World J Microbiol Biotechnol DOI 10.1007/s11274-015-1840-3
ORIGINAL PAPER
Green synthesis of copper nanoparticles by Citrus medica Linn. (Idilimbu) juice and its antimicrobial activity Sudhir Shende1 • Avinash P. Ingle1 • Aniket Gade1,2 • Mahendra Rai1
Received: 12 August 2014 / Accepted: 6 March 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract We report an eco-friendly method for the synthesis of copper nanoparticles (CuNPs) using Citron juice (Citrus medica Linn.), which is nontoxic and cheap. The biogenic copper nanoparticles were characterized by UV– Vis spectrophotometer showing a typical resonance (SPR) at about 631 nm which is specific for CuNPs. Nanoparticles tracking analysis by NanoSight-LM20 showed the particles in the range of 10–60 nm with the concentration of 2.18 9 108 particles per ml. X-ray diffraction revealed the FCC nature of nanoparticles with an average size of 20 nm. The antimicrobial activity of CuNPs was determined by Kirby-Bauer disk diffusion method against some selected species of bacteria and plant pathogenic fungi. It was reported that the synthesized CuNPs demonstrated a significant inhibitory activity against Escherichia coli followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Propionibacterium acnes and Salmonella typhi. Among the plant pathogenic fungi tested, Fusarium culmorum was found to be most sensitive followed by F. oxysporum and F. graminearum. The novelty of this work is that for the first time citron juice was used for the synthesis of CuNPs. Keywords Citron juice (Citrus medica Linn.) Antimicrobial agent Copper nanoparticles Non-toxic Eco-friendly Economical
& Mahendra Rai [email protected] 1
Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati 444 602, Maharashtra, India
2
Department of Biology, Utah State University, Logan, UT 84322, USA
Introduction Copper nanoparticles (CuNPs) have recently attracted special attention because of their low-cost and novel optical, mechanical, catalytic, electrical and thermal conduction properties, which are different from that of their bulk metals (Brust and Kiely 2002; Lee et al. 2009). Due to their potential applications, there has been a special focus on the synthesis of CuNPs for the past few decades. Many approaches have been used for the synthesis of CuNPs, which include physical, chemical and biological. Out of these three approaches biological synthesis of CuNPs has been always a challenge for researchers due to the major problem of oxidation. CuNPs of size about 20–80 nm have been formed by irradiating copper salts by gamma rays, using Poly N-vinyl pyrrolidone and Poly vinyl alcohol as capping agents (Joshi et al. 1998). Nanocopper has also been produced through solid-state reactions activated by high energy ball milling (Sheibani et al. 2007). Ponce and Klabunde (2005) demonstrated the metal vapor synthesis technique to produce CuNPs by various groups. Reduction of metallic salts in water-in-oil microemulsions or reverse micelles, consisting of nanosized water droplets, has been demonstrated for Pt, Pd, Rh, and Ir (Boutonnet et al. 1982). The same technique has also been used for the preparation of CuNPs (Capek 2004). Controlled current electrolysis using surfactants, thermal reduction, sono-chemical reduction, aquous phase synthesis by using organic nanoparticles, liquid phase plasma reduction method, one-pot synthesis, thermal decomposition and controlled chemical reduction were other methods reported so far (Betancourt-Galindo et al. 2014; Dhas et al. 1998; Huang et al. 2006; Lee et al. 2014; Pileni 1997; Saldanha et al. 2014; Singh et al. 2014; Yang et al. 2003).
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Most of the current methods used for the synthesis of nanoparticles use either toxic chemicals and/or complex processes, which require high amounts of energy (Sheibani et al. 2007). The toxic chemicals serve either as reducing agents for various metal salts to their corresponding zero valent metallic nanoparticles or as stabilizing agents to prevent nanoparticles from agglomeration. Hydrazine, N,Ndimethylformamide and sodium borohydride are the popularly used reducing agents (Feitz and Waite 2004), which are highly toxic to living organisms and the environment. This drives the researchers to search for the clean, non-toxic and environmentally acceptable routes for the production of nanoparticles. Lee et al. (2013) synthesized the stable CuNPs by the treatment of aqueous solution of copper sulphate pentahydrate solution with Magnolia kobus leaf extract with evaluation of antibacterial potential against E. coli which showed significant antibacterial activity. Kulkarni and Kulkarni (2013) developed stable CuNPs by green route using Ocimum sanctum leaf extract. Similarly, Mittal et al. (2014) reviewed that plant can be used as nanofactories for the synthesis of metal nanoparticles like CuNPs. However, there are some reports on the use of plant systems for the synthesis of CuNPs which includes Capparis zeylanica (Saranyaadevi et al. 2014), Gloriosa superba L. (Naika et al. 2015), Vitis vinifera (Angrasan and Subbaiya 2014), Nerium oleander (Gopinath et al. 2014), Artabotrys odoratissimus (Kathad and Gajera 2014), etc. Varshney et al. (2011) synthesised the CuNPs by using Pseudomonas stutzeri bacterial strain isolated from the wastewaters of electroplating industry. Salvadori et al. (2014) developed a biological system for the biosynthesis of CuNPs by removal of copper from wastewater by dead biomass of the yeast Rhodotorula mucilaginosa. CuNPs can be the alternative to Ag and Au nanoparticles and have potential applications in many industrial areas like bio-fungicide and bio-pesticides. Many synthetic routes have been documented for the preparation of CuNPs, but a few routes are eco-friendly and scalable. There has been some activity in this direction, resulting in various synthesis methods that make use of natural products involving organisms ranging from bacteria to fungi and plants. Recently, Ingle et al. (2013) reviewed the bioactivities of CuNPs, and reported that CuNPs have broad spectrum activities, which includes antibacterial, antifungal, antiviral, antiparasital and anticancerous activities. Yoon et al. (2007) studied the susceptibility of E. coli and B. subtilis to the AgNPs and CuNPs and found that survival rate of bacteria decreased with increasing concentration of nanoparticles. Both the bacteria were completely inhibited at the concentration higher than 70 and 60 lg/mL for AgNPs and CuNPs, respectively. This study proved the effectiveness of CuNPs over AgNPs. Das et al. (2010)
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studied the antibacterial activity of CuNPs against three bacteria namely S. aureus, B. subtilis and E. coli. They reported that CuNPs were effective against these bacteria. In another study by Ramyadevi et al. (2012), it was reported that CuNPs showed potential antibacterial activity against Micrococcus luteus, S. aureus, E. coli, K. pneumoniae and P. aeruginosa. Out of these, E. coli was the most susceptible bacterium followed by S. aureus, M. luteus and K. pneumoniae, while P. aeruginosa was found to be the least sensitive to CuNPs. Similarly, there are few reports available on the antifungal activity of CuNPs. Petranovskii et al. (2003) evaluated the fungicidal action of Cu-based zeolites against three species of fungi viz. Cladosporium cladosporoides, Phaeococcomyces chersonesos and Ulocladium chartarum. They found that zeolite powder was very much effective. Kim et al. (2006) demonstrated antimicrobial activity of the Cu–SiO2 nanocomposite against Candida albicans and Penicillium citrinum and reported the potential activity against both the fungi. Due to significant antimicrobial nature of CuNPs, researchers are trying to use them in different formulations and textile fabrics. Similar attempts have been made by Usha et al. (2010) who developed copper oxi-decoated fabrics and studied its activity against A. niger. Chattopadhyay and Patel (2010) evaluated the antimicrobial activity of CuNPs by soil burial test results clearly showed the CuNPs treatment enhanced the resistance of cotton fabrics to microbial attack. The aim of the present study was to develop a nontoxic and cost-effective biological method for the synthesis of CuNPs. It is a green approach for the synthesis of copper nanoparticles which can be achieved only in 15–20 min.
Materials and methods Plant material Fruits of Citrus medica Linn. were collected from garden of Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India. Preparation of fruit juice of citron and synthesis of CuNPs Fruit juice of C. medica Linn. was extracted at room temperature by squeezing the prewashed mature fruits of citron and filtered through muslin cloth. The known volume of filtered extract of citron was added to CuSO45H2O solution (100 mM) prepared in sterilized distilled water. The solution was mixed thoroughly, poured into an aluminium vessel for the reaction and gradually heated to boiling (60–100°C).
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Detection and characterization of CuNPs Visual observation The primary detection was carried out by visual observation. The change in color of the precursor solution from blue to pale-yellow or colorless with time and deposition of reddish shiny brown colored precipitation on the inner surface of the vessel provides evidence of CuNPs synthesis. Ultraviolet–Visible (UV–Vis) spectroscopy Further, CuNPs were detected by UV–Vis spectrophometer for which the reaction mixture was subjected to optical analysis and the spectra (Shimadzu UV-1700, Japan) were obtained at the resolution of 1 nm from 200 to 800 nm for each sample. Nanoparticle tracking and analysis system (NTA) The CuNPs thus synthesized were later characterized by NanoSight LM-20 to find out the size of the particles. LM 20 is a laser-based light scattering system, in which particles suspended in the liquid medium were injected into the LM viewing unit and viewed in the closed proximity to the optical element. Brownian motion of CuNPs present within the path of the laser beam was observed via a dedicated non-microscope optical instrument (LM-20, NanoSight Pvt. Ltd., UK) having charge-coupled device (CCD) camera. The motions of the particles in the field of view (approximately 100 9 100 lm) were recorded (at 30 fps) and the subsequent sample images and video were captured. X-ray diffraction (XRD) X-ray diffraction (XRD) studies were carried out on a Rigaku Miniflex II dextop X-ray diffractometer instrument using CuKa radiation (0.15418 nm). XRD data analysis was done using ISDD software JCPDS and the average size of CuNPs was calculated by using Debye– Scherrer’s equation (D = 0.9k/b cos h). Assessment of antimicrobial activity of CuNPs Test organisms The pure cultures of bacteria E. coli (ATCC 14948), P. acne (MTCC-1951), K. pneumoniae (MTCC 4030), S. typhi (ATCC 51812) and P. aeruginosa (MTCC 4676) and two plant pathogenic fungi, namely F. oxysporum (MTCC 1755) and F. culmorum (MTCC 349) were procured from culture collection centers, F. graminearum (DBT 4) was isolated from kidney beans. In vitro evaluation of antimicrobial activity The antibacterial and antifungal activity of CuNPs was evaluated against E. coli, P. acne, K. pneumoniae, S. typhi, and P. aeruginosa and plant pathogenic fungi, namely F. oxysporum, F. graminearum and F. culmorum. The overnight grown
cultures of E. coli, P. acne, K. pneumoniae, S. typhi, and P. aeruginosa were used to assess the activity of CuNPs. The activity of CuNPs was evaluated singly by impregnating 20 ll of CuNPs solution in the sterile discs. A disc diffusion assay was performed on Muller–Hinton agar plates for bacterial cultures. A single colony of bacterium was grown overnight in Muller–Hinton broth on a rotary shaker (100 RPM) at 37 °C. The inocula (100 ll) were then spread onto the agar plates and prepared discs containing CuNPs were placed onto agar surface. After incubation at 37 °C for 24 h, the zones of inhibition were measured. Similarly, potato dextrose agar plates were inoculated with the spore suspension (20 ll) of the test fungi (F. oxysporum, F. graminearum and F. culmorum). The discs containing CuNPs were placed on to the surface of plates inoculated with respective test fungi and incubated at 25° ± 2 °C for 52 h followed by measurement of the zones of inhibition. The antimicrobial assays were performed in triplicate.
Results After addition of fruit juice extracted from citron by squeezing the fruit with aqueous copper sulfate solution (100 mM) in an aluminium vessel and heating the mixture to boiling, the blue color of the precursor solution changed to light-yellow or sometimes become colorless with time and the CuNPs were deposited on the inner wall of the vessel (Fig. 1a, b). Deposition of the shiny reddish-brown precipitate on the inner wall of the aluminium vessel, indicates the formation of CuNPs. The synthesized CuNPs were collected, by brushing or scraping the inner walls of the vessel and dissolved in liquid ammonia to make its colloidal solution for further characterization. Surface Plasmon Resonance (SPR) of synthesized CuNPs was detected by using UV–Vis spectrophotometer. The colloidal suspension of CuNPs in liquid ammonia showed the peak at 631 nm (Fig. 1c) which is specific for CuNPs. For the determination of size and concentration of CuNPs, Nanoparticle Tracking and Analysis (NTA) was carried out, which confirmed that CuNPs synthesized were in the range of 10-60 nm with an average size of 33 nm. Further, the total concentration of CuNPs was found to be 2.18 9 108 particles/ml (Fig. 2a, b). XRD patterns obtained for the CuNPs synthesized using citron juice has been shown in Fig. 3. The presence of intense peak corresponds to 111, 111, 200 and 220, indexed a crystalline copper FCC phase (Mott et al. 2007; Sastry et al. 2013; Usman et al. 2012). The average size is calculated by using Debye–Scherrer’s equation (D = 0.9k/b cos h), where, D is the average grain size of crystallite, k is the incident wavelength, h is the Bragg angle, b is the diffracted full
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Fig. 1 (a) Copper Sulfate solution with citron juice in aluminium vessel, (b) Deposited CuNPs on the inner wall of aluminium vessel after synthesis (c) UV–Vis spectrum of the synthesised CuNPs
dissolved in liquid ammonia solution showing absorbance at 631 nm (scanned in 200–800 nm range)
Fig. 2 NTA analysis of synthesized CuNPs (a) histogram showing particle size distribution and the average size of CuNPs (33 nm) (b) 3D view
width at half maximum (in radians), respectively (MontesBurgos et al. 2010), which gives an average size of CuNP 20 nm. The in vitro antimicrobial activity of CuNPs produced in this study was evaluated against bacteria E. coli, P. acne, K. pneumoniae, S. typhi, and P. aeruginosa and three plant pathogenic fungi viz. F. oxysporum, F. graminearum and F. culmorum. The synthesised CuNPs showed significant activity against all the test organisms. It was found that
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E. coli was the most sensitive bacterium followed by K. pneumoniae, P. aeruginosa, P. acne and S. typhi. However, among the plant pathogenic fungi tested, F. culmorum was found to be the most sensitive followed by F. oxysporum and F. graminearum (Figs. 4, 5). Whereas, liquid ammonia and copper sulfate did not show any significant activity against these test organisms (Figs. 4, 5). These results corroborate with the findings obtained in previous studies, which confirmed that metal nanoparticles were effective
World J Microbiol Biotechnol Fig. 3 XRD pattern of CuNPs showing the FCC structure of crystallite and size calculated about 20 nm
Fig. 4 Histogram of antibacterial activity of CuNPs which showed that E. coli was the most sensitive bacterium followed by K. pneumoniae, P. aeruginosa, P. acne and S. typhi
against human as well as plant pathogens (Barik et al. 2008; Gajbhiye et al. 2009; Goswami et al. 2010; Kanhed et al. 2014; Owolade et al. 2008; Rai and Ingle 2012).
Discussion The deposition of copper on aluminum is a well-known cementation process, where a more reactive metal displaces a less reactive metal from its compound in solution
(Sastry et al. 2013). Thus, a single-displacement redox reaction (electroless deposition) can be described as: 2AlðsÞ þ 3Cu2 SO4 ðaqÞ ! 6CuðsÞ þ Al2 ðSO4 Þ3 ðaqÞ As the reaction proceeds, copper gets precipitated out of the solution by forming a thin layer/coating on the wall of the aluminium vessel used. Unlike, noble metals such as Ag and Au, the light transition metal Cu usually cannot be obtained via the reduction of simple copper salts such as copper sulfate in aqueous solution (Hirai et al. 1986). The
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World J Microbiol Biotechnol Fig. 5 Histogram of antifungal activity of CuNPs which showed F. culmorum was found to be the most sensitive followed by F. oxysporum and F. graminearum
reduction usually stops at Cu2O stage due to the presence of a large number of water molecules (Zhu et al. 1994). So it requires reagent carrying functional groups that could form complexes with copper ions. The surfactants used in the solution acted as capping agents to get the CuNPs. Without presence of capping agents, the particle size would be in the micron range (Song et al. 2004) which may be because of the agglomeration or formation of aggregates of CuNPs. Song et al. (2004) suggested that the metallic CuNPs were stable in nonpolar solvents only. In this method, the organic hydrocarbon part of capping agent used facilitate the protection of copper. The antioxidant nature and acidic property of citron also prevents oxidation of copper because all the protons present in the medium interfere with electro-deposition of copper at low pH range. It was reported that citron juice (acts capping agent) had weak acidic constituent as ascorbic acid, saponins and flavonoids (Gattuso et al. 2007). NTA (NanoSight-LM 20) is an analytical instrument, which is preferably used for the determination of nanoparticles size. It determines the size of nanoparticles based on Brownian motion of particles present in the samples (Raheman et al. 2011). Kanhed et al. (2014) reported the in vitro antifungal activity of chemically synthesized CuNPs with a commercially available antifungal agent against four different plant pathogenic fungi, viz., F. oxysporum, C. lunata, A. alternata and P. destructiva. CuNPs showed activity against all the plant pathogenic fungi used in the experiment. The results demonstrated that C. lunata and A. alternata were comparatively resistant to the commercial
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antifungal agent (bavistin) but showed sensitivity towards CuNPs. In this study, CuNPs demonstrated the maximum antifungal activity against C. lunata followed by A. alternata, and the minimum activity was reported against P. destructiva. It was also found that antifungal activity of bavistin increases in combination with CuNPs in case of F. oxysporum. The present study also confirmed the antifungal nature of CuNPs against plant pathogenic fungi tested. Kabra et al. (2012) reported that the ethanolic extract of peels of C. medica L. showed in vitro antimicrobial activity against bacteria such as Staphylococcus aureus, Proteus vulgaris, K. pneumoniae, E. coli, B. subtilis and P. aeruginosa. Sah et al. (2011) reported that the fruit juice of C. medica L. inhibited all tested Gram-positive, Gram-negative bacteria and two fungi A. niger and A. flavus with zone of inhibition compared to standard drugs. In the present study also, the Citron juice showed some efficacy against the test bacteria but it was significantly lower than that of activity of CuNPs (Fig. 4). On the other hand, in case of efficacy of citron juice against fungi only F. graminearum was found to be less susceptible while rest of two fungi (F. oxysporum and F. culmorum) did not show any susceptibility (Fig. 5). In another study, Usman et al. (2013) reported the synthesis of pure CuNPs in the presence of a chitosan stabilizer through chemical means with the evaluation of antimicrobial activity of the nanoparticles by using several test microorganisms like methicillin-resistant S. aureus, B. subtilis, P. aeruginosa, Salmonella choleraesuis and Candida albicans. Sampath et al. (2014) synthesized jasmine bud-shaped CuNPs by a green chemical reduction method using polyvinylpyrrolidone (PVP) as a capping agent,
World J Microbiol Biotechnol Fig. 6 Schematic representation of hypothetical mechanism of action for CuNPs. 1 Accumulation of CuNPs on the cell surface form pits which causes cell leakage; 2 interaction of CuNPs with cell membrane decrease the transmembrane electrochemical potential, which affects membrane integrity; 3 DNA damage due to the interaction with CuNPs; 4 interaction of Cu2? with sulfhydryl group of protein, inactivates the proteins; 5 entry of CuNPs and Cu2? inside the cell develop oxidative stress, which leads to cell death
L-ascorbic acid (AA) as a reducing agent and antioxidant agent, isonicotinic acid hydrazide (INH) as a reducing agent. The antibacterial activity of the Cu nano buds was evaluated by testing against E. coli and S. aureus. Agriculture is the backbone of India and most of the developing countries, with more than 60 % of the population depending on it for their livelihood. The infections caused by various bacterial and fungal species, are increasing with a fast pace in the crop plants and thereby limiting crop productivity by yield loss of around 25 %. So it is necessary to use the modern technology to control the diseases and enhance yields. Among the inorganic antimicrobial agents, copper compounds have been commonly used in agriculture practices as a fungicide (Garcia et al. 2003), pesticides (CDPR 2009), algaecide (De Oliveira-Filho et al. 2004) and herbicide (Mastin and Rodgers 2000) as well as in animal husbandry as a disinfectant (Mortazavi and Ahmad 2009). As the biogenic CuNPs showed potent activity against a wide range of microbes it can be used as alternative antimicrobials specially against crop pathogens (Lee et al. 2011; Majumder 2012). Different formulations of CuNPs as nanofungicides, nanoantimicrobials or nanofertilizers serve as a dual purpose to control the infections as well as a nutrient for plants. It is widely demonstrated and also proved from the present study, that CuNPs have significant antimicrobial activity, but its mechanism of action is yet unknown. There are only few reports available on mechanism of action of CuNPs but still the mechanism is not completely understood. According to Das et al. (2010) CuNPs enter into the cell due to their small size and inactivate their proteins/
enzymes, generating hydrogen peroxide, which causes bacterial cell death. In another study, it was reported that inactivation of proteins occur due to the interaction of CuNPs with its –SH (sulfhydryl) group (Schrand et al. 2010). Similarly, CuNPs also interact with DNA molecules leading to its degradation by disturbing its helical structure (Kim et al. 2000). In fact, the cell membrane integrity depends on its electrochemical potential, but Deryabin et al. (2013) reported that CuNPs decrease the electrochemical potential of the cell membrane, which ultimately affects cell membrane integrity. It was also believed that metal nanoparticles release their respective ions and such heavy metal ions demonstrated adverse effect on bacterial cell (Cioffi et al. 2005). Accumulation of CuNPs and Cu ions on cell surface form pits in the membrane, which mainly leads to the leakage of cellular component from cell and eventually causes cell death. Another important reason proposed for the cell death due to action of CuNPs is development of oxidative stress (Deryabin et al. 2013). Considering all the above possibilities here, we have proposed a schematic representation (Fig. 6) for hypothetical mechanism of action of CuNPs in bacteria.
Conclusions Use of citron juice for the synthesis of CuNPs is a novel step towards the biogenic synthesis of CuNPs. It is an ecofriendly, non-toxic and rapid approach. Further, the use of biogenic CuNPs against different human and plant pathogens confirmed its effectiveness against wide range of microorganisms. Due to the antimicrobial property of
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CuNPs, it can be used in different formulations like nanofungicides, nanoantimicrobials and nanofertilizers, which could serve dual purpose, by protecting the crop plants from its pathogens and also providing nutrients to the plants. Finally, and most important is that it is a costeffective approach as raw materials involved in the synthesis are very cheap. Acknowledgments The authors are thankful to University Grants commission, New Delhi for financial support under UGC-SAP and API is highly thankful to Department of Science and Technology, New Delhi for providing financial assistance under DST Fast Track Scheme for Young Scientists.
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Saranyaadevi K, Subha V, Ravindran RSE, Renganathan S (2014) Synthesis and characterization of copper nanoparticle using Capparis zeylanica leaf extract. Int J ChemTech Res 6(10):4533–4541 Sastry ABS, Aamanchi RBK, Rama Linga Prasad CS, Murty BS (2013) Large-scale green synthesis of Cu nanoparticles. Environ Chem Lett 11:183–187 Schrand AM, Rahman MF, Hussain SM, Schlager JJ, Smith DA, Syed AF (2010) Metal-based nanoparticles and their toxicity assessment. WIREs Nanomed Nanobiotechnol 2:554–568 Sheibani S, Ataie A, Manesh SH, Khayati GR (2007) Structural evolution in nano-crystalline Cu synthesized by high energy ball milling. Mater Lett 61:3204–3207 Singh A, Kaur S, Kaur A, Aree T, Kaur N, Singh N, Bakshi M (2014) Aqueous-phase synthesis of copper nanoparticles using organic nanoparticles: application of assembly in detection of Cr3?. ACS Sustain Chem Eng 2:982–990 Song X, Sun S, Zhang W, Yin Z (2004) A method for synthesis of spherical copper nanoparticles in the organic phase. J Colloid Interface Sci 273:463–469 Usha R, Prabu E, Palaniswamy M, Venil CK, Rajendran R (2010) Synthesis of metal oxide nanoparticles by Streptomyces sp. for development of antimicrobial textiles. Global J Biotechnol Biochem 5:153–160 Usman MS, Ibrahim NA, Shameli K, Zainuddin N, Yunus WM (2012) Copper nanoparticles mediated by chitosan: synthesis and characterization via chemical methods. Molecules 17:14928–14936 Usman MS, Zowalaty MEE, Shameli K, Zainuddin N, Salama M, Ibrahim NA (2013) Synthesis, characterization, and antimicrobial properties of copper nanoparticles. Int J Nanomed 8:4467–4479 Varshney R, Bhadauria S, Gaur MS, Pasricha R (2011) Copper nanoparticles synthesis from electroplating industry effluent. Nano Biomed Eng 3:115–119 Yang X, Chen S, Zhao S, Li D, Ma H (2003) Synthesis of copper nanorods using electrochemical methods. J Serb Chem Soc 68:843–847 Yoon KY, Byeon JH, Park JH, Hwang J (2007) Susceptibility constants of Escherichia coli and Bacillus subtilis to silver and copper nanoparticles. Sci Total Environ 373:572–575 Zhu YJ, Qian YT, Zhang MW, Chen ZY, Xu DF (1994) Preparation and characterization of nanocrystalline powders of cuprous oxide by using C-radiation. Mater Res Bull 29:377–383
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ORIGINAL PAPER
Green synthesis of copper nanoparticles by Citrus medica Linn. (Idilimbu) juice and its antimicrobial activity Sudhir Shende1 • Avinash P. Ingle1 • Aniket Gade1,2 • Mahendra Rai1
Received: 12 August 2014 / Accepted: 6 March 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract We report an eco-friendly method for the synthesis of copper nanoparticles (CuNPs) using Citron juice (Citrus medica Linn.), which is nontoxic and cheap. The biogenic copper nanoparticles were characterized by UV– Vis spectrophotometer showing a typical resonance (SPR) at about 631 nm which is specific for CuNPs. Nanoparticles tracking analysis by NanoSight-LM20 showed the particles in the range of 10–60 nm with the concentration of 2.18 9 108 particles per ml. X-ray diffraction revealed the FCC nature of nanoparticles with an average size of 20 nm. The antimicrobial activity of CuNPs was determined by Kirby-Bauer disk diffusion method against some selected species of bacteria and plant pathogenic fungi. It was reported that the synthesized CuNPs demonstrated a significant inhibitory activity against Escherichia coli followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Propionibacterium acnes and Salmonella typhi. Among the plant pathogenic fungi tested, Fusarium culmorum was found to be most sensitive followed by F. oxysporum and F. graminearum. The novelty of this work is that for the first time citron juice was used for the synthesis of CuNPs. Keywords Citron juice (Citrus medica Linn.) Antimicrobial agent Copper nanoparticles Non-toxic Eco-friendly Economical
& Mahendra Rai [email protected] 1
Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati 444 602, Maharashtra, India
2
Department of Biology, Utah State University, Logan, UT 84322, USA
Introduction Copper nanoparticles (CuNPs) have recently attracted special attention because of their low-cost and novel optical, mechanical, catalytic, electrical and thermal conduction properties, which are different from that of their bulk metals (Brust and Kiely 2002; Lee et al. 2009). Due to their potential applications, there has been a special focus on the synthesis of CuNPs for the past few decades. Many approaches have been used for the synthesis of CuNPs, which include physical, chemical and biological. Out of these three approaches biological synthesis of CuNPs has been always a challenge for researchers due to the major problem of oxidation. CuNPs of size about 20–80 nm have been formed by irradiating copper salts by gamma rays, using Poly N-vinyl pyrrolidone and Poly vinyl alcohol as capping agents (Joshi et al. 1998). Nanocopper has also been produced through solid-state reactions activated by high energy ball milling (Sheibani et al. 2007). Ponce and Klabunde (2005) demonstrated the metal vapor synthesis technique to produce CuNPs by various groups. Reduction of metallic salts in water-in-oil microemulsions or reverse micelles, consisting of nanosized water droplets, has been demonstrated for Pt, Pd, Rh, and Ir (Boutonnet et al. 1982). The same technique has also been used for the preparation of CuNPs (Capek 2004). Controlled current electrolysis using surfactants, thermal reduction, sono-chemical reduction, aquous phase synthesis by using organic nanoparticles, liquid phase plasma reduction method, one-pot synthesis, thermal decomposition and controlled chemical reduction were other methods reported so far (Betancourt-Galindo et al. 2014; Dhas et al. 1998; Huang et al. 2006; Lee et al. 2014; Pileni 1997; Saldanha et al. 2014; Singh et al. 2014; Yang et al. 2003).
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Most of the current methods used for the synthesis of nanoparticles use either toxic chemicals and/or complex processes, which require high amounts of energy (Sheibani et al. 2007). The toxic chemicals serve either as reducing agents for various metal salts to their corresponding zero valent metallic nanoparticles or as stabilizing agents to prevent nanoparticles from agglomeration. Hydrazine, N,Ndimethylformamide and sodium borohydride are the popularly used reducing agents (Feitz and Waite 2004), which are highly toxic to living organisms and the environment. This drives the researchers to search for the clean, non-toxic and environmentally acceptable routes for the production of nanoparticles. Lee et al. (2013) synthesized the stable CuNPs by the treatment of aqueous solution of copper sulphate pentahydrate solution with Magnolia kobus leaf extract with evaluation of antibacterial potential against E. coli which showed significant antibacterial activity. Kulkarni and Kulkarni (2013) developed stable CuNPs by green route using Ocimum sanctum leaf extract. Similarly, Mittal et al. (2014) reviewed that plant can be used as nanofactories for the synthesis of metal nanoparticles like CuNPs. However, there are some reports on the use of plant systems for the synthesis of CuNPs which includes Capparis zeylanica (Saranyaadevi et al. 2014), Gloriosa superba L. (Naika et al. 2015), Vitis vinifera (Angrasan and Subbaiya 2014), Nerium oleander (Gopinath et al. 2014), Artabotrys odoratissimus (Kathad and Gajera 2014), etc. Varshney et al. (2011) synthesised the CuNPs by using Pseudomonas stutzeri bacterial strain isolated from the wastewaters of electroplating industry. Salvadori et al. (2014) developed a biological system for the biosynthesis of CuNPs by removal of copper from wastewater by dead biomass of the yeast Rhodotorula mucilaginosa. CuNPs can be the alternative to Ag and Au nanoparticles and have potential applications in many industrial areas like bio-fungicide and bio-pesticides. Many synthetic routes have been documented for the preparation of CuNPs, but a few routes are eco-friendly and scalable. There has been some activity in this direction, resulting in various synthesis methods that make use of natural products involving organisms ranging from bacteria to fungi and plants. Recently, Ingle et al. (2013) reviewed the bioactivities of CuNPs, and reported that CuNPs have broad spectrum activities, which includes antibacterial, antifungal, antiviral, antiparasital and anticancerous activities. Yoon et al. (2007) studied the susceptibility of E. coli and B. subtilis to the AgNPs and CuNPs and found that survival rate of bacteria decreased with increasing concentration of nanoparticles. Both the bacteria were completely inhibited at the concentration higher than 70 and 60 lg/mL for AgNPs and CuNPs, respectively. This study proved the effectiveness of CuNPs over AgNPs. Das et al. (2010)
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studied the antibacterial activity of CuNPs against three bacteria namely S. aureus, B. subtilis and E. coli. They reported that CuNPs were effective against these bacteria. In another study by Ramyadevi et al. (2012), it was reported that CuNPs showed potential antibacterial activity against Micrococcus luteus, S. aureus, E. coli, K. pneumoniae and P. aeruginosa. Out of these, E. coli was the most susceptible bacterium followed by S. aureus, M. luteus and K. pneumoniae, while P. aeruginosa was found to be the least sensitive to CuNPs. Similarly, there are few reports available on the antifungal activity of CuNPs. Petranovskii et al. (2003) evaluated the fungicidal action of Cu-based zeolites against three species of fungi viz. Cladosporium cladosporoides, Phaeococcomyces chersonesos and Ulocladium chartarum. They found that zeolite powder was very much effective. Kim et al. (2006) demonstrated antimicrobial activity of the Cu–SiO2 nanocomposite against Candida albicans and Penicillium citrinum and reported the potential activity against both the fungi. Due to significant antimicrobial nature of CuNPs, researchers are trying to use them in different formulations and textile fabrics. Similar attempts have been made by Usha et al. (2010) who developed copper oxi-decoated fabrics and studied its activity against A. niger. Chattopadhyay and Patel (2010) evaluated the antimicrobial activity of CuNPs by soil burial test results clearly showed the CuNPs treatment enhanced the resistance of cotton fabrics to microbial attack. The aim of the present study was to develop a nontoxic and cost-effective biological method for the synthesis of CuNPs. It is a green approach for the synthesis of copper nanoparticles which can be achieved only in 15–20 min.
Materials and methods Plant material Fruits of Citrus medica Linn. were collected from garden of Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India. Preparation of fruit juice of citron and synthesis of CuNPs Fruit juice of C. medica Linn. was extracted at room temperature by squeezing the prewashed mature fruits of citron and filtered through muslin cloth. The known volume of filtered extract of citron was added to CuSO45H2O solution (100 mM) prepared in sterilized distilled water. The solution was mixed thoroughly, poured into an aluminium vessel for the reaction and gradually heated to boiling (60–100°C).
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Detection and characterization of CuNPs Visual observation The primary detection was carried out by visual observation. The change in color of the precursor solution from blue to pale-yellow or colorless with time and deposition of reddish shiny brown colored precipitation on the inner surface of the vessel provides evidence of CuNPs synthesis. Ultraviolet–Visible (UV–Vis) spectroscopy Further, CuNPs were detected by UV–Vis spectrophometer for which the reaction mixture was subjected to optical analysis and the spectra (Shimadzu UV-1700, Japan) were obtained at the resolution of 1 nm from 200 to 800 nm for each sample. Nanoparticle tracking and analysis system (NTA) The CuNPs thus synthesized were later characterized by NanoSight LM-20 to find out the size of the particles. LM 20 is a laser-based light scattering system, in which particles suspended in the liquid medium were injected into the LM viewing unit and viewed in the closed proximity to the optical element. Brownian motion of CuNPs present within the path of the laser beam was observed via a dedicated non-microscope optical instrument (LM-20, NanoSight Pvt. Ltd., UK) having charge-coupled device (CCD) camera. The motions of the particles in the field of view (approximately 100 9 100 lm) were recorded (at 30 fps) and the subsequent sample images and video were captured. X-ray diffraction (XRD) X-ray diffraction (XRD) studies were carried out on a Rigaku Miniflex II dextop X-ray diffractometer instrument using CuKa radiation (0.15418 nm). XRD data analysis was done using ISDD software JCPDS and the average size of CuNPs was calculated by using Debye– Scherrer’s equation (D = 0.9k/b cos h). Assessment of antimicrobial activity of CuNPs Test organisms The pure cultures of bacteria E. coli (ATCC 14948), P. acne (MTCC-1951), K. pneumoniae (MTCC 4030), S. typhi (ATCC 51812) and P. aeruginosa (MTCC 4676) and two plant pathogenic fungi, namely F. oxysporum (MTCC 1755) and F. culmorum (MTCC 349) were procured from culture collection centers, F. graminearum (DBT 4) was isolated from kidney beans. In vitro evaluation of antimicrobial activity The antibacterial and antifungal activity of CuNPs was evaluated against E. coli, P. acne, K. pneumoniae, S. typhi, and P. aeruginosa and plant pathogenic fungi, namely F. oxysporum, F. graminearum and F. culmorum. The overnight grown
cultures of E. coli, P. acne, K. pneumoniae, S. typhi, and P. aeruginosa were used to assess the activity of CuNPs. The activity of CuNPs was evaluated singly by impregnating 20 ll of CuNPs solution in the sterile discs. A disc diffusion assay was performed on Muller–Hinton agar plates for bacterial cultures. A single colony of bacterium was grown overnight in Muller–Hinton broth on a rotary shaker (100 RPM) at 37 °C. The inocula (100 ll) were then spread onto the agar plates and prepared discs containing CuNPs were placed onto agar surface. After incubation at 37 °C for 24 h, the zones of inhibition were measured. Similarly, potato dextrose agar plates were inoculated with the spore suspension (20 ll) of the test fungi (F. oxysporum, F. graminearum and F. culmorum). The discs containing CuNPs were placed on to the surface of plates inoculated with respective test fungi and incubated at 25° ± 2 °C for 52 h followed by measurement of the zones of inhibition. The antimicrobial assays were performed in triplicate.
Results After addition of fruit juice extracted from citron by squeezing the fruit with aqueous copper sulfate solution (100 mM) in an aluminium vessel and heating the mixture to boiling, the blue color of the precursor solution changed to light-yellow or sometimes become colorless with time and the CuNPs were deposited on the inner wall of the vessel (Fig. 1a, b). Deposition of the shiny reddish-brown precipitate on the inner wall of the aluminium vessel, indicates the formation of CuNPs. The synthesized CuNPs were collected, by brushing or scraping the inner walls of the vessel and dissolved in liquid ammonia to make its colloidal solution for further characterization. Surface Plasmon Resonance (SPR) of synthesized CuNPs was detected by using UV–Vis spectrophotometer. The colloidal suspension of CuNPs in liquid ammonia showed the peak at 631 nm (Fig. 1c) which is specific for CuNPs. For the determination of size and concentration of CuNPs, Nanoparticle Tracking and Analysis (NTA) was carried out, which confirmed that CuNPs synthesized were in the range of 10-60 nm with an average size of 33 nm. Further, the total concentration of CuNPs was found to be 2.18 9 108 particles/ml (Fig. 2a, b). XRD patterns obtained for the CuNPs synthesized using citron juice has been shown in Fig. 3. The presence of intense peak corresponds to 111, 111, 200 and 220, indexed a crystalline copper FCC phase (Mott et al. 2007; Sastry et al. 2013; Usman et al. 2012). The average size is calculated by using Debye–Scherrer’s equation (D = 0.9k/b cos h), where, D is the average grain size of crystallite, k is the incident wavelength, h is the Bragg angle, b is the diffracted full
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Fig. 1 (a) Copper Sulfate solution with citron juice in aluminium vessel, (b) Deposited CuNPs on the inner wall of aluminium vessel after synthesis (c) UV–Vis spectrum of the synthesised CuNPs
dissolved in liquid ammonia solution showing absorbance at 631 nm (scanned in 200–800 nm range)
Fig. 2 NTA analysis of synthesized CuNPs (a) histogram showing particle size distribution and the average size of CuNPs (33 nm) (b) 3D view
width at half maximum (in radians), respectively (MontesBurgos et al. 2010), which gives an average size of CuNP 20 nm. The in vitro antimicrobial activity of CuNPs produced in this study was evaluated against bacteria E. coli, P. acne, K. pneumoniae, S. typhi, and P. aeruginosa and three plant pathogenic fungi viz. F. oxysporum, F. graminearum and F. culmorum. The synthesised CuNPs showed significant activity against all the test organisms. It was found that
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E. coli was the most sensitive bacterium followed by K. pneumoniae, P. aeruginosa, P. acne and S. typhi. However, among the plant pathogenic fungi tested, F. culmorum was found to be the most sensitive followed by F. oxysporum and F. graminearum (Figs. 4, 5). Whereas, liquid ammonia and copper sulfate did not show any significant activity against these test organisms (Figs. 4, 5). These results corroborate with the findings obtained in previous studies, which confirmed that metal nanoparticles were effective
World J Microbiol Biotechnol Fig. 3 XRD pattern of CuNPs showing the FCC structure of crystallite and size calculated about 20 nm
Fig. 4 Histogram of antibacterial activity of CuNPs which showed that E. coli was the most sensitive bacterium followed by K. pneumoniae, P. aeruginosa, P. acne and S. typhi
against human as well as plant pathogens (Barik et al. 2008; Gajbhiye et al. 2009; Goswami et al. 2010; Kanhed et al. 2014; Owolade et al. 2008; Rai and Ingle 2012).
Discussion The deposition of copper on aluminum is a well-known cementation process, where a more reactive metal displaces a less reactive metal from its compound in solution
(Sastry et al. 2013). Thus, a single-displacement redox reaction (electroless deposition) can be described as: 2AlðsÞ þ 3Cu2 SO4 ðaqÞ ! 6CuðsÞ þ Al2 ðSO4 Þ3 ðaqÞ As the reaction proceeds, copper gets precipitated out of the solution by forming a thin layer/coating on the wall of the aluminium vessel used. Unlike, noble metals such as Ag and Au, the light transition metal Cu usually cannot be obtained via the reduction of simple copper salts such as copper sulfate in aqueous solution (Hirai et al. 1986). The
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World J Microbiol Biotechnol Fig. 5 Histogram of antifungal activity of CuNPs which showed F. culmorum was found to be the most sensitive followed by F. oxysporum and F. graminearum
reduction usually stops at Cu2O stage due to the presence of a large number of water molecules (Zhu et al. 1994). So it requires reagent carrying functional groups that could form complexes with copper ions. The surfactants used in the solution acted as capping agents to get the CuNPs. Without presence of capping agents, the particle size would be in the micron range (Song et al. 2004) which may be because of the agglomeration or formation of aggregates of CuNPs. Song et al. (2004) suggested that the metallic CuNPs were stable in nonpolar solvents only. In this method, the organic hydrocarbon part of capping agent used facilitate the protection of copper. The antioxidant nature and acidic property of citron also prevents oxidation of copper because all the protons present in the medium interfere with electro-deposition of copper at low pH range. It was reported that citron juice (acts capping agent) had weak acidic constituent as ascorbic acid, saponins and flavonoids (Gattuso et al. 2007). NTA (NanoSight-LM 20) is an analytical instrument, which is preferably used for the determination of nanoparticles size. It determines the size of nanoparticles based on Brownian motion of particles present in the samples (Raheman et al. 2011). Kanhed et al. (2014) reported the in vitro antifungal activity of chemically synthesized CuNPs with a commercially available antifungal agent against four different plant pathogenic fungi, viz., F. oxysporum, C. lunata, A. alternata and P. destructiva. CuNPs showed activity against all the plant pathogenic fungi used in the experiment. The results demonstrated that C. lunata and A. alternata were comparatively resistant to the commercial
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antifungal agent (bavistin) but showed sensitivity towards CuNPs. In this study, CuNPs demonstrated the maximum antifungal activity against C. lunata followed by A. alternata, and the minimum activity was reported against P. destructiva. It was also found that antifungal activity of bavistin increases in combination with CuNPs in case of F. oxysporum. The present study also confirmed the antifungal nature of CuNPs against plant pathogenic fungi tested. Kabra et al. (2012) reported that the ethanolic extract of peels of C. medica L. showed in vitro antimicrobial activity against bacteria such as Staphylococcus aureus, Proteus vulgaris, K. pneumoniae, E. coli, B. subtilis and P. aeruginosa. Sah et al. (2011) reported that the fruit juice of C. medica L. inhibited all tested Gram-positive, Gram-negative bacteria and two fungi A. niger and A. flavus with zone of inhibition compared to standard drugs. In the present study also, the Citron juice showed some efficacy against the test bacteria but it was significantly lower than that of activity of CuNPs (Fig. 4). On the other hand, in case of efficacy of citron juice against fungi only F. graminearum was found to be less susceptible while rest of two fungi (F. oxysporum and F. culmorum) did not show any susceptibility (Fig. 5). In another study, Usman et al. (2013) reported the synthesis of pure CuNPs in the presence of a chitosan stabilizer through chemical means with the evaluation of antimicrobial activity of the nanoparticles by using several test microorganisms like methicillin-resistant S. aureus, B. subtilis, P. aeruginosa, Salmonella choleraesuis and Candida albicans. Sampath et al. (2014) synthesized jasmine bud-shaped CuNPs by a green chemical reduction method using polyvinylpyrrolidone (PVP) as a capping agent,
World J Microbiol Biotechnol Fig. 6 Schematic representation of hypothetical mechanism of action for CuNPs. 1 Accumulation of CuNPs on the cell surface form pits which causes cell leakage; 2 interaction of CuNPs with cell membrane decrease the transmembrane electrochemical potential, which affects membrane integrity; 3 DNA damage due to the interaction with CuNPs; 4 interaction of Cu2? with sulfhydryl group of protein, inactivates the proteins; 5 entry of CuNPs and Cu2? inside the cell develop oxidative stress, which leads to cell death
L-ascorbic acid (AA) as a reducing agent and antioxidant agent, isonicotinic acid hydrazide (INH) as a reducing agent. The antibacterial activity of the Cu nano buds was evaluated by testing against E. coli and S. aureus. Agriculture is the backbone of India and most of the developing countries, with more than 60 % of the population depending on it for their livelihood. The infections caused by various bacterial and fungal species, are increasing with a fast pace in the crop plants and thereby limiting crop productivity by yield loss of around 25 %. So it is necessary to use the modern technology to control the diseases and enhance yields. Among the inorganic antimicrobial agents, copper compounds have been commonly used in agriculture practices as a fungicide (Garcia et al. 2003), pesticides (CDPR 2009), algaecide (De Oliveira-Filho et al. 2004) and herbicide (Mastin and Rodgers 2000) as well as in animal husbandry as a disinfectant (Mortazavi and Ahmad 2009). As the biogenic CuNPs showed potent activity against a wide range of microbes it can be used as alternative antimicrobials specially against crop pathogens (Lee et al. 2011; Majumder 2012). Different formulations of CuNPs as nanofungicides, nanoantimicrobials or nanofertilizers serve as a dual purpose to control the infections as well as a nutrient for plants. It is widely demonstrated and also proved from the present study, that CuNPs have significant antimicrobial activity, but its mechanism of action is yet unknown. There are only few reports available on mechanism of action of CuNPs but still the mechanism is not completely understood. According to Das et al. (2010) CuNPs enter into the cell due to their small size and inactivate their proteins/
enzymes, generating hydrogen peroxide, which causes bacterial cell death. In another study, it was reported that inactivation of proteins occur due to the interaction of CuNPs with its –SH (sulfhydryl) group (Schrand et al. 2010). Similarly, CuNPs also interact with DNA molecules leading to its degradation by disturbing its helical structure (Kim et al. 2000). In fact, the cell membrane integrity depends on its electrochemical potential, but Deryabin et al. (2013) reported that CuNPs decrease the electrochemical potential of the cell membrane, which ultimately affects cell membrane integrity. It was also believed that metal nanoparticles release their respective ions and such heavy metal ions demonstrated adverse effect on bacterial cell (Cioffi et al. 2005). Accumulation of CuNPs and Cu ions on cell surface form pits in the membrane, which mainly leads to the leakage of cellular component from cell and eventually causes cell death. Another important reason proposed for the cell death due to action of CuNPs is development of oxidative stress (Deryabin et al. 2013). Considering all the above possibilities here, we have proposed a schematic representation (Fig. 6) for hypothetical mechanism of action of CuNPs in bacteria.
Conclusions Use of citron juice for the synthesis of CuNPs is a novel step towards the biogenic synthesis of CuNPs. It is an ecofriendly, non-toxic and rapid approach. Further, the use of biogenic CuNPs against different human and plant pathogens confirmed its effectiveness against wide range of microorganisms. Due to the antimicrobial property of
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CuNPs, it can be used in different formulations like nanofungicides, nanoantimicrobials and nanofertilizers, which could serve dual purpose, by protecting the crop plants from its pathogens and also providing nutrients to the plants. Finally, and most important is that it is a costeffective approach as raw materials involved in the synthesis are very cheap. Acknowledgments The authors are thankful to University Grants commission, New Delhi for financial support under UGC-SAP and API is highly thankful to Department of Science and Technology, New Delhi for providing financial assistance under DST Fast Track Scheme for Young Scientists.
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