Biosynthesis of metal nanoparticles by plant extracts is currently under exploitation. The .... Copper (Cu) is a transition metal with a distinct red-orange colour and ...
BIOSYNTHESIS OF COPPER NANOPARTICLES USING SOME PLANT LEAF EXTRACTS, THEIR CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY
M.Sc. THESIS
G/EGZIABHER H/MICHAEL
OCTOBER 2012 HARAMAYA UNIVERSITY
BIOSYNTHESIS OF COPPER NANOPARTICLES USING SOME PLANT LEAF EXTRACTS, THEIR CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY
A Thesis Submitted to College of Natural and Computational Sciences, Department of Chemistry, School of Graduate Studies
HARAMAYA UNIVERSITY
In Partial Fulfillment of the Requirements for the Degree OF MATSER OF SCIENCE IN CHEMISTRY (PHYSICAL CHEMISTRY)
By
G/Egziabher H/Michael
October 2012 Haramaya University ii
SCHOOL OF GRADUATE STUDIES
HARAMAYA UNIVERSITY As thesis research advisor, we hereby certify that we have read and evaluated this thesis prepared, under my guidance, by G/Egziabher H/Michael entitled: Biosynthesis of Copper Nanoparticles Using Some Plant Leaf Extracts, their Characterization and AntiBacterial Activity. I recommended as fulfilling the thesis requirement.
Prof. O.P. Yadav Major Adivisor Dr. Tesfahun Kebede Co-Advisor
____________________ Signature ______________________ Signature
_____________ Date _________________ Date
As members of the board of examiners of the M.Sc. thesis Open defense examination, we certify that we have read and evaluated the thesis prepared by G/Egziabher H/Michael and examined the candidate. We recommended that the thesis be accepted as fulfilling the thesis requirement for the degree of Master of Science in Chemistry. _________________ Chairperson _________________ Internal Examiner ___________________ External Examiner
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Signature
Date
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Signature
Date
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___________________ Date
DEDICATION
This Thesis manuscript is dedicated to my family who encouraged and strengthened me in all my life.
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STATEMENT OF THE AUTHOR First I declare that this thesis is my bonafide work and that all sources of materials used for this thesis have been dully acknowledged. This thesis has been submitted in partial fulfillment of the requirement for M.Sc. degree at Haramaya University and is deposited at the university library to be made available to borrow under rules of library. I solemnly declared that this thesis is not submitted to any other institution anywhere for the award of any academic Degree, diploma, or certificate.
Brief quotation from this thesis is allowed without special permission provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the Dean of school of Graduate Studies or the Head of Chemistry Department when in his or her judgment the proposed use of the material is in the interest of scholarship. In all other instances, however, permission must be obtained from the author. Name of the author: G/Egziabher H/Michael
Signature ____________
Place: Haramaya University, Haramaya Date of submission: __________________
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BIOGRAPHICAL SKETCH The author was born in February 1980 at Hahayle, Centeral zone, Tigray regional State, Ethiopia. He attended his Primary education in Myhanse and Ksadgeba Primary Schools, N/Western zone, and his secondary school in Shire Senior Secondary High School. After completion of his secondary school education, he joined to Addis Ababa University in 2003/2004 and graduated with B.Sc. degree in Applied Chemistry in 2006. After that, he worked in Amhara Region West Gojjam zone Abay Minch High School for one year and seven months; he has then joined Samara University in July 2008. Two years later he joined the School of Graduate Studies of Haramaya University to pursue his M.Sc study in Physical Chemistry.
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ACKNOWLEDGEMENT First and foremost let me praise and honor the Almighty God and his mother St. Mary for bestowing up on me health, strength, patience and protection to realize my hope.
Secondly I would like to express my deepest gratitude to my advisors Prof. O.P. Yadav and Dr. Tesfahun Kebede for their unreserved cooperation, constructive suggestions, supervision, appreciable encouragement and fatherly consultancy.
I would also like to convey my special thanks to my beloved parents for their unreserved moral throughout my study and I would like to extend my gratitude to Samara University for providing me financial support to accomplish my study.
Last, but not least, my heartfelt gratitude goes to my colleagues and friends for their generous support and contribution in the accomplishment of this work.
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LIST OF ABBREVIATIONS AFM
Atomic Force Microscopy
DDW
Doubly De-ionized Water
DMSO
Dimethylsulfoxide
FCC
Face Centred Cubic
FTIR
Fourier Transform Infrared
FWHM
Full Width at Half Maxima
IR
Infra-Red
LCuNPs
Latex Copper Nanoparticles
MLCC
Multi-Layer Ceramic Capacitor
MBC
Minimum Bactericidal Concentrations
MIC
Minimum Growth Inhibitory Concentrations
Nm
Nanometer
Nps
Nanoparticles
PVP
Poly-vinylpyrrolidone
SEM
Scanning Electron Microscopy
SERS
Surface Enhanced Raman Scattering
SPs
Surface Plasmons
SPR
Surface Plasmon Resonance
TEM
Transmission Electron Microscopy
UV-Vis
Ultra Violet Visible
XRD
X-Ray Diffraction
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TABLE CONTENTS STATEMENT OF THE AUTHOR
v
BIOGRAPHICAL SKETCH
vi
ACKNOWLEDGEMENT
vii
LIST OF ABBREVIATIONS
viii
TABLE CONTENTS
ix
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF TABLES IN THE APPENDIX
xiii
LIST OF FIGURES IN THE APPENDIX
xiv
ABSTRACT
xv
1. INTRODUCTION
1
2. LITERATURE REVIEW
6
2.1. Nanotechnology 2.2. Copper Nanoparticles 2.3. Applications of Copper Nanoparticles 2.4. Kinetics and Stability of Colloidal Solution 2.5. Methods of Synthesis
6 7 8 10 11 12 13
2.5.1 Chemical reduction method 2.5.2. Biosynthesis method
2.6. Characterization Techniques
14
2.6.1. UV-Visible absorption techniques 2.6.2. X-ray diffraction (XRD) techniques 2.6.3. Fourier transform infrared (FTIR) spectroscopy
3. MATERIALS AND METHODS
14 15 16
21
3.1. Experimental Site 3.2. Materials and Apparatus 3.2.1. Apparatus and instruments 3.2.2. Chemicals and reagents 3.2.3. Test organisms 3.3.1. Preparation of plant leave extracts 3.3.2. Biosynthesis of copper nanoparticles ix
21 21 21 21 22 22 22
TABLE CONTENTS (COTINUED) 3.3.3. Kinetics of copper ion reduction 3.4.1. X-ray diffraction (XRD) studies 3.4.2. Fourier transform infrared (FTIR) spectroscopic studies 3.5. Antibacterial Activity Studies
23 23 24 24
3.5.1. Preparation of inoculums 3.5.2. Preparation of test solutions
24 25
3.6. Methods of Data Analysis
25
4. RESULTS AND DISCUSSION
26
4.1. Synthesis of Copper Nanoparticles 4.2. UV-Visible Absorption Spectroscopic Study 4.3. Fourier Transform Infrared Spectroscopic Study 4.4. X-Ray Diffraction Studies 4.5. Antibacterial Activities of Copper Nanoparticles 5. SUMMARY, CONCLUSION AND RECOMMENDATION 5.1. Summary and Conclusion 5.2. Recommendation
26 27 29 31 32 38 38 39
6. REFERENCES
40
7. APPENDIX
48
7.1. Appendix Tables 7.2. Appendix Figures
49 51
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LIST OF TABLES Table
Page
1. Comparison activities of copper nanoparticles on Gram (-) and Gram (+) bacteria ............... 19 2. FTIR absorption frequencies of plant leaf extract mediated copper nanoparticles. ................ 29 3. Average crystalline size of the as-synthesized copper nanoparticles using leaf extracts ........ 31 4. Zone of inhibition (mm) of KH (Catha edulis), CO (Ricinus communis) and DH (Prosopis juliflora) mediated copper nanoparticles ....................................................... ……………….33
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LIST OF FIGURES Figure
Page
1. Schematic view of the processes occurring during the formation of Cu nanoparticles in a dendritic structure. ........................................................................................................... 12 2. UV-Vis absorption spectra of yellow, light orange, orange and red color (λmax= 564 nm) reaction mixture ............................................................................................................... 15 3. XRD patterns of copper nanoparticles ............................................................................... 16 4. FTIR spectra of vacuum dried powder of (a) Euphorbiaceae stem latex (lower) and (b) LCuNPs (upper). .............................................................................................................. 17 5. UV-visible spectra of aqueous copper sulfate pentahydrate solution mixed with Khat (Catha edulis) as a function of time. ................................................................................. 27 6. UV-visible spectra of aqueous copper sulfate pentahydrate solution mixed with Castor oil (Ricinus communis) as a function of time. ........................................................................ 28 7. UV-visible spectra of aqueous copper sulfate pentahydrate solution mixed Dergihara (Prosopis juliflora) as a function of time. ......................................................................... 28 8. FTIR spectra of (a) Khat (Catha edulis), (b) Castor Oil (Ricinus communis) and (c) Dergihara (Prosopis juliflora) plant leaf extract mediated copper nanoparticles ................ 30 9. XRD pattern of KH = Khat (Catha edulis), CO = Castor oil (Ricinus communis) and DH = Dergihara (Prosopis juliflora) leaf extract mediated copper nanoparticles ........................ 32 10. Antibacterial activity of copper nanoparticles (10 μL) on Escherichia coli ...................... 34 11. Antibacterial activity of copper nanoparticles (20 μL) and on Escherichia coli. .............. 35 12. Antibacterial activity of copper nanoparticles (10 μL) on Staphylococcus aureus ............ 36 13. Antibacterial activity of copper nanoparticles (20 μL) on Staphylococcus aureus. ........... 37
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LIST OF TABLES IN THE APPENDIX Appendix Table
page
1. Values of surface plasmon absorbance and their corresponding
at different time of
reaction for KH mediated reduction of copper ions. .............................................................. 49 2. Values of surface plasmon absorbance and their corresponding
at different time of
reaction for CO mediated reduction of copper ions. .............................................................. 49 3. Values of surface plasmon absorbance and their corresponding
at different time of
reaction for DH mediated reduction of copper ions. .............................................................. 50
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LIST OF FIGURES IN THE APPENDIX Figure
Page
1. XRD spectra of Khat (Catha edulis) leaf extract mediated copper nanoparticles ................... 51 2. XRD spectra of Castor oil (Ricinus communis) leaf extract mediated copper nanoparticles ... 51 3. XRD spectra of Dergihara (Prosopis juliflora) leaf extract mediated copper nanoparticles .... 52
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BIOSYNTHESIS OF COPPER NANOPARTICLES USING SOME PLANT LEAF EXTRACTS, THEIR CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY ABSTRACT This study presents the biological synthesis of copper nanoparticles using plant leaf extracts of Khat (Catha edulis), Castor oil (Ricinus communis) and Derjihara (Prosopis juliflora) as reducing and stabilizing agents. On treatment of aqueous solutions of CuSO4.5H2O with the leaf extracts, stable copper nanoparticles were formed. UV-Visible spectroscopy was used to monitor the quantitative formation of copper nanoparticles. The as-synthesized nanoparticles were characterized by XRD and FTIR spectroscopy. The XRD analysis of copper nanoparticles indicated that they ranged in size from 22.32 to 29.05 nm and FTIR measurements suggests that materials present in the leaf extracts have ability to bind metal particles indicating that the proteins could possibly form a layer encapsulating the metal (capping of copper nanoparticles) to prevent from agglomeration and thereby stabilize the nanoparticles. Antibacterial tests of the as-synthesized nanomaterials were carried out on Gram-negative bacteria Escherichia coli and Gram-positive bacteria staphylococcus aureus by impregnating the as-synthesized copper nanoparticles using micropipette on paper discs of 6 mm in diameter and zones of inhibition were measured after 24 h of incubation. The result showed that the as-synthesized copper nanoparticles exhibited a strong antibacterial activity against both Escherichia coli and staphylococcus aureus. Keywords: Antibacterial, Biosynthesis, Characterization, Copper Nanoparticles, Surface Plasmon Resonance.
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1. INTRODUCTION The field of nanotechnology is one of the most active areas of research in modern materials science. Nanoparticles usually referred to as particles with a size approximately extending from 1 nm up to 100 nm in length in at least one dimension (Hutchison, 2008), exhibit completely new or improved properties based on specific characteristics such as size, distribution and morphology. The application of nanoscale materials and structures is an emerging area of nanoscience and nanotechnology. Nanomaterials provide solutions to technological and environmental challenges in the areas of solar energy conversion, catalysis, medicine, and water-treatment (Sangiliyandi et al., 2009). Nanomaterials particularly metallic nanomaterials have assumed a great deal of importance as they often display unique and considerably modified physical, chemical and biological properties as compared to their counterparts of the macroscale (Fayaz et al., 2011).
In recent years, much attention has been paid to metal nanoparticles, which exhibit novel chemical and physical properties owing to their extremely small dimensions and high specific surface area. Specifically, these small particles are interesting materials for research on catalysts with specific activity and selectivity (Song et al., 2004). So, a burst of research activity is witnessed in recent years in the area of synthesis and fabrication of different sizes and shapes of metal nanoparticles. Nanometer sized particles display many interesting optical, electronic, magnetic and chemical properties yielding applications in several fields: biomedicine, diagnostics (Panyam et al., 2003), drug delivery systems (Park et al., 2009), sensors (Uludag et al., 2010), catalysis (Cho et al., 2005) and optics (Kvitek et al., 2008) and antimicrobial activities (Sanpui et al., 2008).
In the field of nanotechnology, the development of reliable and eco-friendly techniques for the controlled synthesis of metal nanoparticles of well defined size, shape and composition, to be used in many fields is a big challenge. During the past decade, nanomaterials have received increasing attention due to their excellent physical and chemical properties different from their corresponding bulk counterparts (Eustis and El-Sayed, 2006). It is widely accepted that
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these properties are often determined by their size, shape, composition, crystallinity, and structure. Therefore, controlling the synthesis of nanomaterials with definite morphology and uniform size is one of the most effective ways to achieve desirable properties.
Amongst many metals like Au, Ag, Pd, Pt, towards which research is directed, copper and copper based compounds are the most important materials. The metallic Cu plays a significant role in modern electronic circuits due to its excellent electrical conductivity and low cost nanoparticles (Schaper et al., 2004). So Cu will gain increasing importance as is expected to be an essential component in the future nanodevices due to its excellent conductivity as well as good biocompatibility and its surface enhanced Raman scattering (SERS) activity (Pergolese et al., 2006). Metallic copper nanocrystals homogeneously dispersed in silica layers, which have attracted great attention recently and in comparison with bulk copper, are potentially suitable materials for using in printed electronics and are good substitutes for conductive and expensive noble metals like gold and silver in the chemical and metallurgical processes (Song et al., 2004). Copper nanoparticles have been used to make conductive pastes for formation of thick film conductors such as electrodes or conductive patterns in multilayered electronic parts, printed circuit boards, hybrid integrated circuit and metallization of multilayer ceramic capacitor (MLCC) in the electronic industries (Yu et al., 2007).
The synthesis of metallic nanoparticles is an active area of academic and, more importantly, application research in nanotechnology. A variety of chemical and physical procedures such as chemical reduction (Prakash et al., 2009), electrochemical reduction (Zhang et al., 2008), chemical vapor deposition (Coulombe et al., 2007), thermal decomposition (Kim et al., 2006) solvothermal reduction (Tang et al., 2006) have been reported for synthesis of metallic nanoparticles. However, these methods are fraught with many problems including use of toxic solvents, generation of hazardous by-products, and high energy consumption. Accordingly, there is an essential growing need to develop clean, reliable, biocompatible, cost-effective, environmentally benign, and sustainable procedures for synthesis of metallic nanoparticles that donot use toxic chemicals which encouraged more and more researchers to exploit biological systems as possible eco-friendly nano-factories. A promising approach to achieve 2
high yield, low cost, environment-friendly and sustainable procedures for the synthesis of metal nanoparticles is to exploit the array of biological resources in nature. Indeed, over the past several years, plants, algae, fungi, bacteria, and viruses have been used for production of low cost, energy-efficient, and nontoxic metallic nanoparticles.
Biological systems using microorganisms such as algae (Singaravelu et al., 2007), fungi (Fayaz et al., 2009a), bacteria (Sangiliyandi et al., 2009), enzymes (Narayanan and Sakthivel, 2011), mushrooms (Philip, 2009) and plant leaf extracts (Narayanan and Sakthivel, 2010b) have been suggested as possible eco-friendly alternatives to chemical and physical methods for the synthesis of low-cost, energy efficient, and non toxic metallic nanoparticles. Use of plant extracts for synthesis of nanoparticles could be advantageous over other environmentally benign biological processes as this eliminates the elaborate process of maintaining cell culture (Taleb et al., 1998) and the advantage of using plants for the synthesis of nanoparticles is that they are easily available, safe to handle and possess a broad variability of metabolites that may aid in reduction.
Biosynthesis of metal nanoparticles by plant extracts is currently under exploitation. The uses of Azadirachta indica (Neem) (Shankar et al., 2004), Emblica officinalis (amla, Indian Gooseberry) (Amkamwar et al., 2005), mangosteen leaf (Veerasamy et al., 2011), Chenopodium album (Dhanaraj, 2011) have already been reported. Studies have indicated that biomolecules like proteins, phenols, and flavonoids not only play a role in reducing the ions to the nanosize, but also play an important role in the capping of the nanoparticles (Vedpriya, 2010).
Furthermore, the emergence of nanoscience and nanotechnology in the last decade presents opportunities for exploring the bactericidal effect of metal nanoparticles. Microbial contamination of water poses a major threat to public health. With the emergence of microorganisms resistant to multiple antimicrobial agents (Kolar et al., 2001), there is a growing interest in developing new bactericides based on inorganic
materials to
substitute the traditional organic agents, as organic agents have limited their applications due to their low heat resistance, high decomposability and short life. Metal nanoparticles 3
with bactericidal activity can be immobilized and coated on to surfaces, which may find application in various fields and their anti-microbial effect has been attributed to their small size and high surface area to volume ratio, which allows them to interact closely with microbial membranes (Morones, 2005). Although only a few studies have been reported on the antibacterial properties of copper nanoparticles, they show that copper nanoparticles have a significant promise as bactericidal agent. Yoon et al., (2007) reported the antibacterial effects of silver and copper nanoparticles on single representative strains of E. coli and Bacillus subtilis, where the copper nanoparticles demonstrated superior antibacterial activity compared to the silver nanoparticles.
The dispersion of metal nanoparticles displays intense colors due to the plasmon resonance absorption. The surface of metals like Cu can be treated as free-electron systems called plasma, containing equal numbers of positive ions (fixed in position) and conduction electrons (free and highly mobile). Under the irradiation of an electromagnetic wave, the free electrons are driven by the electric field to oscillate coherently at a plasma frequency relative to positive ions (Xia, and Halas, 2005). Surface plasmon resonance is, therefore, a collective excitation of the electrons to the conduction band around the nanoparticle surface. Electrons conform to a specific vibration mode by particle size and shape. Therefore, metallic nanoparticles display characteristic optical absorption spectra in the UV–Visible region (Fayaz et al., 2009b). Biosynthesis of metal nanoparticles, using plant leaf extracts both as reductants and capping agent, is currently under exploitation. It is an eco-friendly, cost effective and more efficient alternative method for the large scale synthesis of metal nanoparticles. However, only a very little work has been done for the synthesis of copper nanoparticles using plant leaf constituents (materials). Under this study, copper nanoparticles were synthesized using some plant leaf extracts as reducing agents and capping materials.
4
General objective To synthesize copper nanoparticles using some plant leaf extracts, characterize them and evaluate their antibacterial activity.
Specific objectives To synthesize copper nanoparticles using some plant leaf extracts of Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) To characterize the as-synthesized copper nanoparticles using UV-Vis spectroscopy, XRD and FTIR techniques To test the antibacterial activity of the as-synthesized copper nanoparticles
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2. LITERATURE REVIEW 2.1. Nanotechnology Nanoscience involves the study of materials on the nanoscale level between approximately 1 and 100 nm in length in at least one dimension (Hutchison, 2008) and the study of how to control the formation of two- and three-dimensional assemblies of molecular scale building blocks into well-defined nanostructures or nanomaterials (Rosi and Mirkin, 2005). Nanotechnology is the application of science and technology to control matter at the molecular level, which is also referred to as the ability for designing, production, characterization and application to structures, devices and systems by controlling shape and size at the nanometer scale (Uskokovic, 2008). Nanotechnology emerges from the physical, chemical, biological and engineering sciences where novel techniques are being developed to probe and manipulate single atoms and molecules. The technology springs from advancements in material science-the ability to fabricate nanoscale materials in a uniform and reliable manner, at reasonable scale and cost and has turned many of our dreams true by enabling construction of micro/nanodevices.
Nanomaterials have broad applications in a variety of fields because of their unusual and size dependent optical, magnetic, electronic and chemical properties (Burda et al., 2005). Nanoparticles are characterized by an extremely large surface area to volume ratio, and their properties are determined mainly by the behaviour of their surface (Hodes, 2007). The applications of nanoparticles are well known in the fields of cosmetics and pharmaceutical products, coatings, electronics, polishing, semiconductors and catalysis. The design and preparation of novel nanomaterials with tunable physical and chemical properties remains a growing area.
Nanobiotechnology is an emerging area of opportunity that seeks to fuse nano/micro fabrication and biosystem to the benefit of both. Increasing awareness towards green chemistry and other biological processes has led to the development of simple and ecofriendly approaches towards the synthesis of nanomaterial (Hsiao et al., 2006). There has 6
been a considerable amount of recent interest in using biotechnological approaches to achieve scaleable, cost-effective bioproduction options.
2.2. Copper Nanoparticles Copper (Cu) is a transition metal with a distinct red-orange colour and metallic luster having atomic number 29 and atomic mass 63.546. It is relatively more abundant metallic element of the Earth’s crust (the 8th) having special properties of high electrical conductivity, high thermal conductivity, high corrosion resistance, good ductility and malleability, and its reasonable tensile strength makes it an essential element to the functioning of society and has played several important roles in society for thousands of years. Copper is a good conductor, can joined to itself very easily and has better corrosion resistance and is a more abundant hence cheaper material to use. This properties, has made copper the number one material used in modern household water piping and associated plumbing and the metal of choice for most vehicle radiators and air conditioners.
Copper has been used as a biocide for centuries (Dollwet and Sorenson, 2001); copper was used to sterilize water and wounds in ancient Egypt (2000BC), copper was prescribed for pulmonary diseases and for purifying drinking water in ancient Greeks (400BC) and copper cooking utensils were used to prevent the spread of disease (during the Roman Empire). Copper is one of a relatively small group of metallic elements that are essential to human health as it is a constituent of many enzymes involved in numerous body functions and is a constituent of hair and of elastic tissue contained in skin, bone and other body organs (Goyer, 1997). A number of recent studies have explored the potential benefit of using copper in place of stainless steel on surfaces to reduce bacterial loads in a number of settings including hospitals and the food industry (Airey and Verran, 2007; Mehtar et al., 2008.).
Gold, silver, and copper have been used mostly for the synthesis of stable dispersions of nanoparticles, which are useful in areas of photography, catalysis, biological labeling, photonics, optoelectronics, and surface-enhanced Raman scattering (SERS) detection (Smith et al., 2006). In recent years, the preparation of copper nanoparticles has received increasing 7
attention from many researchers since copper nanoparticles are viewed as possible replacements for Ag and Au nanoparticles because of their useful anti-microbial, anti-biotic and anti-fungal (fungicide) agent and for their potential electrical, dielectric, magnetic, optical, imaging, catalytic, biomedical and bioscience properties (Theivasanthi and Alagar, 2011a).
Although copper is one of the most widely used materials in various applications, its synthesis in nano sizes is challenging due to its high tendency for oxidation. Unlike gold and silver, copper is extremely sensitive to air, and the oxide phases are thermodynamically more stable (Jeong et al., 2008). Therefore, the formation of a surface oxide layer on copper nanoparticles is inevitable. The presence of copper oxides on the surface of nanoparticles is not desirable for many industries, such as electronics that count on copper as a good alternative for current expensive metals. The electrical conductivity of copper nanoparticles decreases dramatically if they become impure with oxide phases. One can rarely find a method in literature that produces pure copper nanoparticles, unless the whole procedure was done under an inert atmosphere (Mott et al., 2007). Khanna et al.(2007) described their achievement in synthesis of pure copper nanoparticles by reducing copper salt in the presence of surfactant.
2.3. Applications of Copper Nanoparticles Copper nanoparticles, due to their unique physical and chemical properties and low cost preparation, have been of great interest recently. Copper nanoparticles have great applications as heat transfer systems (Eastman et al., 2001), antimicrobial materials (Esteban-Cubillo et al., 2006), superstrong materials (Guduru et al., 2007), sensors (Kang et al., 2007), and catalysts (Kantam et al., 2007). Copper nanoparticles can easily oxidize to form copper oxide. If the application requires the copper nanoparticles to be protected from oxidation, the copper nanoparticles are usually encapsulated in organic or inorganic materials such as carbon and silica (Moya et al., 2006).
Copper nanoparticles due to their high surface to volume ratio are very reactive, can easily interact with other particles and increase their antimicrobial efficiency. Colloidal copper has 8
been used as an antimicrobial agent for decades. Copper monodispersed nanoparticles (2 - 5 nm) embedded into a polysilicate called sepiolite (Mg8Si12O30 (OH)4(H2O)4.8H2O) have revealed a strong antibacterial activity and were able to decrease the microorganism concentration by 99.9% (Esteban-Cubillo et al., 2006). Copper nanoparticles (about 6 nm) embedded in polyvinylmethylketone films exhibit a noticeable inhibitory effect on the growth of microorganisms (E.coli and S. cerevisiae) (Cioffi et al., 2005). Due to the stability of copper nanoparticles supported on a matrix, and their disinfecting properties, copper nanoparticles can be used in paint or plaster as a bactericide agent to coat hospital equipment.
Metallic nanoparticles can be used in heat transfer systems to improve efficiency. Fluids containing metallic nanoparticles with a thermal conductivity of about three times that of a pure fluid could double the fluid’s heat transfer rate. It is reported that adding only 0.3 volume percent of copper nanoparticles, with average diameter of less than 10 nm, to ethylene glycol increased its thermal conductivity up to 40% (Eastman et al., 2001).
A major problem facing fuel-cell technologies is formation of high levels of carbon monoxide (CO) which is produced during hydrogen production. One way to eliminate the CO byproduct is to combine it with water to produce hydrogen gas and carbon dioxide (CO2) in a process known as the “water- gas shift” reaction (See reaction below).
CO + H2O → H2 + CO2 With the assistance of proper catalysts, the water-gas shift reaction can convert a large portion of carbon monoxide into carbon dioxide. For this purpose, to achieve greatest catalytic activity, nanoparticles (2 - 4 nm) of either gold or copper supported on a metal oxide (zinc oxide, ZnO and cerium oxide, CeO2) have been used. Although, gold nanoparticles show the greatest catalytic activity in water-gas shift reaction, copper is almost as reactive, and its cost is much lower (Rodriguez et al., 2007).
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2.4. Kinetics and Stability of Colloidal Solution
For most real world applications, experimental conditions need to be tightly controlled in order to obtain nanoparticles with at least the following characteristics: identical particles in terms of size (a uniform size distribution), shape or morphology, chemical composition and crystal structure (ideally, core and surface composition must be the same, unless specifically designed for other purposes), and monodispersity (no aggregation) (Rotello, 2003).
Stabilization of metal nanoparticles in the dispersing medium is crucial in colloid chemistry because of small metal particles are unstable with respect to agglomeration to the bulk which is caused by the attractive Van der Waals force and/or the driving force that tends to minimize the total surface energy of the system. So special precautions have to be taken to avoid their aggregation or precipitation during the preparation of such colloidal particles in solution. Obtaining stable nanomaterials that consist of exclusively chemically pure, without using any stabilizing agent, is an unresolved issue that remains the subject of contrasting opinions (Ott and Finke, 2007). Therefore, to obtain stable copper colloids, the most effective and common strategy is the introduction of a protective agent in the reaction system.
Recently, Zhu et al. (2008) synthesized well-dispersed copper nanoparticles with controllable shapes (rods, needles, etc) using non-aqueous liquid (polyol) as a solvent and reducing agent and stabilizing agents, such as poly-vinylpyrrolidone, (PVP) to prevent the nanoparticles from agglomeration and oxidation. The stabilizing agent determines the shape, size and uniformity of the resulting nanoparticles, related with the PVP/M2+ molar ratio.
More stable dispersions were obtained by the use of polymers as capping agents. Jeong et al. (2008) followed by Engels et al. (2010) evaluated the effect of the well established stabilizer, poly(N-vinylpyrrolidone) (PVP), at various molecular weights (10,000, 29,000 and 40,000 g/mol). They found that the particle size increases as the PVP molecular weight increases. A reaction mechanism between copper ions and PVP was proposed. The first step was the formation of a coordinative bonding between PVP and copper ions, forming a Cu2+PVP complex. The formed complex was present in the solution and when the current pulse was 10
applied, the Cu2+ was reduced to CuO on the polymer preventing the agglomeration of the metallic nanoparticles. IR studies showed that the PVP is coordinated with Cu through C-N and C=O bonds.
2.5. Methods of Synthesis
Copper nanoparticles have been synthesized via various techniques, typically categorized as physical and chemical processes. Physical methods, such as laser ablation (Yeh et al., 1999), vacuum vapor deposition (Liz-Marzán, 2004), and radiation methods (Joshi et al., 1998) are capable of producing a wide range of metal nanoparticles with little effort being required to modify them for each material. However, the quality of produced particles is not as high as chemically synthesized ones. These physical methods usually require expensive vacuum systems to generate plasmas.
Niu and Crooks (2003) synthesized copper nanoparticles using wet-chemical method encapsulating the nanoparticles by dendritic molecules. Interestingly, they found that dendrimer behaves as a monodisperse nanoreactor and allows the pre-organization of the metal into its inner part based on a strong interaction between Cu (II) ions and the dendrimer core and it has been shown that the number of metal atoms adsorbed into the dendrimer inner part corresponds to the number of tertiary amines present therein, although nonspecific surface complexation may occur leading to undesired phenomena such as nanoparticle aggregation. Subsequently, the metal ions become entrapped in the template structure, while a conventional chemical reduction of the metal – dendrimer complex (example by means of NaBH4) induces the formation of a nanosized inorganic core inside the dendrimer shell (Figure 1)
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Figure 1. Schematic view of the processes occurring during the formation of Cu nanoparticles in a dendritic structure.
2.5.1 Chemical reduction method
Chemical reduction is the most frequent applied method as the growth and assembly of copper nanoparticles can be controlled by optimizing reaction parameters, such as temperature and the concentration of surfactant, reducing agent, solvent, and precursor (Xie et al., 2004). In the chemical reduction technique a copper salt is reduced by a reducing agent such as sodium borohydride (Chen et al., 2006), hydrazine (N2H4) (Zhu et al., 2005), ascorbate (Wang et al., 2006), polyol (Park et al., 2007) as well as glucose (Panigrah et al., 2006).
Interestingly, processes involving the use of polyol/alcohol solvents appear as a form of modified/improved version of the polyol method, in which the polyol/alcohol solvent is assisted by a specific (and generally stronger) reducing agents with the simultaneous presence of PVP and solvents with alcoholic moieties made it possible to obtain monodisperse copper nanocubes (Wang et al., 2006), nanoparticle arrays and aggregates or nanorodes (Liu et al., 2003), as a function of the experimental conditions.
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2.5.2. Biosynthesis method
The need for biosynthesis of nanoparticles rose as the physical and chemical processes were costly and often physical and chemical synthesis methods lead to the presence of some toxic chemical species adsorbed on the surface that may have adverse effects in medical applications (Parashar et al., 2009). So in the search for cheaper pathways for nanoparticle synthesis, scientists used microorganisms and then plant extracts for synthesis. Nature has devised various processes for the synthesis of nano- and micro- length scaled inorganic materials which have contributed to the development of relatively new and largely unexplored area of research based on the biosynthesis of nanomaterials (Mohanpuria et al., 2007).
Nanoparticles exhibit many unique properties, for which they are intensely being studied in a number of research fields. Although a very little work has been done on the bio-reduction of copper nanoparticles, but still they show a promising result. An easy and rapid synthesis of copper nanoparticles capped with peptides present in the latex of plant Euphorbianivulia (Euphorbiaceae) that acts as both reducing and capping agent is reported (Valodkar et al., 2011).
Narayanan and Sakthivel, (2011) had shown that NADH-dependent enzymes are responsible for the biosynthesis of nanoparticles. The reduction mechanism seems to be initiated by electron transfer from the NADH by NADH-dependent reductase as electron carrier. One mechanism of metal nanoparticles biosynthesis by microorganisms is bioreduction, in which myriads of proteins, carbohydrates and biomembranes are involved (Moghaddam, 2010). Nanoparticles are formed on cell wall surfaces, and the first step in bioreduction is the trapping of the metal ions on this surface which probably occurs due to the electrostatic interaction between the metal ions and the negatively charged groups in enzymes present at the cell wall. This may be followed by enzymatic reduction of the metal ions, leading to their aggregation and the formation of nanoparticles (Bansal et al., 2004).
Recently, Varshney et al. (2010) reported a novel biological method using non-pathogenic bacterial strain Pseudomonas stutzeri isolated from soil for synthesizing copper nanoparticles. 13
It is, therefore, important to develop synthetic strategies which are simple, cost-effective, environment friendly, easily scalable and at the same time with parameters to control size and shape of the materials.
2.6. Characterization Techniques Nanoparticles are characterized using UV-Vis absorption spectroscopy, FTIR, XRD, SEM, TEM etc. techniques.
2.6.1. UV-Visible absorption techniques The formation of metal nanoparticles is monitored by UV-Visible spectroscopy as colloidal dispersions of metal show absorption bands in the UV-Visible range due to the excitation of plasma resonances or interband transitions, characteristic properties of the metallic nature of the particles. Bahadory (2008) prepared copper nanoparticles by chemical reduction method using sodium borohydride as reducing agent and first yellow, light orange, then orange, and finally red color formation was observed upon mixing sodium borohydride and copper (II) solutions. Plasmon absorbance (near 560 nm) appears only when the color of solution is red which containing 52 nm particles measured by AFM with plasmon absorbance near 560 nm. Figure 3 shows the spectra of the yellow, light orange, orange and red reaction mixture.
14
Figure 2. UV-Vis absorption spectra of yellow, light orange, orange and red color (λmax= 564 nm) reaction mixture.
The plasmon absorbance cannot be seen for the yellow color reaction mixture and the absorbance is very weak when it turns orange which probably indicates onset of particle formation. Surface plasmon absorbance is very broad for the small particles with diameters below 4 nm. As the particles grow and the number of the particles increases, the surface plasmon absorbance narrows and increases in intensity of the color and becomes orange and finally red for the sols that absorb near 560 nm (Pileni, 2004).
2.6.2. X-ray diffraction (XRD) techniques
XDR is a technique used for determining the size and crystal structure of the as-synthesized nanomaterial. Theivasanthi and Alagar, (2011b) have been synthesized copper nanoparticles by an electrolysis method and characterize them for their properties using XDR. The X-ray diffraction (XRD) spectral data of face centred cubic (FCC) Cu corresponding to 2θ values of 43.64, 50.80, and 74.42o corresponding to (111), (200) and (220) planes of copper is observed (Figure 3). No peaks of impurities were observed in XRD data. The crystallite size was found by applying Sherrer’s equation and the average crystallite size is found to be 24 nm.
15
Figure 3. XRD patterns of copper nanoparticles
2.6.3. Fourier transform infrared (FTIR) spectroscopy FTIR measurements help to elucidate the possible molecules responsible for capping thereby stabilizing the metal nanoparticles. Valodkar et al. (2011) synthesized Cu nanoparticles using peptides present in stem latex of a plant, Euphorbiaceae and FTIR measurements were carried out to identify the possible interactions between the nanoparticles core (copper) and the shell (latex) since the amide linkages in proteins and polypeptides give well-known signature in IR region. Figure 4 shows the FTIR spectra of vacuum dried powder of Euphorbiaceae stem latex and latex stabilized Cu nanoparticles.
16
Figure 4. FTIR spectra of vacuum dried powder of (a) Euphorbiaceae stem latex (lower) and (b) LCuNPs (upper).
The changes in the transmittances related to the bonds with N atoms reveal that nitrogen atoms are the binding sites for metal on latex. The strong broad peak at 3300 to 3500 cm−1 is characteristic of the N–H stretching vibration. Compared to pure latex, this peak shows a shift to lower frequency and a decrease in intensity on binding with the copper. IR active modes attributed to side chain vibrations include aliphatic C–H stretching modes at 2934 cm−1 while carbonyl stretching frequency appears at about 1735 cm−1. Although not many changes were observed at these frequencies, the significant changes observed for peaks at 1382 and 1457 cm−1 are indicative of the role of protein in capping the Cu nanoparticles. The two peaks are closely merged in pure latex but are more individualized after interaction with the metal. The band at 1457 cm−1 is assigned to methylene scissoring vibrations from the proteins in the solution. 1639 cm−1 is related to the maximum absorbance of the amide I band of proteins with predominantly beta sheet structure. After the synthesis of LCuNPs, this peak also shifted to 1595 cm−1. This indicated that the secondary structure of the proteins is affected as a consequence of binding with the copper NPs.
17
2.7. Medicinal Value of Copper Nanoparticles Microbial contamination of water poses a major threat to public health. With the emergence of microorganisms resistant to multiple antimicrobial agents (Kolar et al., 2001) there is increased demand for improving disinfection methods. Researchers have recommended the use of silver and copper ions as superior disinfectants for waste water generated from hospitals containing infectious microorganisms (Lin et al., 1998). However, residual copper and silver ions in the treated water may adversely affect human health (Blanc et al., 2005). The emergence of nanoscience and nanotechnology in the last decade presents opportunities for exploring the bactericidal effect of metal nanoparticles.
The antibacterial efficiency of the metal nanoparticles was investigated by introducing the particles into a media containing bacteria. The antibacterial investigations were performed in solutions and on Petri dishes. People have used copper for its antibacterial qualities for many centuries. However, copper nanoparticles have showed antibacterial activities more than copper in a bulk form because copper nanoparticles have very specific large surface area to volume ratio, having high surface area to volume ratio in nanocrystals can lead to unexpected properties, increases their reactivity tremendously as they have a greater number of reaction sites. Surfaces of copper nanoparticles affect/interact directly with the bacterial outer membrane, causing the membrane to rupture and killing bacteria. Theivasanthi and Alagar, (2011a) reported the synthesis of copper nanoparticles using electrolysis and chemical reduction methods, in both cases more antibacterial activities were shown for Escherichia coli (Table 1)
18
Table 1. Comparison of activities of copper nanoparticles on Gram (-) and Gram (+) bacteria.
Copper nanoparticles synthesis Name of bacteria
Variety of Inhibition
method
bacteria
Electrolysis method
Chemical reduction method
zone
diameter (mm)
Escherichia coli
Gram (-)
15
Bacillus megaterium
Gram (+)
5
Staphylococcus aureus
Gram (+)
6-8
Salmonella typhimurium
Gram (-)
6-8
Pseudomonas aeruginosa Gram (-)
6-8
Escherichia coli
8 - 10
Gram (-)
.Although only few studies have reported the antibacterial properties of copper nanoparticles, they show copper nanoparticles have a significant promise as bactericidal agent.
2.8. Implications of Nanoparticles to Human Health and Environment Nanoparticles are special and interesting because their chemical and physical properties are different from their macro counterparts. Almost all properties of nanoparticles are due to their small sizes and they are attracting a great deal of attention because of their potential for achieving specific processes and selectivity, especially in biological and pharmaceutical applications. (Theivasanthi and Alagar, 2011a).
However, it is also recognized that nanoparticles may have many undesirable and unforeseen effects on the environment and in the eco-system (Long et al., 2006). Some metallic nanoparticles are showing increased toxicity, even if the same material is relatively inert in its bulk form (example, Ag, Au and Cu). Nanoparticles also interact with proteins and enzymes within mammalian cells and they can interfere with the antioxidant defense mechanism leading to reactive oxygen species generation. Increasingly, study of their fate and impact in the environment is becoming important due to the discharges already occurring to the environment, the likely increase in discharges as the industry grows dramatically, the known 19
toxicity of nanoparticles and the immense gaps in our knowledge leading to difficulties in risk assessment and management (Handy et al., 2008), so that still there is a need for economic, commercially viable and environmentally benign route to synthesize the metal nanoparticles.
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3. MATERIALS AND METHODS
3.1. Experimental Site
The synthesis of copper nanoparticles and their antimicrobial tests were conducted at the Chemistry Research Laboratory and Plant Pathology Microbiology Laboratory, respectively of Haramaya University. Besides, some plants leaves of Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) were collected from Haramaya and Awash areas. UV-Vis and FTIR spectroscopic data of the synthesized samples were obtained at Chemistry Research Laboratory of Haramaya University and Chemistry research laboratory of Addis Ababa University, respectively and XRD measurements were done at Geosciences laboratory of Ethiopian Geological Survey in Addis Ababa.
3.2. Materials and Apparatus 3.2.1. Apparatus and instruments UV-Visible spectrophotometer (SANYO SP65 GALANAKAMP, UK); FTIR spectrometer (SHIMADZU 1730, JAPAN);
XRD (BRUKER D8 Advanced XRD, West Germany);
Analytical balance (OHAUS, Switzerland), Hot plate (SM6, from UK), Deionizer (ELGACAN, Cartridge type C114, UK), Autoclave, Incubator; Centrifuge (K2 series, CENTERION SCIENTIFIC LTD, west Sussex UK), Furnace (BiBBY, Stuart, UK); Oven (OV150C, England), Pyrex glass beakers, mortar and pestle, Ceramic crucibles, Volumetric flasks, Pipettes, Magnetic stirrer, Erlenmeyer flasks, water bath were used for the present study.
3.2.2. Chemicals and reagents
Laboratory reagent copper (II) sulfate pentahydrate (CuSO4.5H2O) as chemical precursor (HiMedia Laboratories, PVt. Ltd.), extra pure (98.5%), MW = 249.68 g/mol, nutrient agar media for bacteria growth; potassium bromide (KBr, 99.5%, BDH chemicals Ltd Poole, 21
England), sterilized water, de-ionized water, plant leave extracts of Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) as reducing and stabilizing agent were used for synthesis of copper nanoparticles.
3.2.3. Test organisms Antibacterial activity of the as-synthesized copper nanoparticles was investigated for clinical isolates
of Gram-negative
bacteria
Escherichia
coli
and
Gram-positive
bacteria
Staphylococcus aureus were used as a test organism. These human pathogenic bacteria were grown in plant pathology microbiology laboratory.
3.3. Experimental Methods and Procedures
3.3.1. Preparation of plant leave extracts
25 gm fresh leaves of each plant of Khat (Catha edulis), Castor oil (Ricinus communis and Dergihara (Prosopis juliflora) were separately washed thoroughly with de-ionized water to remove dirt particles if any adsorbed on the surface of the leaves and the washed samples were air-dried. The dried leaves were crushed with mortar and pestle. The mashed sample of fresh leaves was then mixed with 100 mL of sterile double distilled water (DDW) in a 250 mL Erlenmeyer flask and kept at 650C for 30 min and then filtered off using Whatmann No.1 filter paper. The resulting sample leaf extract was stored at 40C (Hailemariam, 2011).
3.3.2. Biosynthesis of copper nanoparticles
In a typical synthesis of copper nanoparticles, 10 mL of each fresh leaf extracts was added to 100 mL of 0.01 M CuSO4.5H2O aqueous solution and the mixture was kept at 560C with constant stirring on a magnetic stirrer for 6 h. The suspension produced was centrifuged at 3000 rpm for 10 min and the supernatant liquid was decanted off and the residue was repeatedly washed with 10 mL of de-ionized water. Centrifugation-decantation-washing processes were repeatedly done six times to remove impurities if any on the surface of the 22
copper nanoparticles. The obtained precipitate was dried in an oven at 500C for 24 h. The assynthesized copper nanoparticles were then kept for further characterization by FTIR, XRD and antibacterial studies.
3.3.3. Kinetics of copper ion reduction
2.5 mL of the as-prepared leaf extract was added to 30 mL of 0.01 M CuSO4.5H2O aqueous solution. The reaction mixture was stirred at 560C on a magnetic stirrer. For measuring the absorbance of copper colloidal solution a 0.5 mL of the aliquot suspension was diluted in quartz cuvette with de-ionized water and its UV-Visible spectrum was measured at the specified temperature (Hailemariam, 2011).
The kinetics of Cu2+ ions reduction was
monitored by measuring the absorbance periodically of UV-Visible over the range 200 - 800 nm.
3.4. Methods of Characterization
3.4.1. X-ray diffraction (XRD) studies For XRD analysis, the dried sample of the as-synthesized copper nanoparticles was calcined at 4000C for 4 h in furnace. The XRD pattern of the as-synthesized nanomaterial was then recorded using an X-ray diffractometer. A thin film of the sample was made by dipping a glass plate for XRD studies. The diffraction pattern was recorded with Cu targeted Κ radiation at a wavelength of 1.5405 Å. The scanning was done over 2 value range of 4o to 80o at 0.02 min-1 and at 1 second time constant. The instrument was operated at a current of 30 mA and voltage of 40 kV. The crystalline domain size was calculated using Scherer formula.
D=
.
Where D = Average crystallite size, λ = X-ray wavelength β = Full width at half maxima (FWHM) of XRD spectral peak (in radians) and θ = Bragg’s angle.
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3.4.2. Fourier transform infrared (FTIR) spectroscopic studies
For FTIR measurements, the precipitate of copper nanoparticles obtained using each plant leaf extract of Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) were dried in an oven at 500C for 24 h. The dried as-synthesized copper nanoparticles were then ground with KBr and casted into pellet and made ready for analysis on FTIR spectrophotometer in the diffuse reflectance mode operating at a resolution of 4 cm1
(Hailemariam, 2011).
3.5. Antibacterial Activity Studies The antibacterial assays were done for Gram-negative Escherichia coli and Gram-positive bacteria Staphylococcus aureus by paper disc diffusion method. Nutrient agar media were used to cultivate bacteria.
3.5.1. Preparation of inoculums The test bacterial strains were transferred from the stock cultures as streaked on nutrient agar (NA) plates and incubated for 24 h. Well separated bacterial colonies were then used as inoculums. Bacteria were transferred using bacteriological loop to autoclaved nutrient agar that was cooled to about 450C in a water bath mixed by gently swirling the flasks. The medium was then poured to sterile Petri plates, allowed to solidify and used for the biotest (Jain et al., 2009)
A fresh culture of inoculums of each culture was streaked on nutrient agar media in a petri dish. 10 and 20 μL aliquots containing 5 mg/mL as-synthesized copper nanoparticles were impregnated using micropipette on paper discs of 6 mm in diameter.
24
3.5.2. Preparation of test solutions Three samples were prepared for bacterial test and labeled as 1, 2 and 3 for Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) mediated Cu nanoparticles, respectively. Then the three samples of the as-synthesized copper nanoparticles solution were prepared at concentration of 5 mg/mL by dissolving them in DMSO (dimethylsulfoxide). Zones of inhibition were measured after 24 h of incubation. The magnitude of antibacterial effect against, gram negative Escherichia coli and gram positive bacteria Staphylococcus aureus was determined based on the inhibition zone measured in the disk diffusion test.
3.6. Methods of Data Analysis
Origin version six software was used to analyze the data collected from UV-Visible and FTIR spectrophotometeric measurement.
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4. RESULTS AND DISCUSSION
The present study reports the biological synthesis of copper nanoparticles by reduction of aqueous copper ions using leaf extracts of Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) and the progress of formation of copper nanoparticles was monitored by UV-Visible spectroscopy.
4.1. Synthesis of Copper Nanoparticles
When copper nanoparticles were synthesized using materials from leaf extracts of Khat (Catha edulis) as reducing and stabilizing agent, the aqueous copper sulfate pentahydrate solution was turned to red color within 33 min. Intensity of the red color increased in direct proportion to the reaction time. It may be due to the excitation of surface plasmon resonance (SPR) effect and reduction of copper ions whereas in case of Castor Oil (Ricinus communis) leaf extract mediated synthesis of copper nanoparticles, aqueous copper sulfate solution was turned to red color within 44 min, it took 67 min before the copper sulphate aqueous solution turned red when extracts of Dergihara (Prosopis juliflora) was used as reductant. The appearance of characteristic absorption peak of copper nanoparticles near 565 nm (Figures 5 to 7) indicated the formation of copper nanoparticles. As observed from the intensity of the surface Plasmon absorption peak at around 565 nm as a function of time, the rate of reduction of copper ions using different leaf extracts are in the order of: Dergihara (Prosopis juliflora) < Castor oil (Ricinus communis) < Khat (Catha edulis).
Surface plasmon resonance (SPR) patterns that are characteristics of metal nanoparticles, strongly dependent on nanoparticle size, presence of stabilizer molecules and the dielectric constant of the medium. Observed surface plasmon resonance bands, which appeared with increase in the reaction time may indicate the formation of anisotropic molecules that later stabilized in the medium (Krishnaraj et al., 2010). The results are in agreement with the previous observations (Dadgostar, 2008; and Bahadory 2008).
26
4.2. UV-Visible Absorption Spectroscopic Study UV-Visible spectra of aqueous CuSO4.5H2O mediated with three leaf extracts as a function of reaction time are shown in Figure 5, 6 and 7 respectively. Bioreduction of Cu2+ ions to Cu nanoparticles using leaf extracts were indicated by the change of color from greenish to red. The progress in reduction of copper ions to Cu nanoparticles using the three leaf extracts was indicated by the enhanced intensity of surface Plasmon absorption peak observed within 554 nm to 567 nm. Time taken for attaining maximum reduction of copper ions to form Cu nanoparticles for Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) were recorded as 165, 220 and 335 min, respectively.
Figure 5. UV-visible spectra of aqueous copper sulfate pentahydrate solution mixed with Khat (Catha edulis) as a function of time.
27
Figure 6. UV-visible spectra of aqueous copper sulfate pentahydrate solution mixed with Castor Oil (Ricinus communis) as a function of time.
Figure 7. UV-visible spectra of aqueous copper sulfate pentahydrate solution mixed Dergihara (Prosopis juliflora) as a function of time.
Strong absorption of visible radiation is shown by the metal nanoparticles due to its induced polarization in its conduction electrons with respect to the immobile nucleus. When a particular wavelength is matched to the size of a nanoparticle, dipole oscillation is generated 28
in the compensated form of the induce polarization and the electrons in the nanoparticle resonate, introducing a strong absorption (Moskovits and Vlckova, 2005).
4.3. Fourier Transform Infrared Spectroscopic Study
IR spectroscopic measurement was carried out to elucidate the possible biomolecules present in the leaves of Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora), which may be responsible for capping and stabilizing of the Cu nanoparticles. IR spectra of copper nanoparticles observed by the reduction of copper ions using each of these three leaf extracts are presented in Table 2 and in Figure 8.
Table 2. FTIR absorption frequencies of plant leaf extract mediated copper nanoparticles.
Sample
Wave number (cm-1)
CO
3406.66, 2920.48, 1703.42,1604.26,1474.72,1324.39, 1188.05,1066.90
KH
3421.06, 2920.48, 1618.66,1439.54, 1381.96, 1074.90
DH
3406.66, 2920.48, 2848.51, 1639.45, 1381.96, 1246.02, 1039.72
Note: CO = Castor Oil (Ricinus communis), KH = Khat (Catha edulis) and DH = Dergihara (Prosopis juliflora)
Table 2 shows FTIR absorption frequencies of the three plant leaf extract mediated copper nanoparticles. The main differences between the three spectra are the fact that peaks at 1703.42 cm-1and 2848.51 cm-1 appear only on the spectra of CO and DH, respectively and peaks at 1474.72 cm-1 and 1439.54 cm-1 appear only on CO and KH, respectively. .
29
Figure 8. FTIR spectra of (a) Khat (Catha edulis), (b) Castor Oil (Ricinus communis) and (c) Dergihara (Prosopis juliflora) plant leaf extract mediated copper nanoparticles. The peaks near 3400 and 2920 cm-1 are characteristics of O-H (or N-H) and aldehydic C-H stretching, respectively. The bands at 1703.42, 1639.45 cm-1 and 1618.66 cm-1 are corresponding to amide, arising due to carbonyl stretching in proteins and the bands at 1604.26 cm-1 is characteristics of N-H bending. The peak at 1474.72 and 1039.72 - 1381.96 cm-1 correspond to methylene scissoring vibrations from the proteins in the solution and C-N stretching vibrations of amine, respectively. Although not many changes were observed at these frequencies but all peaks show a shift to lower frequency and a decrease in intensity on binding with the copper nanoparticles. This suggests that free carbonyl and NH2 groups from amino acid residues and proteins have ability to bind a metal indicating that the proteins could possibly form a layer encapsulating the metal (capping of copper nanoparticles) to prevent agglomeration and thereby stabilize the nanoparticles. FTIR spectra show that it is the protein molecule in the leaf extract, which possibly causes the reduction of copper ions and stabilize the Cu nanoparticles leading to their stabilization, which are in agreement with the previous reports (Dadgostar, 2008; Hailemariam, 2011; Valodkar et al., 2011).
30
4.4. X-Ray Diffraction Studies
The crystalline nature of Copper nanoparticles was confirmed from the analysis of the X-ray diffraction (XRD) pattern. XRD pattern of the as-synthesized Cu nanoparticles obtained by bioreduction of copper ions using Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) leaf extracts are presented in Figure 9 and their average crystalline size, which were calculated using Scherer’s formula are 22.32 nm, 27.96 nm and 29.06 nm, respectively. These are given in Table 3 and for convenience the samples which were prepared for XRD measurements were labeled as KH, CO and DH for Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) mediated copper nanoparticles, respectively.
Table 3. Average crystalline size of the as-synthesized copper nanoparticles using leaf extracts.
Sample
FWHM (degree)
Average crystallite size (nm)
KH
0.401
22.32
CO
0.320
27.96
DH
0.308
29.06
Note: CO = Castor Oil (Ricinus communis), KH = Khat (Catha edulis) and DH = Dergihara (prosopis juliflora)
31
Figure 9. XRD pattern of KH = Khat (Catha edulis), CO = Castor oil (Ricinus communis) and DH = Prosopis (Prosopis juliflora) leaf extract mediated copper nanoparticles.
The observed diffraction peaks at 2θ = 43.8 o, 50.5 o, and 74.4o which corresponds to (111), (200) and (220) planes of copper nanoparticles, respectively are suggesting the face centered cubic (FCC) crystal structure of as-synthesized Cu nanoparticles and results shown are in agreement with previous observations (Dash and Balto, 2011; Theivasanthi and Alagar, 2011b). The XRD pattern with broadening of the Bragg peaks indicates the formation of nanoparticles.
4.5. Antibacterial Activities of Copper Nanoparticles
The antibacterial activities of Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora) leaf extract mediated copper nanoparticles were performed against two pathogenic bacteria, Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus using the paper disk diffusion method. The mean values of three replicates of zone of inhibition (mm) around all samples of the as-synthesized copper nanoparticles are presented in Table 4.
32
Table 4. Zone of inhibition (mm) of KH (Catha edulis), CO (Ricinus communis) and DH (Prosopis juliflora) mediated copper nanoparticles.
Sample
Concentration of copper nanoparticles (μL)
Test Organism
10 KH = 1
Escherichia coli
7 + 0.58
8.33 + 0.58
Staphylococcus aureus
7 + 0.58
8.33 + 0.58
Escherichia coli
8 + 1.00
12.33 + 1.16
7.33 + 0.58
11.33 + 0.58
Escherichia coli
8 + 1.00
12.33 + 0.58
Staphylococcus aureus
8 + 1.00
13.33 + 0.58
Escherichia coli
20.67 + 1.15
22.33 + 2.52
Staphylococcus aureus
21.33 + 1.15
21.67 + 1.53
CO = 2
Staphylococcus aureus DH = 3
Standard=5 (Ampicillin)
20
Note: values of the mean zone of inhibition in diameter (mm) of KH = Khat (Catha edulis), CO = Castor Oil (Ricinus communis), and DH = Dergihara (Prosopis juliflora) on the test organisms at varying concentrations .
Although the as-synthesized copper nanoparticles of the present study are less active against Escherichia coli and Staphylococcus aureus as compared to the reference standard drug, but still they exhibited good antibacterial activity (Table 4). Results shown that the test solutions are significant indicating that Cu nanoparticles exhibit good biocidal activity. This corroborates with the previous observations of other researchers (Theivasanthi and Alagar, 2011a; Hailemariam, 2011).
The study shows that copper nanoparticles synthesized via green route are promising antibacterial agent against the pathogens which are highly toxic to multidrug resistant bacteria hence have a great potential in biomedical applications. There is a variation in the measured 33
zones of inhibition as a function of applying concentrations of copper nanoparticles suspension and nature of the bacteria employed. These results clearly demonstrate that the assynthesized copper nanoparticles are promising antibacterial agents against the pathogenic bacteria (Figures 10, 11, 12 and 13).
Figure 10. Antibacterial activity of copper nanoparticles (10 μL) on Escherichia coli Note: 1 = Khat (Catha edulis) mediated copper nanoparticles 2 = Castor Oil (Ricinus communis) mediated copper nanoparticles 3 = Dergihara (Prosopis juliflora) mediated copper nanoparticles 4 = Control (DMSO) 5 = Standard (Ampicillin)
34
Figure 11. Antibacterial activity of copper nanoparticles (20 μL) and on Escherichia coli. Note: 1 = Khat (Catha edulis) mediated copper nanoparticles 2 = Castor Oil (Ricinus communis) mediated copper nanoparticles 3 = Dergihara (Prosopis juliflora) mediated copper nanoparticles 4 = Control (DMSO) 5 = Standard (Ampicillin)
35
Figure 12. Antibacterial activity of copper nanoparticles (10 μL) on Staphylococcus aureus Note: 1 = Khat (Catha edulis) mediated copper nanoparticles 2 = Castor Oil (Ricinus communis) mediated copper nanoparticles 3 = Dergihara (Prosopis juliflora) mediated copper nanoparticles 4 = Control (DMSO) 5 = Standard (Ampicillin)
36
Figure 13. Antibacterial activity of copper nanoparticles (20 μL) on Staphylococcus aureus. Note: 1 = Khat (Catha edulis) mediated copper nanoparticles 2 = Castor Oil (Ricinus communis) mediated copper nanoparticles 3 = Dergihara (Prosopis juliflora) mediated copper nanoparticles 4 = Control (DMSO) 5 = Standard (Ampicillin)
37
5. SUMMARY, CONCLUSION AND RECOMMENDATION 5.1. Summary and Conclusion The present study illustrates simple, convenient and eco-friendly method for the synthesis of copper nanoparticles by using leaf extracts of Khat (Catha edulis), Castor oil (Ricinus communis) and Dergihara (Prosopis juliflora). The reduction of the metal ions through the leaf extracts leading to the formation of copper nanoparticles of fairly well-defined dimensions. Besides, the formation of the as-synthesized nanomaterial was monitored by UVVisible spectrophotometer.
Characterizations of the synthesized copper nanoparticles have been successfully done using XRD and FTIR techniques. FTIR results show that reduction of copper ions and stabilization of copper nanoparticles are thought to occur through possible participation of leaf proteins and other metabolites present in the leaf extracts and X-ray diffraction (XRD) spectral results confirmed the face centered cubic (FCC) structure of nanoparticles with high stability and without any impurity. Investigations on the antibacterial effect of the as-synthesized copper nanoparticles that were performed against pathogenic bacteria, Escherichia coli and Staphylococcus aureus, reveal that high efficacy of copper nanoparticles as a strong antibacterial agent.
In conclusion, this greener approach toward the synthesis of copper nanoparticles, using plant leaf material as reducing and capping agent, has many advantages such as ease with which the process can be scaled up, economic viability, environmentlly benign and renewable, there is no need to use high pressure, energy, temperature and toxic chemicals. Applications of ecofriendly copper nanoparticles in bactericidal, wound healing and other medical and electronic applications are potentially exciting for their large-scale synthesis. Toxicity of copper nanoparticles on human pathogen bacteria opens a door for new range of antibacterial agents.
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5.2. Recommendation
Based on the results obtained from the present study, the following recommendations are made There are several parameters that seem to affect the growth and nucleation of copper nanoparticles, such as reduction time, reduction temperature, pH of the medium, metal ion concentration and amount of plant leaf extract. So, studies on the effect of these and other parameters for the biosynthesis of copper and other nanoparticles should be carried out. Extensive work on the synthesis of copper nanoparticles should be tried with other plant materials.
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7. APPENDIX
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7.1. Appendix Tables
Appendix Table 1. Values of surface plasmon absorbance and their corresponding λmax at different time of reaction for KH mediated reduction of copper ions.
Absorbance
Reaction time(min)
λmax (nm)
0.125
33
553
0.166
66
556
0.192
99
556
0.288
132
559
0.395
165
565
Note: KH = Khat (Catha edulis), λmax (nm) = Maximum wavelength in nanometer. Appendix Table 2 Values of surface plasmon absorbance and their corresponding λ max at different time of reaction for CO mediated reduction of copper ions.
Absorbance
Reaction time(min)
λmax (nm)
0.144
44
557
0.168
88
561
0.215
132
563
0.299
176
565
0.383
220
567
Note: CO= Castor oil (Ricinus communis), λmax (nm) = Maximum wavelength in nanometer.
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Appendix Table 3 Values of surface plasmon absorbance and their corresponding λ max at different time of reaction for CO mediated reduction of copper ions.
Absorbance
Reaction time(min)
λmax (nm)
0.126
67
554
0.149
134
556
0.193
201
558
0.298
268
562
0.388
335
564
Note: DH = Derjihara (Prosopis juliflora), λmax (nm) = Maximum wavelength in nanometer.
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7.2. Appendix Figures
Appendix Figure 1. XRD pattern of Khat (Catha edulis), leaf extract mediated copper nanoparticles.
Appendix Figure 2. XRD spectra of Castor oil (Ricinus communis) leaf extract mediated copper nanoparticles.
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Appendix Figure 3. XRD spectra of Dergihara (Prosopis juliflora) leaf extract mediated copper nanoparticles.
52
