Current Nanoscience, 2012, 8, 000-000
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Rapid Biosynthesis of Silver Nanoparticles using Eichornia crassipes and its Antibacterial Activity S C G Kiruba Daniel1, K Nehru2 and M Sivakumar1* 1
Dept. of Nanoscience and Technology, Anna University of Technology - Tiruchirappalli, Tiruchirappalli – 620 024, India; 2Dept. of Chemistry, Anna University of Technology - Tiruchirappalli, Tiruchirappalli – 620 024, India Abstract: In this paper, we have studied the rapid synthesis and antimicrobial activity of silver nanoparticles synthesized using extract of Eichornia crassipes (Water Hyacinth) which is considered as one of the most notorious aquatic weeds present all over the world. The synthesized nanoparticles exhibited absorption maximum at 420 nm which is a characteristic feature of Surface Plasmon Resonance (SPR) peak for silver nanoparticles. The silver nanoparticles were characterized with Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM) and found the size ranges from 10 to 50 nm. Further Fourier Transformation Infrared Spectrum (FTIR) revealed that the phenolic groups present in the plant extract were responsible for the reduction of silver nitrate into silver nanoparticles and stabilization of formed silver nanoparticles. Finally antibacterial activities of the silver nanoparticles were checked against Bacillus subtilis, Pseudomonas fluorescens and Klebsiela pneumonia and significant zones of inhibition were found.
Keywords: Antimicrobial activity, biosynthesis, silver nanoparticles, water hyacinth. 1. INTRODUCTION The synthesis of inorganic metal nanoparticles and nanostructure materials are attracting much attention in recent research because of their valuable application in biosensors [1], labels for cells and biomolecules [2], peptide probes [3], anti-microbial agents [4], wound healing agent [5] and cancer therapeutics [6]. Silver nanoparticles are widely used in the field of electrocatalysis [7], chemical sensors [8], catalysis [9] and optical devices [10]. Several methods for the synthesis of silver nanoparticles have been proposed by many researchers, such as chemical method [11], Gammairradiation [12], push-pull methods, thermal decomposition methods [13], photochemical methods [14] and biosynthesis [15]. In the biosynthesis of silver nanoparticles, microbes [16,17] and different plant extracts [15] have been widely used in the synthesis of silver nanoparticles. For the synthesis of silver nanoparticles, plant extracts are taken from plants such as Aloe vera [18], Neem [19], Tea [20], Jatropha [21], Acalypha [22], Cactus [23] and Gliciridia [24]. Herein we report the synthesis of silver nanoparticale using Water Hyacinth a noxious weed which will be of great advantage due to high productivity and this may also help in eradicating the aquatic weed. 2. EXPERIMENTAL SECTION 2.1. Synthesis of Silver Nanoparticles Eichornia crassipes leaves were collected from nearby water sources mainly from tropical areas. The plant leaf broth extract was prepared by taking 20 grams of thoroughly washed and finely cut leaves in 100 ml sterile distilled water in a beaker. The mixture was boiled for 10 min. and filtered using filter paper. The filtered broth extract was kept at 4o C for further studies. 10 ml of leaf broth was added to 90 ml of a 1mM solution of AgNO3 for reduction of silver ions and the reduction reaction temperature was optimized at 90oC. The effect of reaction time on the surface plasmon resonance of silver nanoparticles has been *Address correspondence to this author at the Dept. of Nanoscience and Technology, Anna University of Technology - Tiruchirappalli, Tiruchirappalli – 620 024, India; Tel: +91 431 2407959; Fax: +91431 2407999; E-mail:
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1573-4137/12 $58.00+.00
noticed. Effect of pH on SPR of silver nanoparticles is evaluated by adjusting the solutions to different pH levels (2, 4, 6, 8, and 10). 2.2. Characterization 2.2.1. UV Visible Absorption and FTIR Spectroscopy The synthesized silver nanoparticles by reducing silver metal ion solution with leaves extract were initially characterized by UV visible absorption spectroscopy. The samples were taken in a 1cm quartz cuvette and measured in a JASCO V 650 spectrophotometer containing double beam in identical compartments each for reference and test solution from 200 nm to 900 nm. FTIR analysis was done using Perkin Elmer FTIR spectrometer. 2.2.2. Atomic Force Microscopy Atomic Force Microscopy image was taken using Park system AFM XE 100. The aqueous silver nanoparticles were deposited onto a freshly cleaved mica substrate. The sample aliquot was left for 1 min and then washed with deionized water and left to dry for 15 min. The images were obtained by scanning the mica in air in non – contact mode. 2.2.3. Transmission Electron Microscopy Transmission Electron Microscopy image was taken using JOEL JEM SX 100. The sample was placed on a copper grid and left to dry for 60 min under vacuum. The sample was then subjected to transmission electron microscopy studies. 2.3. Antibacterial Activity Silver nanoparticles were tested for their antibacterial activity by the agar diffusion method. The bacterial strains Bacillus subtilis, Pseudomonas fluorescens and Klebsiela pneumonia were utilized for this antibacterial analysis. Wells were made by standard cork borer at equal distance and were filled with silver nanoparticles. The formation of a clearing zone (restricted bacterial growth) around the cavity was measured. 3. RESULTS AND DISCUSSION 3.1. UV Visible Spectroscopy Noble metal nanoparticles exhibit specific Surface Plasmon Resonance (SPR) which shows peak specific characteristic for each metal. The characteristic SPR of colloidal silver nanoparticles ranges between 390 nm to around 420 nm due to Mie scattering [25]. UV – visible absorption spectroscopy is one of the main tech© 2012 Bentham Science Publishers
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niques to examine the size and shape of the nanoparticles in aqueous solutions. It was observed that by heating 10 ml of EC plant extract in 100 ml of 1mM silver nitrate solution at 70oC is sufficient to reduce the aqueous silver nitrate solution to silver nanoparticle with in 5 min. At different time interval, there was a shift in the SPR peaks and absorption Fig. (1). The difference in the intensity of the SPR peak ranged between 420 nm to 440 nm. Normal pH of the colloidal silver nanoparticle solution obtained was found to be 5. Variations in pH (2, 4, 6, 8, and 10) of Ag nanoparticle also exhibited different SPR peaks Fig. (2) - a blue shift because of variation in size of the nanoparticles as reported earlier [22].
Fig. (1). UV- Absorption spectroscopy of silver nanoparticles synthesized at different reaction time of Eichornia crassipes leaf extract in 1 mM silver nitrate solution.
Daniel et al.
3.3. FTIR Spectroscopy And Reducing Mechanism FTIR measurement was made to identify the possible biomolecules, responsible for the stabilization of the silver nanoparticles synthesized by leaf extract. The FTIR spectra of untreated and treated leaf extract samples containing silver nanoparticles are depicted in Fig. (5). The major secondary metabolites present in Eichornia as reported by Sanaa et al. [26] are phenolic compounds (4.35%), terpenoids (1.53%) and alkaloids (0.98%). All the above said compounds were reported to play a role in the synthesis of nanoparticles from different plants. The presences of number of aromatic compounds in the plant were revealed earlier by GC – MS studies [26]. The strong band at 1639 cm-1 is contributed to amide I. When compared to the amide I band of plant extract, the band present in silver nanoparticles had lower absorbance. The band observed at 1368 cm-1corresponds to C - O stretching which may be due to phenolic compounds present in the plant extract. Phenolic compounds are stated to play important role in reduction of metal ions [27]. In addition the observed bands suggest the presence of terpenoids which was in accordance with the studies reported earlier [26]. The terpenoids may also play a role in reduction of metal ions by the oxidation of aldehyde groups in the molecules to carboxylic acids [19]. Water hyacinth has rich sources of phenolic compounds, terpenoids and alkaloids that help in the synthesis of silver nanoparticles by reduction of metal ions and subsequent stabilization. 3.4. Antibacterial Activity Silver nanoparticles exhibited excellent antibacterial activity against the bacteria Bacillus subtilis, Pseudomonas fluorescens and Klebsiela pneumonia. In spite of the presence of an organic protecting layer, the silver nanoparticles were found to exhibit antibacterial activity Fig. (7). The average zone of inhibition for Bacillus subtilis, Pseudomonas fluorescens and Klebsiela pneumonia were found to be 18, 15 and 13 mm respectively Table 1. In earlier study it was found that the crude extract as well as the plant extract after TLC separation of water hyacinth exhibited antimicrobial and antialgal activities [30]. In our study the extract alone kept in well showed no antibacterial activity, but silver nanoparticles synthesized from the extract exhibited good antibacterial activity. This may be due to the presence of silver nanoparticles. Table.1 Values of Zone of Inhibition Seen Around Well Containing Green Synthesized Silver Nanoparticles S.No
Fig. (2). SPR variations of synthesized silver nanoparticles adjusted to different pH exhibiting blue shift
3.2. Particle Size Analysis Particle size was analysed by Atomic Force Microscopy and Transmission Electron Microscopy. AFM was used to view the nanoparticles both in Two Dimensional view and Three Dimensional view Fig. (3). TEM images and measurements of the synthesized nanoparticles also showed that the nanoparticles are relatively uniform in diameter and have spherical shape Fig. (4) and size of the synthesized silver nanoparticles to be between 10 nm and 70 nm.
Bacteria
Zone of Inhibition (mm)
1
Bacillus subtilis
18 + 2
2
Pseudomonas fluorescens
15 + 3
3
Klebsiela pneumonia
13 + 2.5
4. CONCLUSIONS We have developed a simple, cheap, fast and green method to synthesize silver nanoparticles using Eichornia crassipes weed. The synthesized silver nanoparticles were characterized by using UV visible absorption spectroscopy, Atomic Force Microscopy, Transmission Electron Microscopy and FTIR. Antibacterial activity was observed against Bacillus subtilis, Pseudomonas fluorescens and Klebsiela pneumonia. Further work to elucidate the role of silver nanoparticles in the anti-bacterial activity is going on. ACKNOWLODGEMENTS We are very much thankful to Anna University of Technology Tiruchirappalli for the funding of Instrument facilities. The authors gratefully acknowledge the instrumentation support given by
Rapid Biosynthesis of Silver Nanoparticles Using Eichornia Crassipes
Current Nanoscience, 2012, Vol. 8, No. 1
Fig. (3). Atomic Force Microscopy image of silver nanoparticles 2D and 3D view showing uniform sized green synthesized Ag nanoparticles.
Fig. (4). Transmission Electron Microscopy image of green synthesized silver nanoparticles taken at 100,000 X.
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Transmittance
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1368
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1117 40
622 20 Silver Nanoparticle
1639 0 4000
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Wavelength (cm-1) Fig. (5). FTIR spectroscopy of Eichornia crassipes synthesized silver nanoparticles compared with Eichornia crassipes leaf extract.
R
H O
OH + R
OH
HO
AgNO3
OH
H O
R
Ag R
O H
HO
OH
O H
R Fig. (6). Schematic representation of mechanism of reduction and stabilization of silver nanoparticles by Eichornia crassipes leaf extract.
Fig. (7). Antibacterial assay: Zone of Inhibition seen well around green synthesized silver nanoparticles a. Bacillus subtilis, b. Pseudomonas flourescens and c. Klebsiela pneumonia with controls at the centre of each petriplates (C – blank, E – extract alone).
Rapid Biosynthesis of Silver Nanoparticles Using Eichornia Crassipes
Dr. Pushpa Vishwanathan of Electron Microscopy Division, Adyar Cancer Research Institute, Chennai, India for Transmission Electron Microscopy image. REFERENCES [1] [2]
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