Immobilization of silver nanoparticles synthesized using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity

Bioresource Technology 101 (2010) 7958–7965

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Immobilization of silver nanoparticles synthesized using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity Muthuswamy Sathishkumar, Krishnamurthy Sneha, Yeoung-Sang Yun * Environmental Biotechnology National Research Laboratory, School of Chemical Engineering, Research Institute of Industrial Technology, Chonbuk National University, Jeonju 561-756, Republic of Korea

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Article history: Received 27 August 2009 Received in revised form 7 May 2010 Accepted 13 May 2010 Available online 11 June 2010 Keywords: Bioreduction Silver Nanoparticles Curcuma longa Antimicrobial activity

a b s t r a c t The present study reports the synthesis of silver (Ag) nanoparticles from silver precursor using plant biomaterials, Curcuma longa tuber powder and extract. Water-soluble organics present in the plant materials were mainly responsible for the reduction of silver ions to nano-sized silver particles. pH played a major role in size control of the particles. Silver nanoparticle synthesis was higher in tuber extract compared to powder, which was attributed to the large and easy availability of the reducing agents in the extract. Zeta potential studies showed that the surface charge of the formed nanoparticles was highly negative. The minimum bactericidal concentration (MBC) for Escherichia coli BL-21 strain was found to be 50 mg/L. Immobilization of silver nanoparticles on cotton cloth using sterile water showed better bactericidal activity when compared to polyvinylidene fluoride (PVDF) immobilized cloth, but on consecutive washing the activity reduced drastically in sterile water immobilized cloth. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Nanoparticles have a wide range of applications in the field of environmental pollution control, drug delivery system, material chemistry and so on (Gittins et al., 2000). There has been extensive research in chemical synthesis of nanoparticles. The chemical synthesis of nanoparticles has several occupational exposure hazards like carcinogenicity, genotoxicity, cytotoxicity and general toxicity (Mukherjee et al., 2008). So there is a pressing need to develop clean non-toxic and eco friendly procedures for synthesis and assembly of nanoparticles (Mukherjee et al., 2008). The study of microorganisms and its behavior plays a crucial role for synthesis of metal nanoparticles. Since the beginning of this century, researchers have been capaciously working on extracellular and intracellular synthesis of metal nanoparticles from bacteria, fungi, yeast and many other biological sources (Sastry et al., 2003; Shankar et al., 2004a; Binupriya et al., 2010). But the major draw back in using microbes for bioreduction is to maintain the aseptic condition, which not only requires technical labor but also shoots up the production cost to colossal size at industrial level. Nanoparticles have also been synthesized using plants like lemon grass and neem (Shankar et al., 2004a,b). Leaf extracts have been applied for synthesis of silver nanoparticles which has highlighted the possibility of faster rate of synthesis and may also reduce the steps in downstream process* Corresponding author. Tel.: +82 63 270 2308; fax: +82 63 270 2306. E-mail address: [email protected] (Y.-S. Yun). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.05.051

ing, there by making the process cost efficient (Shankar et al., 2003; Huang et al., 2007). Sastry and co-workers (Sastry et al., 2003; Shankar et al., 2003, 2004a,b; Ankamwar et al., 2005) have squeezed out the possibilities of biosynthesizing metal nanoparticles from various plant sources. Huang et al. (2007) have harvested the ability of sun dried Cinnamomum camphora leaf to synthesize quasi-spherical silver nanoparticles and triangular or spherical gold nanoparticles without addition of any protectors or accelerators. They demonstrated that the polyol components and water soluble hydrocyclic compounds present in leaf were mainly responsible for reduction of silver ions or chloroaurate ions. We already have demonstrated the feasibility of using not only the plant material extract for nanoparticle synthesis, but also the material as such in powdered form (Sathishkumar et al., 2009). For thousands of years silver has been used as a healing and antibacterial agent throughout the world (Panacek et al., 2007). But with the discovery of modern medicine, the interest in use of silver had almost become extinct till it was found that silver nitrate kills burn bacteria, thereby enhancing wound healing. Some research has shown silver’s magisterial performance in fighting microbes (Jung et al., 2008). Ionic silver which is a present in industrial waste is highly toxic to most bacterial cells. The accumulation of silver to high concentrations in tissues gives rise to concern about possible sub lethal effects and has toxic effects on terrestrial organisms, freshwater organisms, marine organisms, plants, fungi and animals. It is evident from reports that silver is capable of making a cloth self-sterilized which thereby can be used to make magical textiles with a wide rage of

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applications from bactericidal material to electronic sensors (Lee et al., 2007). Focusing on use of biomaterial for synthesis of nanoparticles, an effort has been made in this experiment, to synthesize silver nanoparticles using Curcuma longa tuber powder and extract as biomaterial. In this study, our objective is to reduce the ionic silver to nanoparticles using biomaterials and this addresses the two major factors which is, being environmental benign since it removes the toxicity from the industrial waste and also the cost effectiveness and easy availability of biomass. This was followed by immobilization of silver nanoparticles on cotton cloth in presence and absence of a binder and testing its bactericidal activity for several cycles with consecutive washing. 2. Methods 2.1. Preparation of C. longa tuber powder (CLP) and extract (CLPE) Tubers of C. longa were purchased from a local market, washed to remove the adhering mud particles and possible impurities. Later it was dried under sunlight for a week to completely remove the moisture. The tuber was cut into small pieces, powdered in a mixer and then sieved using a 20-mesh sieve to get uniform size range. The final sieved powder was used for all further studies. For the production of extract, 2.5 g of CLP was added to a 500 mL Erlenmeyer flask with 100 mL sterile distilled water and then boiled for 5 min. 2.2. Biosynthesis of nano-scale silver particles Silver nitrate was purchased from Sigma–Aldrich, USA. For the silver nanoparticle synthesis from CLP, 100, 500 and 1000 mg of CLP was carefully weighed and added to 50 mL of 1 mM aqueous AgNO3 solution in a 250 mL Erlenmeyer flask. The flasks were then incubated in a rotary shaker at 160 rpm in the dark at 25 °C. For CLPE mediated synthesis, 1, 2.5 and 5 mL extract was added to 50 mL of 1 mM aqueous AgNO3 solution. To study the effect of pH on biosynthesis, the experiments were conducted at various initial pH ranging from 1 to 11 both for CLP and CLPE. Similarly the experiments were also conducted at various temperatures ranging from 20 to 60 °C. 2.3. UV–visible spectra analysis The biosynthesis of silver nanoparticles was monitored periodically in a UV–visible spectrophotometer (Shimadzu, UV-2550, Japan). For the analysis, 0.1 mL of the sample in a cuvette and was diluted to 2 mL with deionized water. The UV–visible spectra of the resulting diluents were monitored as a function of reaction time and biomaterial dosage at a resolution of 1 nm. 2.4. Characterization of nano-scale silver nanoparticles After the reaction, in case of CLP the biomass had settled at the bottom of the conical flasks and the suspension above the precipitate was sampled for transmission electron microscopy (TEM) observation, whereas in case of CLPE, the solution was centrifuged at 10,000 rpm for 15 min and the settled nanoparticles was separated with some solution, which was used for TEM observation. TEM samples of the aqueous suspension of silver nanoparticles were prepared by placing a drop of the suspension on carboncoated copper grids and the films on the TEM grids were allowed to stand for 2 min, after which the extra solution was removed using a blotting paper and the grid was allowed to dry prior to measurement. TEM observations were performed on a HITACHI-JP/ H7600 instrument (Japan) operated at an accelerating voltage of

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100 kV. The size distribution of the resulting nanoparticles was estimated on the basis of TEM micrographs with the assistance of SigmaScan Pro software (SPSS Inc., Version 4.01.003). Energy dispersive X-ray (EDX) analyses were performed on a JEOL JSM6400 microscope (Japan) fitted with Oxford-6506 (England) EDX analyser. For complete recovery of nanoparticles from CLP, the mixture was sonicated for 5 min and centrifuged as 1000 rpm for 5 min and the tuber particles were removed. The procedure was repeated thrice to ensure maximum recovery of the nanoparticles formed. 2.5. Bactericidal studies in broth To test the bactericidal activity of the silver nanoparticles produced, growth inhibition studies against gram-negative bacteria Escherichia coli strain BL-21 were conducted in Luria–Bertani (LB) broth media (Becton–Dickinson and Co., USA). For growth inhibition studies, sterile 250 mL Erlenmeyer flasks, each containing 100 mL LB broth media and the desired amount of silver nanoparticles, were inoculated with 1 mL of the freshly prepared bacterial suspension in order to maintain initial bacterial concentration in the same range in all the flasks. The flasks were then incubated in a rotary shaker at 160 rpm at 37 °C. Bacterial growth was then monitored every hour for 24 h by measuring the increase in absorbance at 600 nm in a spectrophotometer (Shimadzu, UV-2550, Japan). The experiments also included a control flask containing only media and bacteria devoid of silver nanoparticles. Percent inhibit of bacterial growth was calculated based on the difference in growth between the control and media containing silver nanoparticles after 24 h. 2.6. Immobilization of silver nanoparticles on cloth and disk diffusion studies Bacterial sensitivity to antibiotics is commonly tested using a disk diffusion test, employing antibiotic impregnated disks (Case and Jhonson, 1984). A similar test with nanoparticle laden disks was used in this study. Desired amount of nanoparticles were suspended in 10 mL of sterile distilled water and was sprayed over the pre-sterilized white cotton cloth (10 cm2) in aseptic condition. The cloth was then placed in a sterile box and was kept in an oven at 60 °C overnight to evaporate the water content. The nanoparticle laden filter paper was dried in an oven for 1 h and small disks of uniform size (6 mm diameter) were punched out and stored in a desiccator at room temperature. Similar method was followed for another set of disks where the polymer polyvinylidene fluoride (PVDF) in acetone (10% wt/vol) was used as medium instead of sterile water. In this case PVDF was used as binder to avoid the loss of silver nanoparticles at each consecutive cycle and to compare the results with nanoparticles suspended in sterile distilled water. The E. coli strain BL-21 suspension (100 ll of 104–105 CFU/mL) was applied uniformly on the surface of a LB agar plate before placing the disks on the plate (four per plate; one control and three with varied silver nanoparticle concentration). The plates were incubated at 37 °C for 24 h, after which the average diameter of the inhibition zone surrounding the disk was measured with a ruler with up to 1 mm resolution. The mean and standard deviation (SD) reported for each type of nanoparticle and with each microbial strain were based on three replicates. 3. Results and discussion 3.1. Biosynthesis of nano-scale silver particles Bioreduction of silver nanoparticles from aqueous AgNO3 solution can be examined using UV–vis spectroscopy indirectly. Fifty

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milliliters of 1 mM AgNO3 solution was taken as the initial solution for the bioreduction of nano-scale silver particles by CLP and CLPE at different dosages. A visible color change was noticed from colorless to pale yellow after 2 h of the addition of CLP to the AgNO3 solution. The intensity of the color increased with increasing CLP dosage due to excitation of surface plasmon vibrations in the metal nanoparticles (Mulvaney, 1996). In the case of CLPE, it was already yellow in color and the color of the solution was intensified as the reaction progressed after its addition to aqueous AgNO3 solution. The color intensity increased with increasing CLPE dosage. After 2 h, the color intensity was even higher, giving it a darker look. Increase in absorbance with respect to time incase of CLP and CLPE, indicated the increase in productivity of silver nanoparticles (data not shown). The sharp peak at around 435 nm is the absorbance spectra emanating from the silver nanoparticles (Huang et al., 2007). The peak area at 435 nm increased with increase in reaction time for all the concentrations of CLP and CLPE studied. The increase in the absorbance values with increasing CLP and CLPE dosage demonstrates the higher production of silver nanoparticles, which is due to the availability of more reducing biomolecules for the reduction of silver ions at higher dosage, which is on par with the results reported by Huang et al. (2007). Absorbance values were higher for CLPE compared to CLP, which represent the high nanoparticle productivity in CLPE. This phenomenon could be due to easy availability of reductants from CLPE which is in liquid form when compared to CLP in powder form. Moreover, boiling procedure in CLPE preparation would have facilitated the release of some reductants which would have played major role in nanoparticle formation in later stages. Even after 5 months of the synthesis, the silver nanoparticles produced from both CLP and CLPE were observed to be very stable in the solution, which validates the application of CLP and CLPE as biomaterials for the synthesis of nano-sized silver particles. C. longa tuber is rich in terpenoids, including cineol, borneol, etc. and in chemicals, including zingiberene, sabinene, a-phellandrene and sesquiterpines. Most of all it

contains a bio-active poly phenol material called curcumin which contribute to its distinct color and aroma. In addition, some protein is also present in the tuber. Terpenoids are believed to play an important role in silver nanoparticle biosynthesis through the reduction of silver ions. Shankar et al. (2003) reported the possibility of terpenoids from geranium leaf in the synthesis of nano-sized silver particles. Polyols such as terpenoids, flavones and polysaccharides in the Cinnamomum camphora leaf were reported to be the main cause of bioreduction of the silver and chloroaurate ions (Huang et al., 2007). Proteins are reported to bind to the nanoparticles either through free amine groups or cysteine residues in the proteins (Shankar et al., 2003; Gole et al., 2001). A similar mechanism may have operated in the present case where the proteins extracted from the C. longa tuber capped the silver nanoparticles, thereby stabilizing them. Thus the water-soluble fractions comprised of complex polyols in the biomass were believed to have played a major role in the bioreduction of silver ions. TEM images of the silver nanoparticles produced by CLP and CLPE are shown in Fig. 1. Size of the silver nanoparticles formed in CLP and CLPE showed no remarkable differences. Similarly, there was no marked difference in the shape at various initial biomaterial dosages, which was contradictory to the result reported by Huang et al. (2007). But in both cases at initial periods (24 h) of the bioreduction experiment, the size of the nanoparticles formed was smaller when compared to the later stages (168 h) (Fig. 2). This proves the importance of reaction time in controlling the size of the nanoparticles. Terminating the reaction at desired time to get desired size of the nanoparticle is a possibility. The nanoparticles which were stored for 5 months in the CLP and CLPE medium did not show any further increase in size beyond a stage. This shows that as the time progresses, the size of the nanoparticles get larger and after a certain point it remains the same. Nanoparticles formed at lower biomaterial dosages were quantitatively sparse compared to those at higher dosages. Most of the nanoparticles were roughly circular in shape with smooth edges. Some

Fig. 1. TEM images of silver nanoparticles synthesized using (A) 500 mg of CLP and (B) 2.5 mL of CLPE at 30 °C with reaction time of 168 h (bar scale: 200 nm for the left figure of (A); 100 nm for the remaining).

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Fig. 2. TEM images of silver nanoparticles synthesized using (A) 500 mg of CLP and (B) 2.5 mL of CLPE at 30 °C with reaction time of 24 h (bar scale 100 nm).

nanoparticles had anisotropic nanostructures with irregular contours. Few ellipsoidal nanoparticles were observed in any of the CLP and CLPE dosages. These particles were credited with producing the weak absorbance at around 360 nm, in addition to that at about 435 nm. Shankar et al. (2003) reported that the peak at 370 nm corresponds to the transverse plasmon vibration in the silver nanoparticles, whereas the peak at 440 nm was due to the excitation of longitudinal plasmon vibrations. Shankar et al. (2003) also reported quasi-linear superstructures of nanoparticles when geranium leaf extract was used as the biomaterial. But in the present study, most of the nanoparticles formed tended to congregate similar to the results reported by Huang et al. (2007). Different shapes of Ag nanoparticles including, decahedral, triangular, spherical and ellipsoidal were formed by both CLP and CLPE. The organics present in the tuber were believed to be the agents responsible for reducing the Ag+ to Ag0 but the shape is believed to control by some chemical factors. Many triangular structured nanoparticles were observed in all dosages of CLP and CLPE. Huang et al. (2007) reported that nascent silver crystals formed using Cinnamomum camphora leaf powder were gradually enclosed by the protective molecules, which as a result eliminated rapid sintering of smaller nanoparticles, which was responsible for the formation of the nanotriangles. Rai et al. (2006) reported the influence of Cl ions in lemon grass broth assisted formation of gold nanotriangles. In the present study too it is believed that presence of Cl would have influenced the nanotriangle formation, whereas the factors responsible for other shapes are not known. EDX spectroscopy results confirmed the significant presence of 77% silver and 23% Cl with no other contaminants (Fig. 3A). The optical absorption peak is observed approximately at 3 keV, which is typical for the absorption of metallic silver nanocrystallites due to surface plasmon resonance (Kalimuthu et al., 2008).

Phase of the prepared nanoparticles was investigated by X-ray diffraction technique and corresponding XRD-patterns are shown in Fig. 3B. However, silver nanoparticles have shown clear peaks of cubic phases (JCPDS No. 04–0783) at 38.15 (1 1 1), 44.39 (2 0 0), 64.54 (2 2 0) and 77.2 (3 1 1). In addition, some minor peaks representing the hexagonal phases (JCPD No. 41–1402) at 45.9 (1 0 0), 57.9 (1 0 3), 71.6 (0 0 6) and 76.1 (1 0 5) were also noticed. It suggests that the prepared silver nanoparticles are biphasic in nature. The slight shift in the peak positions indicated the presence of strain in the crystal structure which is a characteristic of nanocrystallites. Thus the XRD pattern proves to be a strong evidence in favor of the UV–vis spectra and TEM images for the presence of silver nanocrystals. 3.2. Effect of pH Effect of pH on synthesis of nano-crystalline silver by CLP and CLPE was tested over a wider pH range (1–11). It was observed that after the synthesis of silver nanoparticles, the solution pH lowered in most cases (data not shown). At lower/acidic pH large-sized nanoparticles were formed, whereas, at higher/alkaline pH smallsized nanoparticles were formed and where highly dispersed, comparatively. In acidic pH, the aggregation of silver nanoparticles to form larger nanoparticles was believed to be favored over the nucleation to form new nanoparticles. At alkaline pH, however, the large number of functional groups available for silver binding facilitated a higher number of Ag(I) to bind and subsequently form a large number of nanoparticles with smaller diameters. In addition at higher pH the shape of the nanoparticles formed were more of spherical and decahedral in nature rather than ellipsoidal. This result confirmed the very important role played by pH in controlling the shape and size of the silver nanoparticle synthesis. Andree-

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as biomaterial and described this phenomenon may occur due to the efficient crystal growth of (1 1 1) faces by the deposition of silver atoms on cubic (1 0 0) faces than the nucleation of new silver crystals at elevated temperatures. Similarly, Gericke and Pinches (2006), reported faster rate of gold nanoparticle synthesis at elevated temperatures and also observed nanorod and platelet-like morphologies at higher temperatures and small spherical particles at lower temperatures, thus confirming the importance of temperature on size and shape control. Andreescu et al. (2007) reported a rapid synthesis of silver nanoparticles at higher temperatures.

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The particle size distribution did not show any marginal variation with variation in the dosages of CLP and CLPE. Particle density was higher in case of CLPE compared to CLP, which could be attributed to the possibility of more reductants in CLPE, comparatively as discussed earlier. The number of particles increased with increasing dosage due to the variation in the amount of reductive biomolecules. The average size of the nanoparticles after 168 h of reaction period ranged between 71 and 80 nm for both CLP and CLPE, with a few larger particles exceeding 100 nm both in CLP and CLPE. In lower reaction time, the size of the nanoparticles was much smaller, comparatively. Fig. 2 shows the TEM image and histogram of particle size distribution of the nanoparticles formed after 24 h of reaction time in both CLP and CLPE, which shows the average size ranging between 21 and 30 nm. Nanoparticles in smaller and larger size in early stages and later stages of the reaction show that both nucleation to form new nanoparticles and aggregation to form larger particles happens consecutively. These results were in agreement with those reported by Basavaraja et al. (2008), Huang et al. (2007) and Mukherjee et al. (2008). 3.5. Bactericidal activity

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Fig. 3. Energy dispersive X-ray spectrum (A) and XRD spectrum (B) of silver nanoparticles resulting from experiment using 2.5 mL CLPE.

scu et al. (2007) reported a similar pH effect, in addition to the rapid and complete reduction of the silver species at elevated pH. The synthesized silver nanoparticles exhibited a negative zeta potential at all the pH studied and the isoelectric point was below pH 1 (data not shown). Increase in pH increased the absolute value of the negative zeta potential. As mentioned earlier, the particles were highly dispersed at pH >9. Sadowski et al. (2008) explained that this phenomenon could be due to the electrostatic repulsion at higher pH due to high absolute value of the negative zeta potential and they also reported the instability of the nanoparticles at acidic pH. The absolute value of the zeta potential also increased with increasing dosage due to higher productivity of numerous nanoparticles at higher dosages.

3.3. Effect of temperature Increase in temperature increased the productivity of silver nanoparticles. Increase in absorbance with respect to increase in temperature evidently depicts the higher productivity of silver nanoparticles at elevated temperatures. The absolute value of the negative zeta potential increased with increase in temperature further confirming the higher synthesis of silver nanoparticles at higher temperature. Particle size tends to increase with increasing the temperature from 20 to 60 °C. Lengke et al. (2007) observed the same silver nanoparticle synthesis pattern using a cyanobacterium

3.5.1. In broth cultures E. coli strain (BL-21) was used as model bacterial species to test the bactericidal effect of the nano-crystalline silver particles produced. The various tested concentrations of 2, 5, 10, 25 and 50 mg/L produced inhibition of 11.5%, 31.5%, 50.1%, 79.6% and 99.1%, respectively (Fig. 4). Ruparelia et al. (2008) reported different inhibitory effect towards silver nanoparticles for various strains of the same species. They reported a minimum bactericidal concentration (MBC) ranging from 60 to 220 mg/L for various strains of E. coli. In the present study, the MBC for E. coli BL-21 strain was found to be 50 mg/L. In addition Ruparelia et al. (2008) reported a growth inhibition of around 50% in 18 h for the E. coli MTCC 1302 strain when grown with 100 mg/L of silver nanoparticles. In our case, 50.1% inhibition at 10 mg/L concentration of silver nanoparticles in 18 h was recorded. This vast difference may be due to the susceptibility of the organism used in the present study. Although the exact mechanism for the growth inhibition by silver nanoparticles has not yet been elucidated, many possible mechanisms have been proposed. Lin et al. (1998) explained that in general, silver ions from nanoparticles are believed to become attached to the negatively charged bacterial cell wall and rupture it, which leads to denaturation of protein and finally cell death. The attachment of either silver ions or nanoparticles to the cell wall causes accumulation of envelope protein precursors, which results in dissipation of the proton motive force. On the other hand, Lok (2006) elucidated that silver nanoparticles exhibited destabilization of the outer membrane and rupture of the plasma membrane, thereby causing depletion of intracellular ATP. Another proposed mechanism involves the association of silver with oxygen and its reaction with sulfhydryl (–S–H) groups on the cell wall to form R–S–S–R bonds, thereby blocking respiration and causing cell death (Kumar et al., 2004). Silver ions

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are reported to block the electron transport chain functions most sensitively between cytochrome reductase and cytochrome oxidase, and less sensitively between NADH and succinate dehydrogenase (Santoro et al., 2007). Cho et al. (2005) reported that the nanoparticles were effective at significantly lower concentration than the ions were. In contrast, Morones et al. (2005) proposed distinctly different bactericidal mechanisms for silver nanoparticles and silver ions. In the silver nitrate treatment, a central region of low molecular weight was formed within the cells as a defense mechanism, whereas no such phenomenon was observed in the nanoparticle treatment, although the nanoparticles did penetrate through the cell wall. They also reported that silver nanoparticles with size range of 1–10 nm attach to cell membrane and drastically disturb its proper function, like permeability and respiration, further on penetration, more damage is caused by interacting with sulfur- and phosphorous-containing compounds like DNA. In addition, release of silver ions from silver nanoparticles contributes to bactericidal effect. Sarkar et al. (2007) reported that for E. coli (ATCC 10536) and Staphylococcus aureus (ML 422), silver nanoparticles demonstrated greater bactericidal efficiency compared to penicillin, whereas Li et al. (2005) reported that for E. coli, silver nanoparticles also depicted synergistic bactericidal effects with known antibiotics such as amoxicillin. 3.6. Silver nanoparticle immobilization on cloth and disk diffusion studies The cloth immobilized with sterile water containing silver nanoparticles did not show much difference in texture, whereas

the cloth immobilized with PVDF containing silver nanoparticles turned slightly rigid. This change in texture is due to PVDF occupying the interface area of the fiber and forming a membrane-like structure upon drying. PVDF was selected based on its hydrophobic property, stability at wider range of temperatures and inertness to many chemicals. Fig. 5 shows the disk diffusion studies at various concentrations of silver nanoparticles. The cloth sprayed with sterile water and PVDF devoid of silver nanoparticles served as controls for respective plates. There was totally no inhibition by the cloth sprayed with only sterile water, whereas cloth sprayed with only PVDF showed mild inhibition of bacterial growth. From the Fig. 6, it was clear that increase in silver nanoparticle concentration increased the inhibition zone area on the plate, which directly represents the increase in toxicity. The cloth immobilized with sterile water containing silver nanoparticles showed larger inhibition zone than cloth immobilized with PVDF. This may be attributed to the free availability of the silver nanoparticles in cloth immobilized with sterile water. In cloth immobilized with PVDF, a coat of the polymer might slightly inhibit the availability of the silver nanoparticles and thereby its toxicity. But the inhibition zone around the cloth immobilized with sterile water containing silver nanoparticles decreased vastly in consecutive cycles after washing compared to cloth immobilized with PVDF (Fig. 6). The purpose of using PVDF in the present study is to bind the silver nanoparticles to the cotton cloth, which will decrease the loss of the silver nanoparticles in consecutive washing. Thus as expected the loss of silver nanoparticles was lesser in cloth immobilized with PVDF compared to cloth immobilized with sterile water. In case of the cloth immobilized with sterile water containing silver nanoparticles,

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Fig. 5. Image of Petri plates showing the growth inhibition of E. coli by (A) cloth immobilized with silver nanoparticles using sterile water and (B) cloth immobilized with silver nanoparticles using PVDF.

Fig. 6. Growth inhibition of E. coli in consecutive cycles by (A) cloth immobilized with silver nanoparticles using sterile water and (B) cloth immobilized with silver nanoparticles using PVDF.

74% decrease in inhibition zone was noticed between initial bactericidal activity and after 5th wash, which was due to the loss of nanoparticles at each washing, but during the same period only 28% loss was noticed in the cloth immobilized with PVDF. So this explains the binding property of PVDF. Durán et al. (2007) reported impregnation of cotton fabric with silver nanoparticles without the use of any binder, where 2% of the nanoparticles with initial concentration of 240 mg/L were adhered to the fabric. Silver nanopar-

ticle-immobilized cloth could find a wide application in various fields. In hospitals it could be used by medical practitioners as self-sterilizing coat or apron material since these practitioners are constantly exposed pathogenic organisms potent of causing serious infections. This cotton cloth material immobilized with silver nanoparticles can also be used as an antiseptic bandage material for wound dressing. Lee et al. (2007) recently reported on possibility of using cotton cloth immobilized with silver nanopar-

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ticles synthesized chemically for wound dressing. They reported the bactericidal effect of the silver nanoparticle-immobilized cloth material against gram-positive and -negative and antibiotic-resistant bacteria. Skin-irritation tests of the silver nanoparticle-immobilized cloth material on guinea pigs revealed no side effects (Lee et al., 2007). Interaction of silver nanoparticles with human fibroblasts affected their fission but was not toxic (Wen et al., 2007). But direct administration of silver nanoparticles to BRL 3A rat liver cells showed membrane leakage of lactate dehydrogenase, reduced glutathione levels and subsequently leading to death of the cells (Hussain et al., 2005). Though silver nanoparticles are reported to have no big health problems when administered into the body, argyria is always a problem whether silver is nano-sized or not (Chen and Schluesener, 2008). In daily life, silver nanoparticle-impregnated clothing may find a good applicability in underwear and socks (Lee et al., 2007). Santoro et al. (2007) reported the possibility of using silver nanoparticles impregnated/coated on contact lenses, which could be used as extended-wear or continuous-wear contact lenses to protect the eyes from infectious bacteria colonizing on the lenses. Mammalian cell toxicity was observed at high silver concentrations but still the effects of silver nanoparticle on ocular cells are not known. Since C. longa tuber powder, an abundantly available agricultural material was used in the present study devoid of any harmful and expensive solvents or chemicals for silver nanoparticle synthesis with good bactericidal activity, it proves to be superior in terms of cost factor and eco-friendliness. This validates the use of cotton cloth immobilized with silver nanoparticles synthesized biologically. 4. Conclusions Phyto-reductive synthesis of nano-sized silver particles using novel C. longa tuber powder and extract yielded quasi-spherical, triangular and small rod-shaped silver nanoparticles. Tuber extract produced more silver nanoparticles than powder, which may be attributed to the large and easy availability of reducing agents in the extract. Silver nanoparticles immobilized on cotton cloth using sterile water and PVDF showed good bactericidal activity even with consecutive washing. Thus it can be concluded that C. longa tuber extract and powder can be applied as cheap and environment-friendly bio-resource/biomaterial for synthesis of silver nanoparticles with antimicrobial activity. Acknowledgements The authors thank Mr. Jong-Gyun Kang, EM lab, center for University-wide research facilities, Chonbuk National University, Korea for TEM analysis. This work was supported by NRF Grant funded by the Korean Government (2009-0083194) and in part by a grant operated by KEITI of the Ministry of Environment of Korea. References Andreescu, D., Eastman, C., Balantrapu, K., Goia, D.V., 2007. A simple route for manufacturing highly dispersed silver nanoparticles. J. Mater. Res. 22, 2488– 2496. Ankamwar, B., Damle, C., Ahmad, A., Sastry, M., 2005. Biosynthesis of gold and silver nanoparticles using Emblica officinalis fruit extract, their phase transfer and transmetallation in an organic solution. J. Nanosci. Nanotechnol. 5, 1665–1671. Basavaraja, S., Balaji, S.D., Lagashetty, A., Rajasab, A.H., Venkataraman, A., 2008. Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium semitectum. Mater. Res. Bull. 43, 1164–1170. Binupriya, A.R., Sathishkumar, M., Yun, S.I., 2010. Myco-crystallization of silver ions to nano-sized particles by live and dead cell filtrates of Aspergillus oryzae var viridis and its bactericidal activity towards Staphylococcus aureus KCCM 12256. Ind. Eng. Chem. Res. 49, 852–858. Case, C.L., Jhonson, T.R., 1984. Laboratory Experiments in Microbiology. Benjumin Cummings Pub. Inc., California, USA. pp. 126–129.

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