Biosynthesis, characterization and antimicrobial activity of copper oxide nanoparticles (CONPs) produced using brown alga extract (Bifurcaria bifurcata)
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Recently, biosynthesis of nanoparticles has attracted scientists’ attention because of the necessity to develop new clean, cost-effective and efficient synthesis techniques. In particular, metal oxide nanoparticles are receiving increasing attention in a large variety of applications. However, up to now, the reports on the biopreparation and characterization of nanocrystalline copper oxide are relatively few compared to some other metal oxides. In this paper, we report for the first time the use of brown alga (Bifurcaria bifurcata) in the biosynthesis of copper oxide nanoparticles of dimensions 5–45 nm. The synthesized nanomaterial is characterized by UV–visible absorption spectroscopy and Fourier transform infrared spectrum analysis. X-ray diffraction confirms the formation and the crystalline nature of copper oxide nanomaterial. Further, these nanoparticles were found to exhibit high antibacterial activity against two different strains of bacteria Enterobacter aerogenes (Gram negative) and Staphylococcus aureus (Gram positive).
KeywordsGreen synthesis Copper oxide nanoparticles Brown alga Antimicrobial activity
Unlike bulk materials, nanoparticles have been intensively studied over the last decade due to their characteristics: physical, chemical, electronic, electrical, mechanical, magnetic, thermal, dielectric, optical and biological properties (Schmid 1992; Daniel and Astruc 2004). Therefore, nanoparticles are considered as building blocks of the next generation of technology with applications in many industrial sectors. In particular, metal oxide nanoparticles are receiving increasing attention in a large variety of applications. Metal oxide nanoparticles are of interest because of their unique optical, electronic and magnetic properties. The oxides of transition metals are an important class of semiconductors, which have applications in magnetic storage media, solar energy transformation, electronics, gas sensors and catalysis (Ramgir et al. 2013; Jani et al. 2013; Shalana et al. 2013; Montferrand et al. 2013; Ahmadi et al. 2011). Although various physical and chemical methods have been extensively used to produce nanocrystalline copper oxide such as microemulsion method (Nassar and Husein 2007), arc-submerged nanoparticle synthesis system (Kao et al. 2007), flame-based aerosol methods (Chiang et al. 2012), sonochemical (Vijayakumar et al. 2001), hydrothermal (Zhang et al. 2006) and solid-state techniques (Wang et al. 2004), the stability and the use of toxic chemicals are subjects of paramount concern. The use of toxic chemicals on the surface of nanoparticles and non-polar solvents in the synthesis procedure limits their applications in clinical fields. Therefore, development of clean, biocompatible, nontoxic and eco-friendly methods for nanoparticles synthesis deserves merit. The interest in this field has shifted toward ‘green’ chemistry and bioprocess approach. These approaches focus on utilization of environment-friendly, cost-effective and biocompatible reducing agents for synthesis of copper oxide nanoparticles (CONPs). Review of literature revealed that synthesis of CONPs using microorganisms and plant extract has been unexplored; there are only a very few reports on the use of yeast, fungi, bacteria or plant extract for synthesizing CONPs (Honary et al. 2012; Rahman et al. 2009; Gunalan et al. 2012) compared to the great number of articles for other metals, and none using the brown alga (Bifurcaria bifurcata). Therefore, in the present work, attempts were made to utilize the potential of this brown alga as a biofactory for the CONPs synthesis. Interestingly, this is the first report on the synthesis of highly stable CONPs using marine alga (Bifurcaria bifurcata). The process is described and different analytical techniques were used including UV–visible spectroscopy (UV–vis), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and X-ray diffraction analysis (XRD). Furthermore, the bacterial effect of CONPs was also analyzed by disc diffusion method.
Materials and methods
Preparation of alga extract
Bifurcaria bifurcata, a brown alga, collected on rocks at about 3 m depth from Morocco’s Atlantic Coast in Rose Marie near Rabat, was extensively washed with deionized water, until the pH of the wash solution was equal to deionized water, and subsequently dried in a oven at 60 °C overnight. It is then finely powdered and stored. About 10 g of powdered alga was transferred into a 150 ml beaker containing 50 ml double distilled water, mixed well on rotary shaker for 1 h and then boiled for 15 min. The extract obtained was filtered and used as a reducing agent and stabilizer.
Synthesis of copper oxide nanoparticles
Synthesis of colloidal CONPs has been done using the following procedures; typically 2 ml of alga extract was added dropwise into 20 ml of 1 mM aqueous solution of copper(II) sulfate with constant stirring at 100–120 °C. Within few hours, the deep blue solution gradually became colorless and then turned slowly to brick red coloration which changed to dark after vigorous stirring for 24 h.
Characterization of copper oxide nanoparticles
The bioreduction of copper (II) ion in solution was monitored using Perkin-Elmer Lambda 2 double beam UV–visible spectrometer against distilled water as blank. After that, the solution mixture (alga extract and copper sulfate) was centrifuged at 5,500 rpm for 15 min and subsequently redispersed in distilled water to get rid of any uncoordinated biological molecules. This process of centrifugation was repeated thrice to ensure better separation of the CONPs. The purified dried powders were then used for further characterization. The FTIR spectral measurements were carried out on Bruker Tensor-27 spectrophotometer and the XRD analysis was obtained using Bruker D8 Advance powder X-ray diffractometer. The morphology of the nanoparticles was analyzed using the high-resolution image obtained with Tecnai G2 transmission electron microscope.
Antibacterial activity of synthesized copper nanoparticles
The CONPs synthesized using alga extract were tested for antimicrobial activity by agar disc diffusion method against pathogenic bacteria Enterobacter aerogenes (Gram-negative) and Staphylococcus aureus (Gram-positive). The pure cultures of bacteria were subcultured on nutrient agar medium. Each strain was swabbed uniformly onto the individual plates using sterile cotton swabs. Filter paper discs (Whatman no. 3, 6 mm diameter) were sterilized by autoclaving. 20 μl of nanoparticle solution was loaded onto each paper disc and allowed to air dry. The dry discs were placed on the previously inoculated agar. After incubation at 37 °C for 24 h, the different levels of zone of inhibition of bacteria were measured.
Results and discussion
In the present work, we first report an eco-friendly and convenient method for the synthesis of CONPs using brown alga (Bifurcaria bifurcata) extract. No chemical reagent or surfactant template was required in this method, which consequently enables the bioprocess with the advantage of being environmental friendly. The developed nanoparticles were characterized by UV–vis, TEM, XRD and FTIR measurements and showed good antibacterial activity. An important potential benefit of the described method of synthesis of nanoparticles using marine algae is that they are quite stable in solution and this is a very important advantage over other biological methods currently in use. This biosynthesis technique can be a promising method for the preparation of other metals and metal oxide nanoparticles and can be valuable in environmental, biotechnological, pharmaceutical and medical applications.
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