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Colloids and Surfaces B: Biointerfaces 80 (2010) 4550

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Colloids and Surfaces B: Biointerfaces


journal homepage: www.elsevier.com/locate/colsurfb

Banana peel extract mediated synthesis of gold nanoparticles


Ashok Bankar a , Bhagyashree Joshi a , Ameeta Ravi Kumar a,b , Smita Zinjarde a,b,
a b

Institute of Bioinformatics and Biotechnology, University of Pune, Ganeshkhind Road, Pune 411 007, India DST Unit on Nanoscience, Department of Physics, University of Pune, Pune 411 007, India

a r t i c l e

i n f o

a b s t r a c t
Gold nanoparticles were synthesized by using banana peel extract (BPE) as a simple, non-toxic, ecofriendly green material. The boiled, crushed, acetone precipitated, air-dried peel powder was used to reduce chloroauric acid. A variety of nanoparticles were formed when the reaction conditions were altered with respect to pH, BPE content, chloroauric acid concentration and temperature of incubation. The reaction mixtures displayed vivid colors and UVvis spectra characteristic of gold nanoparticles. Dynamic light scattering (DLS) studies revealed that the average size of the nanoparticles under standard synthetic conditions was around 300 nm. Scanning electron microscopy and energy dispersive spectrometry (EDS) conrmed these results. A coffee ring phenomenon, led to the aggregation of the nanoparticles into microcubes and microwire networks towards the periphery of the air-dried samples. X-ray diffraction studies of the samples revealed spectra that were characteristic for gold. Fourier transform infra red (FTIR) spectroscopy indicated the involvement of carboxyl, amine and hydroxyl groups in the synthetic process. The BPE mediated nanoparticles displayed efcient antimicrobial activity towards most of the tested fungal and bacterial cultures. 2010 Elsevier B.V. All rights reserved.

Article history: Received 24 November 2009 Received in revised form 16 May 2010 Accepted 20 May 2010 Available online 27 May 2010 Keywords: Biosynthesis Banana peel extract Gold nanoparticles SEM (scanning electron microscope) FTIR

1. Introduction Gold nanoparticles are some of the most extensively studied materials. These can be easily synthesized, exhibit intense surface plasmon resonance and display high chemical as well as thermal stability [1]. A variety of gold structures including rods, triangles, hexagons, octagons, cubes and nanowires can be synthesized by using different techniques [25]. Studies on the morphological behavior of gold nanostructures and their evolution into microcrystals or microwires are signicant because of their wide use in catalysis, optics, optical electronics, microelectronics, biodiagnostics, imaging, biological and chemical sensing techniques [1,6,7]. It is thus evident that nanoparticles and microstructures have several applications and their synthesis by using simple techniques is of prime importance. Micro-patterning methods such as photolithography and nano-imprinting are the conventional techniques that are used for the fabrication of microstructures [8,9]. Such methods however, are complex, cost-intensive and often involve multiple steps. The development of robust and costeffective micropatterning methods are thus an important aspect of fundamental research in this area. Self-assembling processes often

Corresponding author at: Institute of Bioinformatics and Biotechnology, University of Pune, Ganeshkhind Road, Pune 411 007, India. Tel.: +91 20 25691333; fax: +91 20 25690087. E-mail address: smita@unipune.ernet.in (S. Zinjarde). 0927-7765/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.05.029

depend on the procedures that are employed or on the nature of the mediating molecules [1012]. For example, a simple physical method using a silicon pore template has been developed for the fabrication of monodisperse gold microwires [13]. In addition, chemicals such as citrate and amines have also been successfully employed for their production [14,15]. In the recent years, the use of natural and modied polysaccharides for the synthesis of gold microcrystals varying in size and shape has become a popular alternative. In particular, chitosan and cellulose have been successfully used for the fabrication of gold nanoparticles and microstructures [16,17]. To the best of our knowledge, the use of naturally available agricultural waste material has not been investigated so far, for such applications. Banana peels are a classical example of such abundantly available natural material. India is the largest producer of bananas and FAO sources estimate that 21.77 million metric tons of bananas are cultivated annually. Such estimates for the worldwide production are several folds higher. The peels of banana are usually discarded. They are mainly composed of natural polysaccharides [18]. Their medicinal value has been explored [19] and they have also been used as substrates for the production of fungal biomass [20]. Another application includes their use in the adsorption of heavy metals from water [21]. In this study, we report a novel green biological route for the synthesis of nanoparticles and also demonstrate their assembly into microcubes and microwires (due to the coffee ring phenomenon) by using the banana peel extract (BPE) powder as a reducing material. The structures have been char-

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acterized by using UVvis spectroscopy, SEM-EDS, XRD and FTIR analysis. In addition, an application (antimicrobial activity) of the synthesized nanoparticles has also been demonstrated. 2. Experimental 2.1. Preparation of banana peel extract (BPE) powder Banana (Musa paradisiaca) peels were collected and washed thoroughly. They were boiled in distilled water at 90 C for 30 min. The boiled banana peels (100 g) were crushed in 100 ml distilled water and the resultant extract was ltered through a clean muslin cloth. The ltrate was precipitated with equal volumes of chilled acetone. The resulting precipitate was collected by centrifugation at 1000 rpm for 5 min, air-dried into a powdered form and used for further experiments. 2.2. Synthesis of gold nanoparticles and their assembly into microcubes and microwires For all the experiments, the source of gold was chloroauric acid (HAuCl4 ) in distilled water. Typical reaction mixtures contained 10 mg of BPE powder in 2 ml of chloroauric acid solution (1 mM) unless otherwise stated. The mixtures were incubated at 80 C in a water bath for 3 min. These were monitored for different time intervals and the nanoparticles and microstructures that were formed were characterized further. The effect of pH on nanoparticle synthesis was carried out by adjusting the pH of the reaction mixtures (10 mg BPE, 1.0 mM chloroauric acid) to 2.0, 3.0, 4.0 or 5.0. The effect of the gold salt was determined by increasing the concentration of HAuCl4 from 0.125, 0.25, 0.5 or 1.0 mM. The BPE powder concentration was varied (0.5, 1.0, 2.0, 4.0 or 10.0 mg) while keeping the chloroauric acid concentration at a level of 1.0 mM. To study the effect of temperature on nanoparticle synthesis, reaction mixtures containing 10 mg BPE, and 1.0 mM HAuCl4 at pH 3.0 were incubated at 40, 60, 80 or 100 C. All experiments were carried out in triplicates and representative data is presented here. 2.3. Characterization of gold nanoparticles, microcubes and microwires The gold nanoparticles were characterized by using a UVvis spectrophotometer (Jasco V-530). The size of particles in the reaction mixtures (containing 10 mg BPE, 1.0 mM chloroauric acid, pH 3.0) was analyzed by using the dynamic light scattering equipment (Beckman Coulter DELSA Nano CNano Particle Size Analyzer). Scanning electron microscopy and elemental analysis were performed on platinum coated samples that were previously air-dried on silicon wafers. An analytical scanning electron microscope (JOEL JSM-6360A) equipped with energy dispersive spectrometer (EDS) was used [22,23]. Control samples lacking the gold salt were also analyzed. All samples were analyzed in triplicates and representative micrographs are included here. X-ray diffraction (XRD) measurements of thoroughly dried thin lms of nanoparticles on Si (1 1 1) wafers were carried out. In addition, samples lacking the gold salt (control samples) were also analyzed. Analysis was carried out in the transmission mode with Cu K radiation using = 1.54 and a D 8 Advanced Brucker instrument. 2.4. FTIR analysis In order to determine the functional groups on the BPE powder surface and their possible involvement in the synthesis of gold microstructures, FTIR analysis was carried out as described earlier by us [24]. Control samples (BPE powder before reaction with HAuCl4 ) and the test samples (BPE powder after reaction with the

Fig. 1. Visual observations and UVvis absorption spectra of reaction mixtures (a) at different pH values [(A) pH 2.0, (B) pH 3.0, (C) pH 4.0, (D) pH 5.0 and (E) HAuCl4 control] and (b) with varying concentrations of gold chloride (mM) [(A) 1.0, (B) 0.5, (C) 0.25 and (D) 0.125].

gold salt) were independently dried and blended with KBr to obtain a pellet. The FTIR spectra were collected at resolution of 4 cm1 in the transmission mode (4000400 cm1 ) using a Shimadzu FTIR spectrophotometer (FTIR 8400). 2.5. Antifungal and antibacterial activity Two pathogenic strains of Candida albicans (BX and BH) were used to determine the antifungal activity of the gold nanoparticles. Stock cultures of fungal strains were maintained on MGYP slants (malt extract, 3.0; peptone, 10.0; dextrose, 10.0 g l1 of distilled water). Different bacterial cultures including Citrobacter kosari (MTCC 1657), Escherichia coli (MTCC 728), Proteus valgaris (MTCC 426), Pseudomonas aeruginosa (MTCC 728), Enterobacter aerogenes (MTCC 111) and Klebsiella sp. were used to determine the antibacterial activity of gold nanoparticles. The bacterial cultures were maintained on Nutrient Agar (NA) slants (peptone, 5.0; meat extract, 1.0; yeast extract 2.0; sodium chloride, 5.0; agar 15.0 g l1 of distilled water). The antimicrobial activity of the nanoparticle was determined by spreading 100 l of fungal or bacterial cultures (containing 104 cells ml1 ) on MGYP or NA plates, respectively. Freshly prepared nanoparticle samples derived from the reaction mixtures containing 10 mg BPE, 1.0 mM chloroauric acid, pH 5.0 incubated at 80 C (50 l) were added into the wells in the seeded agar plates. Control samples lacking the chloroauric acid were used to assess the antimicrobial activity of the BPE. The test and control samples

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Fig. 2. Scanning electron micrographs of gold nanoparticles (a) and (b); microwire networks at the periphery due to coffee ring effect (c) and (d) with 0.25 mM chloroauric acid (e) and (f) with 1.0 mM chloroauric acid (a) is magnied 10,000, inset bar represents 1 m (b) is magnied 30,000, inset bar represents 0.5 m (c) and (e) are magnied 100, inset bar represents 100 m (d) and (f) are magnied 2000, inset bar represents 10 m.

were allowed to diffuse for 15 min at 4 C and the plates were further incubated at 37 C for 2448 h. The test was scored positive when a zone of inhibition was observed around the well after the incubation period. All experiments on antimicrobial activity were carried out in triplicates and representative gures are presented here. 3. Results and discussion 3.1. Visual observations and UVvis spectroscopy The reaction mixtures developed an array of colors after 3 min of incubation under different conditions indicating the synthesis of a variety of gold nanoparticles. The effect of pH on nanoparticle synthesis is shown in Fig. 1a. The yellow color of gold chloride solution turned to brown when the reaction was carried out at pH 2.0 [Fig. 1a (A)]. A purplish-pink color was observed at pH 3.0 [Fig. 1a (B)]. A ruby red color was obtained at pH 4.0 [Fig. 1a (C)] and a dark reddish color developed at pH 5.0 [Fig. 1a (D)]. The UVvis spectra of the reaction mixtures are also shown in Fig. 1a. In each case, a peak was observed in the range of 500600 nm suggesting the synthesis of gold nanoparticles as also reported earlier for other biological systems [25]. The control chloroauric acid solution (without BPE) did not display the charac-

teristic peak [Fig. 1a (E)] indicating that abiotic reduction did not occur. Fig. 1b shows the effect of varying chloroauric acid concentrations on nanoparticle synthesis. A purplish-pink color was observed with 1.0 mM concentration of the gold salt [Fig. 1b (A)]. This is consistent with the visual observations made in Fig. 1a (B) wherein, the reaction mixture composition was similar (10 mg BPE, 1.0 mM chloroauric acid, pH 3.0). The UVvis spectra of the tubes also displayed a peak in range of 510600 nm. With 0.5, 0.25 and 0.125 mM of gold chloride, varying shades of purple were observed [Fig. 1b (BD)]. There was a variation in nanoparticle synthesis with increasing contents of the BPE. As stated earlier, with 10 mg ml1 of BPE, a purplish-pink color was observed [as also seen in Fig. 1a (B) and b (A)]. Dark and light ruby red colors were observed at a concentration of 1.0 or 2.0 mg ml1 . The reaction mixture turned dark reddish brown with 4 mg ml1 and a pink color was developed with 5 mg ml1 of the BPE powder. Signature peaks indicative of nanoparticles were also observed. The incubation temperature affected the process of gold reduction. For example, an orangish-brown color was observed at 40 C. A yellowish-brown color was developed at 60 C. Purplish-pink and pinkish-brown colors were observed at 80 C and 100 C, respec-

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tively. The peak characteristics for gold nanoparticles were also observed. The visual observations and UVvis spectra were thus indicative of nanoparticle synthesis. Gold nanoparticles are known to display vivid colors due to the phenomenon of surface plasmon resonance (SPR) as also reported earlier [16,22,23]. 3.2. Scanning electron microscope-energy dispersive spectrum (SEM-EDS) analysis The SEM analysis was used to determine the structure of the reaction products that were formed. Representative SEM micrographs of air-dried reaction mixtures containing 10 mg of BPE powder and 0.25, 0.5 or 1.0 mM of chloroauric acid incubated at pH 3.0 are shown in Fig. 3af. Fig. 3a shows the presence of nanoscale gold particles (white arrows) and nanoplates (black arrows) at a magnication of 10,000. At a magnication of 30,000, their structures were distinct (Fig. 3b). This is in agreement with earlier reports on biological material such as the lemon grass extract mediating the reduction of chloroaurate ions into triangular structures [26]. In the present study, the particle size distribution was also conrmed by DLS studies. The results of these experiments revealed that the average particle size was around 300 nm. This is consistent with the SEM results obtained in Fig. 2a and b. The reaction mixtures were air-dried on silicon wafers when the sample was prepared for the SEM studies. This resulted in a coffee ring phenomenon. When liquids containing ne particles are evaporated on a at surface, the particles tend to accumulate along the outer edge and form typical structures [27]. Gold nanoparticles are also known to display this phenomenon. For example, there is a report on the formation of colloidal gold lms that exhibit the characteristic coffee ring feature [28]. In the present study, the gold nanoparticles accumulated towards the periphery of the dried drop. They formed micronetworks as well as dendrite-like structures (white arrows) that could be observed even at low magnications (Fig. 3c and e). High magnications images (1000 or 2000) revealed that the networks were composed of gold microcubes arranged in a specic array (Fig. 3d and f). Fig. 3f also shows the presence of triangles and hexagons in the patterned microwires (black arrows). BPE thus consistently mediated the synthesis of gold nanoparticles which due to the coffee ring phenomenon aggregated into microcubes and networks. There are a few reports on the biological synthesis of nanocubes. For example, there is a report on the production of such structures (sized 10100 nm) by the bacterium, Bacillus licheniformis [29]. In addition, cysteine grafted chitosan has also been used to synthesize gold cubes. These cubes had an edge length in the range of 12 m [16]. In comparison to these two reports, the size of the microcubes due to the coffee ring effect with BPE were much larger and in the range of 1020 m (Fig. 2b, d and f). Moreover, unlike the earlier two reports [16,29], the cubes formed in this study (due to the coffee ring effect) aggregated into networks that were very long (Fig. 2a, c and e). There is a report on the formation of gold nanowire networks by the cell-free supernatant of the bacterium Rhodopseudomoas capsulata [30]. Such structures were particularly observed with higher concentrations of the gold salt and the diameters of these polycrystalline gold nanowires were in the nanometer range (5060 nm) unlike the results obtained in the current study. There is a recent report on the use of peach fruit extract for the synthesis of hexagonal or triangular gold nanoplates [31]. An advantage of the present study is the use of waste banana peels rather than the edible fruit for the synthetic process. There has been an increasing interest in the use of soluble polymers including biopolymers as soft templates for directing crystal growth and controlling self-assembly of inorganic nanoparticles [32]. Banana peels are largely composed of hot water soluble pectin, cellulose

Fig. 3. (a) Representative spot EDS prole conrming the presence of gold in microcubes and microwire networks. (b) Representative XRD prole of thin lm gold microcubes and microwire networks.

and hemicelluloses that constitute nearly 80% of the mass [33]. Cellulose is known to mediate nanoparticle synthesis [17]. Pectin, on the other hand, has been used to stabilize chemically synthesized bimetallic nanoparticles of gold and platinum [34]. These natural polymers present in BPE may be providing a template for the assembly of nanoparticles into micronetworks. Literature survey has shown that biosynthesis of gold nanoplates and nanocubes are a common feature. However, the formation of large microcubes (1020 m) and their assembly into long networks is an unusual phenomenon that has not been hitherto reported. The micronetworks so assembled in turn, may have applications in a variety of elds. The EDS attachment on the SEM provided chemical analysis of the eld of view as well as spot analyses of minute particles and conrmed the presence of specic elements. Fig. 3a is a representative plot of the spot EDS analysis. The SEM-EDS analysis displayed signature spectra for gold and thus convincingly evidenced the presence of this noble metal in the microcubes and microwires. These results are consistent with other reports on the EDS analysis of gold structures synthesized by using extracts derived from the fruits of pear or the leaves of Magnolia kobus or Diopyros kaki [31,35]. 3.3. X-ray diffraction (XRD) analysis A representative XRD prole of the gold microcubes displaying the structural information and crystallinity are shown in Fig. 3b. A control thin lm of gold chloride before reaction did not show the characteristic peaks. After reaction, the diffraction peaks at 2 = 38.1 , 44.5 , 64.21 and 77.78 assigned to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of a faced centre cubic (fcc) lattice of gold were obtained. The XRD analysis showed predominant peaks at (1 1 1) and (2 0 0) indicative of the presence of microcubes

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Fig. 4. FTIR spectra of BPE powder () before and after (. . . ) reaction.

and microwires displaying fcc lattice structure. The XRD patterns displayed are consistent with earlier reports on microstructures [17,31]. 3.4. FTIR analysis FTIR has emerged as a valuable tool for understanding the involvement of surface functional biological groups in metal interactions. This technique was applied to determine the groups that were present on the BPE powder and to possibly predict their role in the synthesis of gold microstructures. Control spectra of BPE powder (not treated with chloroauric acid) displayed a number of peaks reecting the complex nature of the powder. There was a variation in the intensity of bands in different regions when test samples (after reaction with chloroauric acid) were analyzed (Fig. 4). A major peak was observed at 2930 cm1 that could be assigned to the CH stretching vibrations of methyl, methylene and methoxy groups [36]. This peak shifted from 2930 to 2888 cm1 suggesting the possible involvement of the aforementioned groups in nanoparticle synthesis. The peak located at around 2353 cm1 was attributed to the NH stretching vibrations or the C=O stretching vibrations [37]. The peak shift from 2353 to 2357 cm1 revealed that these groups could also be involved in the nanoparticle synthetic processes. The peak located at 1640 cm1 was assigned to the C=O stretching in carboxyl or C=N bending in the amide group [38,39]. A shift in this peak (from 1640 to 1670 cm1 ) indicated the possible involvement of carboxyl or amino groups of the BPE powder in nanoparticle synthesis. The vibration shift around 14451443 cm1 was suggestive of the involvement of aliphatic and aromatic (CH) plane deformation vibrations of methyl, methylene and methoxy groups in the process [36]. The band observed at 1375 cm1 was assigned to CN stretching or the OH bending [38] and its shift to 1333 cm1 implicated the role of these groups in the interaction with chloroauric acid. These results also indicate that the proteinaceous matter in the peels may also be participating in the process of nanoparticle synthesis. As stated earlier, banana peels are mainly composed of pectin, cellulose and hemicellulose [18,33]. Functional groups associated with these polymers and the proteinaceous matter may thus be involved in reducing the gold salt and stabilizing microstructures. 3.5. Antifungal and antibacterial activity of gold nanoparticles In addition to the several possible applications that the patterned microcubes may display, we have investigated the use of these BPE mediated gold nanoparticles as possible antimicrobial agents. Such BPE mediated gold nanoparticles were immediately

Fig. 5. Representative results of antimicrobial activity of nanoparticles against (a) Candida albicans BX, (b) C. albicans BH, (c) Shigella sp., (d) Enterobacter aerogenes, (e) Klebsiella sp. and (f) Pseudomonas aeruginosa.

tested for antimicrobial activity towards test fungal and bacterial strains. BPE on its own did not display antimicrobial activity. Fig. 5a and b shows the zones of inhibition that were observed with the two strains of C. albicans. In all these gures, the white arrows indicate the wells and the black arrows represent the inhibition zones. Antibacterial activity was observed against Shigella sp., C. kosari, E. coli, P. valgaris and E. aerogenes. Fig. 5c and d is representative inhibition zones observed with Shigella sp. and E. aerogenes, respectively. This is consistent with an earlier report on the antimicrobial activity of gold nanoparticles biosynthesized by the fungus Rhizopus oryzae [40] as well as those synthesized chemically [41]. In the present study, however, antibacterial activity was not observed with all the test cultures. For example, Klebsiella sp. and Ps. aeruginosa were not inhibited by the gold nanoparticles (Fig. 5e and f). There are a few reports in the literature on the inability of gold nanoparticles being effective against test bacteria [42]. The nanoparticles thus synthesized could also be applied as antifungal agents and as selective antibacterial agents. 4. Conclusions In conclusion, BPE could be used as an efcient green material for the rapid and consistent synthesis of gold nanoparticles. A variation in reaction conditions brought about the synthesis of a variety of nanoparticles displaying vivid colors and typical UVvis spectra. BPE mediated structured patterning of the nanoparticles into microcubes and microwire networks. The BPE derived gold nanoparticles displayed antifungal and antibacterial activity towards the test pathogenic fungi and most of the bacterial cul-

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tures. This simple, low cost, non-toxic, eco-friendly, abundantly available green agricultural waste material could thus be used as an efcient alternative to the cost intensive conventional micropatterning methods. The microcrystals and networks generated by this non-conventional method could have a variety of applications in the future. Moreover, this system could also be used as a model for understanding the mechanism of microstructure evolution mediated by biological systems. Acknowledgements AB wishes to thank Council of Scientic and Industrial Research, India for Senior Research Fellowship (CSIRSRF). The authors thank Department of Science and Technology, India for funding the Nanoscience Unit at University of Pune and University Grants Commission, India, for nancial support to the Institute of Bioinformatics and Biotechnology. References
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