In this study, synthesis of copper nanoparticles was performed using organoclay as a support to stabilize the nanoparticles. Organoclay amount was gradually increased, which had an effect on the morphology of the resultant nanoparticles. Low amount of organoclay added resulted in larger and agglomerated copper nanoparticles whereas increased amount of organoclay gave smaller sized nanoparticles. The hybrid materials were characterized using the SEM and TEM for morphology, XRD and FT-IR spectroscopy for structural elucidation, thermal analysis using TGA and also studying their antibacterial effect on the two well-known gram negative bacteria of E. coli and P. Aeruginosa . The synthesized nanoparticles were found to be crystalline Cu nanoparticles with a mix of CuO. Larger sized copper nanoparticles and agglomerates showed the higher thermal behaviour as compared with smaller nanoparticles with higher organoclay loading. The hybrid showed an improved antibacterial activity as compared with organoclay alone. The hybrid showed the higher antibacterial effect against the P. aeruginosa microorganism as compared with the E. coli microorganism.
Since the exploitation of nanotechnology, synthesis of nanoparticles has been the heart of nanotechnology research. With various methods of nanoparticles synthesis been developed to exploit this field of research, number of nanomaterials can be found in many applications at the moment. The synthesis of nanoparticles is of interest due to their wide variety of applications in fields such as electronics [
In particular, copper nanoparticles have been used with promising results as bactericides [
In this study, the nanoparticle growth substrate comes from a known commercial compound of organo-montmorillonite, commercially known as Cloisite 30B. This montmorillonite is modified with methyl, tallow, bis-2 hydroxyethyl, quaternary ammonium, where tallow is 65% C18, 30% C16, and 5% C14. We present results on the synthesis of copper nanoparticles using this organoclay as a substrate surface for the synthesis of the nanoparticles. Study the effect of increasing the organoclay in the solution on the resultant nanoparticles. We have characterized the resulting nanoparticles by SEM, TEM, XRD and FT-IR spectroscopy, TGA and study their antibacterial effect on the two well-known gram negative bacteria of Escherichia coli and Pseudomonas aeruginosa. To the best of our knowledge, this is the first report in the literature on copper nanoparticle synthesis using commercially available organoclay such as Cloisite 30B.
Cupric sulphate pentahydrate (CuSO4∙5H2O) and tri-Sodium Citrate (Na3C6H5O7) were purchase from Associated Chemical Enterprises (ACE, South Africa). Sodium borohydride powder (NaBH4) was obtained from Sigma-Aldrich Chemical Co. (South Africa). The organically modified clay, Cloisite 30B (Organoclay) obtained from Southern Clay Products (USA) was used as a substrate. Cloisite 30B is a montmorillonite organically modified with dimethyl-dihydrogenated tallow quaternary ammonium, in a concentration of 90 meq/100 g clay. The solvent used is N,N-dimethyl acetamide (DMAc) purchased from Sigma-Aldrich Chemical Co. (South Africa).
The antibacterial activity of prepared nanocomposites was determined using two gram negative bacterial strains of Escherichia coli (E. coli) was obtained from Centre for Metal Drug Discovery (CMDD) group at Mintek and Pseudomonas aeruginosa (P. aeruginosa) was obtained from Biolabels group at Mintek.
Copper nanoparticles were prepared following a modified method of Samim et al. [
0.075 M CuSO4∙5H2O | Organoclay added (%) | designated name |
---|---|---|
100 | 1 | CloCu-1 (A) |
100 | 2 | CloCu-2 (B) |
100 | 3 | CloCu-3 (C) |
100 | 4 | CloCu-4 (D) |
100 | 5 | CloCu-5 (E) |
Scanning electron microscopy (SEM) [FEI Novanano230, Netherlands] was employed to observe the morphology of the organoclay/Cu hybrid nanoparticles. The instrument is equipped with an X-ray energy dispersive spectroscopy system (EDS) [AMETEK GmbH, Germany] for compositional analysis. The high resolution Transmission Electron Microscope (TEM) [JEOL JEM 2100F, United Kingdom] was used to determine the morphology of the organoclay and nano- particles. Information about the phases and crystallinity was obtained using the Bruker D8 X-Ray Diffraction (XRD) [Bruker, South Africa] patterns which were recorded in the diffraction angular range 5˚ - 80˚ 2θ using a Bruker Advance 8 diffractometer, working in the reflection geometry and equipped with a graphite monochromator on the diffracted beam (CoKα radiation). FTIR spectroscopy analysis was performed on a PerkinElmer Spectrum 2000 spectrophotometer [PerkinElmer, South Africa] with a resolution of 1 cm−1. Infrared spectra were obtained for the organoclay and organoclay-copper nanoparticles from 600 - 4000 cm−1 using attenuated total internal reflection (ATR). The thermal stability of the synthesized polymer nanocomposite was carried out with a Simultaneous Thermal Analyser (STA) [Netzsch STA 429 Netzsch-Gratebau GmbH, Germany] by heating in nitrogen atmosphere from 25˚C to 1000˚C with heating rates 10˚C/min. Heating was followed under a continuous nitrogen purge of 20 mL/min.
The antibacterial activity of the organoclay/Cu was tested against the two gram negative bacteria of E. coli and P. aeruginosa microorganism zone of inhibition (ZOI) method. This is a simple method of measuring the efficiency of an antibacterial agent against the above mentioned bacterial growth. The suspension of the bacteria cultures were prepared as follows: the Rapid E. coli chromogenic agar powder was used as a culture medium for E. coli bacterial growth and Pseudomonas chromogenic agar powder was used as culture medium for the P. aeru- ginosa bacterial growth. The 37 g of each of the agar was dissolved in 1000 ml of distilled water; then the clear brown solvent was obtained. Both agar media were then sterilized at 120˚C for 60 min in autoclave and then cooled to room temperature, briefly 20 mL of each of the agar medium was poured onto disposable sterilized Petri dishes and allowed to solidify. The surfaces of the solidified agar plates were allowed to dry in the incubator prior to streaking of microorganisms onto the surface of the agar plates. Next, 100 μL of the microbial culture suspension in broth containing colony was streaked over the dried surface of the agar plate and spread uniformly using a sterilized plastic rod and allowed to dry before loading the hybrid materials as disks. The organoclay/Cu hybrid materials were made into 3 mm disks. The loaded disks were applied carefully to the surface of the seeded agar plates using sterile forceps. The diameters of the zones of inhibition were measured after 24 h of incubation at 37˚C.
Organoclay/Cu nanoparticles were successfully synthesized using chemical reduction method. The introduction of NaBH4 solution, acting as a reducing agent, caused the reaction colour to change from blue to dark brown which is an indication of the formation of Cu nanoparticles. The effect of organoclay on the size, shape, stability and antibacterial properties of the synthesized organoclay/ Cu hybrid nanoparticles was evaluated. Prior to testing the nanocomposites for antibacterial effect, they were subjected to different characterization methods to determine their physical and chemical properties. In this instance, SEM was used to study the morphology of organoclay/Cu hybrid nanoparticles and distributions of nanoparticles on the surface of the organoclay.
As the organoclay amount is increased from 1% - 5%, less agglomerated particles are distributed on the organoclay surface as seen on the images. This shows that increased organoclay content provides more surface for nanoparticles to form and therefore are separated enough to minimize agglomeration to some degree. The agglomerates are also mostly observed when they are not formed on the organoclay surfaces. This confirms that the organoclay acts as a good support for nanoparticles formation allowing good dispersion of nanoparticles on the surface.
Energy Dispersive X-Ray Spectroscopy (EDS) analysis from each of the SEM images confirms that the organoclay composition is dominated by C, O, Si and Al peaks, with minor peaks of Mg and Fe observed, representing the structure of the organoclay moiety in the hybrid. Similar peaks were reported by Bhattacharya and Mandot [
Confirmation of the size of copper nanoparticles was achieved through the TEM analysis.
brid materials (A-E). The images show that the copper nanoparticles are supported on the organoclay surface as observed in the SEM analysis. As stated previously from the SEM images, the supported nanoparticles are less aggregated. The organoclay platelets can be seen on these TEM images. Particle size distribution of the nanoparticles supported on organoclay is given alongside each respective micrographs with an average size ranging from 10 - 80 nm for the copper nanoparticles. Increasing organoclay leads to a slight decrease in the average copper nanoparticles size as well as leading to less agglomeration.
The crystal structure of the nanoparticles was verified using XRD technique. According to
An additional confirmatory test was performed by studying the molecular interaction between the organoclay and the synthesized copper nanoparticles using FT-IR technique.
as observed previously by Hadj-Hamou et al. [
The weight percentages of copper nanoparticles-organoclay hybrids were determined by TGA analysis under inert (N2) atmosphere in each of the as-synthe- sized hybrid materials (A-E) as shown in
Kobayashia et al. [
The organoclay stabilized copper nanoparticles exhibited both antibacterial activities against Gram-negative bacteria of E. coli and P. aeruginosa. The organoclay material supported the efficiency of the synthesized copper nanoparticles. As shown in
with the organoclay/Cu hybrid antibacterial activity, Cu nanoparticles showed higher bactericidal effect with the P. aeruginosa microorganism than with the E. coli, in which the hybrid showed a higher activity.
The bacterial effect of metal nanoparticles has been attributed to their size and high surface to volume ratio, which allows them to interact with microbial membranes and is not merely due to the release of metals ions in solution as reported previously by Jung et al. [
Copper nanoparticles supported on organoclay surfaces were successfully synthesized via a chemical method. The organoclay material acted as a good support for nanoparticles formation. Transmission electron micrographs for the CloCu- 5, the highest organoclay loading, indicate the size of the nanoparticles to be smaller as compared with the other hybrid materials. XRD data showed that the copper nanoparticles formed contained a mixture of Cu˚ and CuxOy crystalline phases. The antimicrobial activity of the nanoparticles was determined according to the organoclay loading using a couple of bacterial species. The CloCu-5, which is the highest organoclay concentration, was determined to be optimal, due to its higher activity against the microbial specie tested. This can be attributed to the smaller size of nanoparticles formed as observed in the TEM micrographs. Our results indicate the future potential of these organoclay/Cu hybrids materials for combating pathogenic microorganisms. Further work still needs to be carried out (in vivo studies) to determine the toxicity of these nanomaterials which will allow for the application and use of these nanoparticles.
M. F. B. would like to thank DST/Mintek Nanotechnology Innovation Center to allow the publication of this work.
Bambo, M.F., Krause, R.W.M. and Moutloali, R.M. (2017) Facile Method for the Synthesis of Copper Nanoparticles Supported on the Organoclay Material. Journal of Biomaterials and Nanobiotechnology, 8, 144-158. https://doi.org/10.4236/jbnb.2017.82010