inhibit the growth of nanoparticles by adsorption to their surface (Esumi, Takei, & Yoshimura, 2003).
The effect of silver and copper nitrate concentration on the average particle size of the nanoparticles is shown in Figure 4. As the concentration of silver and copper nitrate increased from 10 to 50 mM, the size of the nanoparticles increased nearly. as corresponding. At the same chitosan concentration, with increased metal salt concentration, less protonated chitosan is absorbed into the pre-formed nanoparticles, and thus larger nanoparticles are formed (Leung, Wong, & Xie, 2010).
Statistical analysis showed that the mean particle size of the nanoparticles was significantly different and influenced by the concentration of chitosan and metal salts. Therefore, the size of the nanoparticles can be controlled by adjusting the concentration of metal ions or the concentration of chitosan during the synthesis.
3.3 The Zeta potential
The zeta potential is an important parameter for determining the stability of the nanoparticle suspension. For a physically stable nanoparticle suspension to be stabilized only by electrostatic repulsion, a minimum zeta potential of ± 30 mV must be obtained (Singare et al., 2010). The mutual repulsion of the nanoparticles is dependent on having a large positive or negative zeta potential.
The Zeta potential measurements of the nanoparticles are shown in Figure 5. This shows that the Ag nanoparticles with a positive surface charge increase with the chitosan concentration from +23.8 mV at 1% (w / v). ) to +32.1 mV at 3% w / v chitosan solution.
The Cu nanoparticles had a slightly higher positive surface charge, ranging from +26.4 mV at 1% (w / v) to +33.9 mV at 3% (w / v), though in terms of No statistically, zeta potentials of Ag and Cu nanoparticles differ significantly.
The zeta potential of the nanoparticles increased with the chitosan concentration due to the greater availability of protons – NH3 + on the surface of the formed nanoparticles. This will create a greater electrostatic repulsion between the particles and thus a lower rate of agglutination and lead to a more stable nanoparticle dispersion.
Figure 5b shows the zeta potential of Ag, Cu, Ag + Cu and nano silver copper (Ag/Cu bimetallic) nanoparticles prepared from 50 mM metal salt solution and 3% chitosan solution (w / v) respectively. In this case, the nano silver had a slightly higher surface charge (+37.8 mV) than the Cu nanoparticles (+35.5 mV).
Mixed nanoparticles (Ag + Cu) give +39.1 mV value and Ag / Cu nanoparticles are one of +35.2 mV. These differences are not statistically significant. Since there may be an effect of particle size on the zeta potential of four types of nanoparticles (Ag, Cu, Ag + Cu and Ag / Cu), the zeta potential is compared for the selected sample that corresponds closest to the particle size. average of 200 nm.
They are synthesized in 3% chitosan (w / v) and correspond to metal ion concentrations of 12.1, 32.0 and 18.2 mM for Ag, Cu and Ag / Cu respectively. Ag + Cu was generated by mixing Ag prepared at 12.1 mM with Cu prepared at 32.0 mM. The corresponding zeta potentials are +33,8, 33,1, 32,6 and 33,3 mV for Ag, Cu, Ag + Cu and Ag / Cu (Figure 5c).
They are very similar to what is shown in Figure 5b where all samples were prepared with a 50 mM solution (corresponding to average particle sizes of 793, 292, 542 and 633 nm respectively). This finding showed that the metal salt concentration used in the preparation did not significantly affect the charge of the nanoparticles.
The results obtained here are consistent with those of Xiong et al. (2013), in which it was reported that the zeta potential of Cu nanoparticles is +32 mV, indicating that the value is high enough to maintain stable colloidal dispersion.
3.4 Antimicrobial properties
MIC and MBC tests were performed to evaluate the antibacterial activity of the nanoparticles against B. subtilis and E. coli. MIC is defined as the lowest concentration at which no visible growth while MBC is defined as the lowest concentration at which no colonies are observed (Wei et al., 2009 ).
MIC and MBC values for B. subtilis and E. coli of Ag, Cu, nano silver copper (Ag/Cu bimetallic) and Ag + Cu nanoparticles prepared from a 50 mM metal salt solution are shown in Table 1. MIC or MBC Lower corresponds to a higher antimicrobial effect. As a control, to assess the possible antibacterial effect of ascorbic acid, separate tests were performed in which samples were prepared according to the method in Section 2.3.1, but without chitosan and silver or copper. and nitrate is tested against both E. coli and B. subtilis.
These samples did not show bactericidal effects when undiluted, but had an inhibitory effect at maximum intensity and 1/2 dilution. However, under such conditions, the ascorbic level is usually one thousand times greater than the level contained in the nanoparticle suspension detailed in Table 1, and therefore all antimicrobial effects are directly attributable to the presence of Ascorbic acid in our experiments can be ignored.
The results showed that Ag nanoparticles had MIC and MBC values for B. subtilis and E. coli significantly higher than the Cu nanoparticles at the same concentration of chitosan (3% w / v) and metal salts ( 50 mM). The results presented here demonstrate that at the same metal salt concentration, Cu nanoparticles have a characteristic smaller particle size than Ag nanoparticles.
Smaller particle sizes tend to enhance antibacterial properties because as the size decreases, there is a greater number of atoms on the surface available to interact with the bacteria (Marambio-Jones & Hoek, 2010). A mixture of Ag and Cu nanoparticles at 50 mM showed intermediate behavior although this was statistically similar to Ag nanoparticles for both bacteria. The 50 mM copper silver bimetallic nanoparticles showed the highest antibacterial efficiency.
All nanoparticles showed very similar values for MIC and MBC, which indicated that the nanoparticles had bactericidal rather than bacteriostatic effect on these two bacteria. Valodkar et al., 2011 reported MIC and MBC values of 0.26 and 0.78 mg / L for Ag nanoparticles of 10 mM and MIC and MBC as 0.23 and 0.65 mg / L for nanoparticles. 10 mM Ag / Cu nano silver copper (Ag/Cu bimetallic) resistant to the lower bacterial concentrations (104 CFU / mL) of E. coli than used here (108 CFU / mL).
Taner et al., 2011 reported MIC values of only> 150 mg / L for Ag nanoparticles and identical values for MIC and MBC of copper silver nanoparticles as 0.5 mg / L compared with High concentration (108 CFU / mL) of E. coli. The reported MIC / MBC values here were lower than those obtained by Valodkar et al., 2011 and Taner et al., 2011 showed better antimicrobial activity and suggested that chitosan, play a role. Being a stabilizer also contributes to the antibacterial effect.
The results obtained here cannot be directly compared with those of Said-Galiev et al. (2011) because they did not report their findings on MIC or MBC. Huang et al. (2004) synthesized nanoparticles using chitosan but did not report the antimicrobial activity of their nanoparticles. The findings reported here show that the MIC / MBC value of the bimetallic alloy nanoparticles is substantially lower than that of the metal pure metal nanoparticles.
Since particle size will influence antimicrobial activity, due to differences in specific surface areas, the samples compared with each other in Table 2 keep the average particle size almost constant across all samples. Samples of approximately 200 nm (as performed for the zeta potential are shown in Figure 5c.
On this basis, the Ag nanoparticles showed significantly lower MIC and MBC values than the Cu nanoparticles for both types of bacteria. Therefore, it can be surmised that Ag has a greater intrinsic antimicrobial activity than Cu. Lowest MIC and MBC values were observed copper silver nanoparticles exposed to both B. subtilis and E. coli at the same metal salt concentration and chitosan or fixed nanoparticle size.
The Cu nanoparticles show statistically the same antibacterial effects as obtained with the mixture of Ag and Cu nanoparticles towards both B. subtilis and E. coli. Bacteria are negatively charged due to an excess of carboxylic numbers and other groups that cause the cell surface to become negative (Stoimenov, Klinger, Marchin, & Klabunde, 2002). The nanoparticle suspension produced here has a positive charge as revealed by zeta potential measurements.
The electrostatic force between positively charged nanoparticles and negatively charged bacterial cells enhances the effect of antimicrobial activity. Adhesion of the nanoparticles to the surface of the bacteria changes its membrane properties, ultimately causing death (Li et al., 2008). MIC and MBC of synthetic nanoparticles against both B. subtilis and E. coli showed that Gram-positive bacteria were more sensitive than Gram-negative bacteria to nanoparticles. This is most likely due to the structural difference between Gram-positive and Gram-negative bacterial cell walls with Gram-negative cell walls having a more complex structure than Gram-positive cell walls.
4. CONCLUSION
Ag and Cu nanoparticles were synthesized using ascorbic acid as a reducing agent in chitosan solution by efficient microwave heating method. Moreover, the synthesis is quick, inexpensive, environmentally benign, energy-saving and does not generate harmful waste. It turns out that the nanoparticle size can be controlled by adjusting the concentrations of chitosan and silver and copper nitrate used in their synthesis.
The particle size can be increased by reducing the concentration of chitosan or increasing the concentration of metal ions. The generated nanoparticles have a positive surface charge and the chitosan used in their synthesis contributes to the stability of those particles’ suspensions and prevents agglomeration.
MIC and MBC tests showed a strong bactericidal effect with Ag nanoparticles showing higher killing efficiency when compared with Cu nanoparticles at the same average particle size. All nanoparticles showed very similar values for MIC and MBC, which indicated that the nanoparticles had bactericidal rather than bacteriostatic effect on these two bacteria. The greatest antibacterial effect is seen when Ag and Cu are combined during synthesis to form alloy particles. The latter use in medical applications is currently under investigation.
LIST OF FIGURES
Figure 1. Nanoparticle suspension of (a) Ag and (b) Cu synthesized into 10 mM metal salts and chitosan with different concentrations of 1, 2 and 3% (w / v), respectively (from left to right) (c) Left to right: Ag, Cu, Ag + Cu and Ag / Cu nano suspensions were synthesized in 50 mM metal salts and 3% (w / v) chitosan.
Figure 2 Absorption spectrum of the nanoparticles of (a) Ag and (b) Cu synthesized in 10 mM metal salts and the different concentrations of chitosan are 1, 2 and 3% (w / v, respectively). ), (c) Ag, Cu, Ag + Cu and nano silver copper (Ag/Cu bimetallic) in 50 mM metal salt and 3% (w / v) of chitosan, respectively.
Figure 3. The average particle size of (a) Ag (b) Cu nanoparticles synthesized in 10 mM metal salts and different chitosan concentrations. Error bars show standard deviation from the mean in the three-fold tests. Different letters indicate a significant difference at p ≤ 0.05.
Figure 4. Average particle size of nanoparticles prepared with different concentrations of (a) silver nitrate (b) copper nitrate (c) silver and copper nitrate (combined concentration) at 3% (w / v) chitosan. Error bars represent standard deviations from the mean of the three tests. Different letters indicate a significant difference at p ≤ 0.05.
Figure 5. Zeta potential of nanoparticles of (a) Ag and Cu of 10 mM metal salts and different concentrations of chitosan (b) Ag, Cu, Ag + Cu and Ag / Cu of 50 metal salts mM and 3% (w / v) of chitosan (c) Ag, Cu, Ag + Cu and nano silver copper (Ag/Cu bimetallic) with nitrate concentration adjusted for a mean particle size of 200 nm (with 3% w / v chitosan) . Error bars represent standard deviations from the mean of the three tests. Different letters indicate a significant difference at p ≤ 0.05.
LIST OF TABLES
Table 1: MIC and MBC values of nano silver (Ag), Cu, nano silver copper (Ag/Cu bimetallic) and Ag + Cu nanoparticles synthesized in 50 mM and 3% (w / v) metal salts of chitosan for B. subtilis and E. coli. Identical underwritten letters denote no statistically significant difference (p> 0.05) between MIC and MBC in a column-based comparison, and letters written above identical denote There was no statistically significant difference (p> 0.05) in the row-based comparison as indicated by an ANOVA manner with Bonferroni multiples on the comparison test.
Table 2: MIC and MBC values of Ag, Cu, nano silver copper (Ag/Cu bimetallic) and Ag + Cu nanoparticles with average particle size of 200 nm for B. subtilis and E. coli. Identical underwritten letters denote no statistically significant difference (p> 0.05) between MIC and MBC in a column-based comparison, and letters written above identical denote there was no statistically significant difference (p> 0.05) in row-based comparison as indicated by an ANOVA way with Bonferroni multiples of the comparison test..
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