Open access peer-reviewed chapter

Comparative Antibacterial Effects of a Novel Copper and Silver- Based Core/Shell Nanostructure by Sonochemical Method

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Selcan Karakus, Ezgi Tan, Merve Ilgar, Ismail Sıtkı Basdemir and Ayben Kilislioglu

Submitted: August 28th, 2018 Reviewed: September 19th, 2018 Published: November 5th, 2018

DOI: 10.5772/intechopen.81588

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In this study, the antibacterial effect of novel copper (Cu) and silver (Ag) metal-based core-shell nanostructures against Escherichia coli (E. coli-Gram negative) was investigated. The novel copper- and silver-based nanostructures were prepared separately by using nontoxic, biodegradable, and biocompatible biopolymers chitosan and guar gum-polyvinyl alcohol (GG-PVA), which were modified by inorganic phases SiO2 and sepiolite. On the other hand, guar gum-PVA (GG-PVA) was modified by sepiolite, and this nanostructure was prepared only for silver. Besides, Cu was dispersed in a different biopolymer chitosan by sonochemical method in the presence and absence of SiO2. X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and X-ray diffraction (XRD) techniques were used to characterize the surface chemistry and morphology of the core/shell nanostructure. Nanoscale zero-valent Cu (NZVCu) was found under thin CuO film according to the XPS results. SEM images showed that spherical Cu/CuO@SiO2 nanostructures (∼100 nm) were homogenously dispersed in the chitosan by using sonochemical method. Antibacterial property of the core-shell nanostructures was analyzed by well-diffusion method against Escherichia coli (E. coli-Gram negative). Cu/CuO@SiO2 nanostructures were found very effective against the E. coli due to high ratio of NZVCu in the nanostructure.


  • sonochemistry
  • chitosan
  • silica
  • core-shell nanostructure
  • guar gum

1. Introduction

Nanostructures are novel materials obtained by dispersing a small amount of nano-sized filler into a biopolymer matrix while preserving the material biodegradability and nontoxicity [1]. Depending on the dispersion and size of the inorganic filler component in nanostructures, they can exhibit improved mechanical, physical, chemical, and barrier properties, and biological reactivity in comparison to pure biopolymers. Because of their nanometer-size dispersion, biopolymer-clay nanocomposites exhibit large-scale advances in the mechanical and physical properties compared with pure biopolymers [2]. Generally, nanoparticles are prepared by chemical reduction, co-precipitation, sol-gel method, hydrothermal synthesis, thermal reduction, microwave process, vacuum vapor deposition, and sonochemical method [3, 4, 5, 6].

The metal core-shell nanoparticle has many significant usages such as particle separation, drug delivery, magnetic resonance imaging, Raman imaging, and biosensor applications [7]. Various multicomponent heterostructured metallic nanoparticles are widely used as antibacterial agents (zinc oxide (ZnO), silver (Ag), Ag@ SiO2, iron (Fe), molybdenum oxide (MoO3), cerium oxide nanoparticles (CeO2), gold-silver (Au-Ag), zirconium oxide ZrO2, aluminum oxide (Al2O3), and magnesium oxide (MgO)) [8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. Rai et al. showed that nanomaterial load, type of substance, size and shape of nanoparticles, surface functional groups, crystallinity concentration are significant factors for their antibacterial effects. In spite of the advantages, their toxicity and safety are barriers that limit their efficient and safe use [18]. Dizaj et al. explained that there are two approaches that explain the antibacterial effect of metal nanoparticles: (a) free metal ion and (b) oxidative stress [19].

The copper-based nanoparticles are preferred to gold or silver nanoparticles because of low cost; high surface area; good thermal, mechanical stability, antimicrobial activity, and UV-light barrier property; high-performance conductive material in various applications; and use in photovoltaic and photocatalytic fields owing to their narrow band gap (1.2 eV) [20, 21, 22, 23, 24, 25].

Chakraborty et al. evidenced that the antibacterial role of the Cu(II) oxide nanoparticle was a “particle-specific effect” which caused cellular DNA damage through phospho-di-ester bond breakage [26]. Gomes et al. demonstrated that Cu-salts were more toxic than Cu-NPs because of the oxidative stress and differential gene expression [27]. Wahid et al. underlined that copper-based nanostructures are easily released out of human body and can be easily mixed with polymers [28]. Gotzmann et al. pointed out that Bacillus subtilis is more sensitive to copper, but E. coli and Staphylococcus aureus are more sensitive to silver and discovered that the silver/copper blend displayed interdependent forceful antibacterial property [29]. Lv et al. provided antibacterial Cu nanoparticle (dosage of 100 g/mL) for disinfection of drinking water. They explained that cell of Escherichia coli were killed because of the reactive oxygen species with H2O2 playing a key role [30].

Chitosan is a natural aminopolysaccharide, nontoxic, biocompatible, biodegradable, derived by the deacetylation of chitin, and widely preferred in the biocomposite material preparation processes [31, 32, 33, 34]. It has antibacterial property in an acidic solution depending on the kind of chitosan, molecular weight, and the degree of polymerization [35, 36]. Tamayo et al. reported that the -OH and -NH2 groups of chitosan can react with H+ ions to generate protonized chitosan with -NH3+ functional groups in acetic acid medium. The size and shape of nanoparticles are associated with protonized -NH3+ chitosan on surfaces and it decreases the amount of agglomeration, so we can synthesize more stable nanostructure [37, 38]. In this study, we determined that the most important condition is to control the size of nanoparticle which increases owing to self-agglomeration and can be intercepted by the addition of chitosan. The chemical bonds (Si-O-Si bonds) on the silica surface are unsaturated because the surface is active during synthesis. When silica functionalized with chitosan, the NH2-functional shell was covalently bounded to the surfaces of silica and the reducing ability of SiO2 increased when functionalized with amino groups [39, 40].

Our hypothesis in this study is acoustic cavitation, and critical amount of the inorganic phase played a key role in synthesizing Chi/Cu/CuO@SiO2 core-shell nanostructure via sonochemistry. These cavitations live through couple of cycles of the ultrasound medium and collapse in a few nanoseconds because of high-temperature (>1000 K) and pressure (>100 atm) conditions in the solution. The chemical bonds (Si-O-Si bonds) on the silica surface are unsaturated because the surface is reactive during the synthesis. When silica functionalized with chitosan, the NH2-functional shell was attached via H-bonds to the surface of silica and the reducing ability of SiO2 increased when functionalized with amino groups. SiO2 was very effective in reduction of Cu2+ into elemental copper. We prepared nanostructures with unique antibacterial properties to destroy pathogenic microorganisms by using sonochemical method. Our aim was the synthesis of the novel core-shell nanoparticle which affects the rate of microbial growth at very low concentrations and produces free radicals (superoxide (˙O2−), hydroxyl radical (˙OH), hydrogen peroxide (H2O2)) [41, 42, 43, 44, 45]. The well-diffusion method was used to determine the antibacterial activity against pathogen bacteria such as Escherichia coli. The growth inhibition zones appeared in the agar layer. We found that the mechanism is founded on self-assembly reduction/oxidation reactions that occur among copper, SiO2, and chitosan.

Silver is commonly used as a well-known antibacterial additive in polymer blends for food packaging and bionanotechnology applications. Guar gum is a biodegradable and eco-friendly biopolymer and is formed of mannose and galactose units [46]. Poly(vinyl alcohol) (PVA) is mixed with guar gum through hydrogen bonds and also has biocompatibility, biodegradability, and good mechanical properties [47]. In this study, Ag@Sepiolite-based nanostructure was encapsulated in a PVA/guar gum/matrix. Antibacterial property of the copper and silver core-shell nanostructures was analyzed by well-diffusion method against Escherichia coli (E. coli-Gram negative). Cu/CuO@SiO2 nanostructures were found to be very effective against the E. coli due to high ratio of NZVCu in the nanostructure. According to this research, the major role of silica in Cu-based core-shell nanostructures under ultrasonic effect was highlighted.


2. Experimental

2.1. Materials

Chitosan (low molecular weight) and cetyltrimethylammonium bromide (CTAB) were purchased from Sigma-Aldrich. Glacial acetic acid, sodium hydroxide, AgNO3, silica gel, NaBH4, and Copper(II) sulfate pentahydrate (CuSO4.5H2O) were purchased from Merck. In the experimental setup, all chemicals and reagents were analytical grade and used without further purification.

2.2. Preparation of the Cu core-shell nanostructure

Chitosan aqueous solutions of low molecular weight, 0.02 g (100 mL), were prepared by dissolving chitosan powder in 5% (v/v) glacial acetic acid. The homogenous solution was obtained by continuous mixing at 25°C. Solutions containing 0.01 M CuSO4.5H2O and 0.02 M NaBH4were prepared separately in a 10-mL deionized water and mixed with a magnetic stirrer drop by drop. Silica gel of weight 0.02 g was added. Then, they were sonicated for 15 minutes (20 kHz) with chitosan solutions under a nitrogen atmosphere by using a Bandelin SONOPULS homogenizer. The samples were dried at 45°C to a constant weight (Figure 1).

Figure 1.

The mechanism of Chi/Cu/CuO@SiO2 core-shell nanostructure.

2.3. Preparation of the Ag core-shell nanostructure

Natural sepiolite of weight 5 g was stirred using 500-mL distilled water to remove the soluble impurities for 1 night. Cetyltrimethylammonium bromide (CTAB) of weight 2 g in 100 mL hot distilled water was added into sepiolite solution and was mixed for 24 hour. Then, the samples were dried overnight at 110°C to a constant weight.

Sepiolite/CTAB of weight 0.0025 g was added to each polymer solution, and they were sonicated for 5 minutes. Solution of 0.133 M, 2.5 mL NaBH4 was added drop by drop to 0.133 M, 2.5 mL AgNO3 under nitrogen atmosphere while it was sonicated. Reduced Ag solution was added drop by drop to each PVA/Guar Gum polymer mixture (1:1) for 5 minutes under sonication under N2 atmosphere. All samples were dried at 60°C. Also, the experiments were carried out without Ag and sepiolite/CTAB.

2.4. Characterization of core-shell nanostructure

2.4.1. X-ray diffraction (XRD) study

The diffraction patterns of the core/shell nanostructure were analyzed by XRD diffractometer (XRD, PAN analytical Xpert-Pro, Cu Ka: 1.5406 Å, a nickel monochromator filtering wave at 40 kV and 40 mA, with a 0.4/min at room temperature, Bruker D8 Advance X-ray Diffractometer).

2.4.2. Scanning electron microscopy (SEM)

The morphological studies were conducted using a Jeol/eo version 1.0 instrument Jsm6390 scanning electron microscope (SEM). The samples were coated with platinum before SEM analysis.


3. Results and discussion

3.1. XRD results

Figure 2 presents the XRD patterns of Chi/Cu/CuO@SiO2, Chi/Cu/CuO, chitosan, and SiO2. SiO2 not only plays a key role in the reduction of copper salt into metallic copper during sonication-assisted mixing of inorganic and organic phases but also leads to the formation of a mineral. According to the XRD results, the diffraction data obtained were well matched with dioptase mineral (ICSD 100077 and PDF 33–487). The XRD pattern of nanostructure indicates the presence of Cu/CuO on the surface of SiO2. A simple scheme was drawn to demonstrate the nanostructure (Figure 3). The XRD pattern of copper displayed characteristic peaks at 2 theta of 22.8, 24.3, 31.5, 34.0, 38.7, 48.9, 53.4, 58.0, 61.0, 66.1, 67.9, 72.2, and 74.9°, respectively [23].

Figure 2.

XRD patterns of (a) Chitosan (b) SiO2 (c) Chi/Cu/CuO (d) Chi/Cu/CuO@SiO2.

Figure 3.

(a) Chi/Cu/CuO core-shell nanoparticle (b) Chi/Cu/CuO@SiO2 core-shell nanoparticle.

Figure 4 presents the XRD patterns of (a) PVA-Guar Gum (b) PVA-Guar Gum-Sepiolite (c) PVA-Guar Gum Ag@Sepiolite core-shell nanoparticle. The XRD pattern of silver displayed characteristic peaks at 2 theta of 38.19, and 44.26°, respectively.

Figure 4.

XRD patterns of (a) PVA-Guar Gum (b) PVA-Guar Gum-Sepiolite (c) PVA-Guar Gum Ag@Sepiolite core-shell nanoparticle.

3.2. SEM results

According to the SEM images of the core-shell nanostructure, a homogenous structure was obtained by using sonication method. The SEM micrographs and EDAX mapping of Cu are shown in Figures 5 and 6.

Figure 5.

SEM images of the surface of Chi/Cu/CuO@SiO2.

Figure 6.

SEM images of the surface of Chi/Cu/CuO.

3.3. X-ray photoelectron spectroscopy analysis

The high-resolution XPS spectra of the Chi/Cu/CuO, Chi/Cu/CuO@SiO2 nanostructures for the copper regions are presented in Figure 7. The intensities of spectra of same elements can be compared with each other in both samples. For the Chi/Cu/CuO@SiO2 sample, elemental Cu shows 2p3/2 peak positioned at 933.6 eV and 2p1/2 peak positioned at 953.4 eV with no satellite. For the Chi/Cu/CuO sample, the 2p3/2 maximum peak was positioned at 932.8 eV and 2p1/2 peak positioned at 952.5 eV. A satellite peak identifies the species as CuO due to surface oxidation of the ZVCu nanoparticles.

Figure 7.

XPS pattern of (a) Chi/Cu/CuO (b) Chi/Cu/CuO@SiO2.

3.4. Antibacterial activity of the core-shell nanoparticle

The Gram-negative Escherichia coli bacteria (NCTC 10538) were cultured at 37°C on a shaking incubator and prepared by spreading of test organism on blood-enriched Mueller-Hinton agar, adjusting 10 mg of sample. The petri dishes were incubated to 35°C at 24 hours (180 rpm). The petri dishes were analyzed for the presence of a clear zone of inhibition. The antibacterial agents inhibited pathogenic bacteria growth leading to a clear, isolated zone in the petri dish. The power of antibacterial properties is proportional to the diameter of inhibition zone (disk diffusion). Figure 8 shows a clear inhibition zone against E. coli microorganisms. The diameter of inhibition zone for nanoparticle against E. coli is approximately 20 mm, so that Chi/Cu/CuO@SiO2 has increased antibacterial performance against the bacteria. Figure 9 shows that the diameter of inhibition zone for nanoparticle against E. coli is approximately 5 mm, so that PVA-Guar Gum Ag@Sepiolite has antibacterial property against the bacteria. Experimental results showed that the size of nanoparticle, amount of nanoparticle, the oxidative stress, and particle-specific effect of SiO2 are responsible factors for their improved antibacterial effect [45].

Figure 8.

The zone of inhibition covered by Chi/Cu/CuO@SiO2 nanoparticles against E. coli. (a) 1 hour; (b) 24 hours.

Figure 9.

The zone of inhibition covered by PVA-Guar Gum Ag@sepiolite nanoparticles against E. coli. (a) 1 hour; (b) 24 hours.


4. Conclusion

A novel antibacterial core/shell nanostructure was prepared by using chitosan which was modified by SiO2 and Cu. Cu was dispersed in chitosan by sonochemical method in the presence and absence of SiO2. When sonication was applied during preparation, the critical amount of SiO2 and Cu caused the formation of dioptase mineral. SiO2 led to the reduction in copper salt into metallic copper during dispersion by sonication and also produced a dioptase mineral.

The specific advantages of the preparation of this novel Cu/CuO@SiO2 core/shell nanostructure included: (i) a low-cost, simple, convenient, and easily feasible sonochemical method, (ii) high content of Cu(0) nanoparticle in the targeting antibacterial agent, (iii) the novel nanoparticle has high bactericidal capacity, and (iv) critical mass production of the antibacterial core-shell nanoparticle. According to the results of the analysis, one could say that the copper-based nanostructure was found more effective against E. coli than the silver-based nanostructure due to the major role of silica in Cu-based core-shell nanostructures by using sonochemical method.



This work was supported by the research fund of Istanbul University project number 13145. The authors acknowledge the support on SEM and XPS analysis from Koc University Surface Science and Technology Center (KUYTAM).


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Written By

Selcan Karakus, Ezgi Tan, Merve Ilgar, Ismail Sıtkı Basdemir and Ayben Kilislioglu

Submitted: August 28th, 2018 Reviewed: September 19th, 2018 Published: November 5th, 2018