Biological Synthesis of Silver Nanoparticles using

Research Journal of Biotechnology

Vol. 10 (2) February (2015) Res. J. Biotech

Biological Synthesis of Silver Nanoparticles using Colocasia Extract and their antimicrobial activity Boruah Himangshu, Talukdar Binita, Parveen Assma, Goswami Gunajit, Barooah Madhumita and Boro Robin Chandra* Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat -13, INDIA *[email protected]

and Gram-positive bacteria14. In gram-negative bacteria three possible mode of antibacterial action have been hypothesized15. First, Ag-NPs nanoparticles in the range of 1–10 nm may attach to the surface of the cell membrane and diminish the permeability and respiration. Secondly, due to their small size they can penetrate inside the bacterial cell and may cause damage to DNA or protein probably by interacting with sulfur and phosphorus. Thirdly the release of silver ions from Ag-NPs and formation of free radicals may have an additional contribution to the bactericidal effect2.

Abstract Silver nanoparticles were synthesized by a simple, non-toxic, eco-friendly biological reduction method using colocasia extract as reducing agent. A variety of nanoparticles were formed when the concentration of the reducing agent was increased with respect to the silver nitrate solution. The reaction mixtures displayed variation in colours and characteristic UVVIS spectra of silver nanoparticles with the increasing concentration of colocasia extract. A single SPR band in between 419 to 438 nm in UV-VIS spectroscopy revealed the formation of silver nanoparticles. TEM analysis of the silver nanoparticles confirmed the spherical shape and size distribution of the nanoparticles found in the range of 10-30nm. Antibacterial activity of the Ag-NPs was confirmed by disk diffusion method against Bacillus flexus, Escherichia coli, Serratia marcescens and Staphylococcus aureus. Keywords: Biosynthesis, Colocasia extract, nanoparticles, TEM, antimicrobial activity.

However the effect of nanoparticles is concentration dependent and is more effective against Gram negative than Gram positive bacteria. It has been observed that Ag-NPs can inhibit the growth of Gram-negative bacteria such as E. coli at a low concentration of Ag-NPs (3.3 nM) but in case of Gram positive bacteria such as S. aureus, growth was inhibited at ten times higher concentrations (33 nM)16. The biggest advantage of nanoparticles is that bacteria cannot build up resistance unlike antibiotics. Efficacy of Ag-NPs depends on the size of the particles and it has been reported that the bactericidal effects of silver nanoparticles are higher than the ionic silver which indicates that smaller particles have greater antimicrobial effect2. Therefore, methods of synthesis are very important to have better efficacy. The methods for synthesis of nanoparticles include chemical, physical, photochemical and biological synthesis. Each method has certain disadvantages such as costs, scalability, particle sizes and size distribution2.

Silver

Introduction Nanoparticles have unique electronic, optical, mechanical, magnetic and chemical properties when compared to larger matter1. They have high reactivity due to the large surface to volume ratio2. A number of metallic nanoparticles have been synthesized in recent years such as gold nanoparticles, titanium oxide nanoparticles, iron nanoparticles, silver nanoparticles etc. Among these, silver nanoparticles (AgNPs) have been most widely studied and employed in various potential applications includingagriculture3, cosmetics4, medicine5, renewable energies6, environmental remediation7, electronic devices8and biomedical 9,10 devices .Because of these properties Ag-NPs have been used in microelectronics and medical imaging.

Although, the chemical methods have been mostly used for production of Ag-NPs, however biological synthesis of AgNPs has become the alternate method as the biological methods are environment friendly, economical, simple, reproducible, requires less energy, reduces the use of hazardous chemicals and has good control over the reaction conditions2. Biological methods employ biological agents such as bacteria9, fungi17, yeast10, algae or plant extracts18 that provide the reducing agent and the stabilizer7 required for the synthesis of Ag-NPs. In this study, we used colocasia stem extracts due to its availability throughout the year and most importantly colocasia contains some microcrystals made up of calcium oxalate which are used by the plant for storing calcium19. We hypothesized that colocasia stem extracts may be used as biological materials to synthesize Ag-NPs and Ag-NPs synthesized from colocasia may have antimicrobial activity against a wide range of microbes.

In addition, Ag-NPs display broad spectrum antimicrobial activities11 and are being incorporated in a wide range of consumer products including soaps, water purification systems, pastes, food and textiles and cosmetics12,13. The antimicrobial activity of silver nanoparticles has been studied by different research groups; however, the actual mode of action is yet to be fully understood. One possible mechanism suggested is that it attacks and causes change in the cell membrane of microbes. Bactericidal activities of Ag-NPs have been demonstrated in both Gram-negative

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Vol. 10 (2) February (2015) Res. J. Biotech Inhibition zones for respective samples were measured after incubation.

Material and Methods Materials: Silver nitrate (AgNO3) was purchased from Sigma-Aldrich, USA. Nutrient agar and Nutrient broths were purchased from Merck, Germany. Colocasia stalk were collected from Assam Agricultural University campus, Jorhat (Assam), India. B. flexus, E. coli, S. marcescens and S. aureus were used to evaluate the antimicrobial activity of the biologically synthesized silver nanoparticles. E. coli and B. flexus are harmless; Staphylococcus aureus is a gram positive coccus bacterium and pathogenic strains cause skin infections, respiratory disease and food poisoning. Serratia marcescens is associated with urinary tract and wound infections in humans.

Results and Discussion Characterization of Silver nanoparticles: Silver nanoparticles exhibit yellowish brown color in aqueous solution due to excitation of surface plasmon resonance. Exposure of colocasia extracts leads to the reduction of silver ion to silver nanoparticles followed by change in color that gave characteristic UV-VIS spectrum (Fig.1). A single SPR band between 400–435nm in the absorption spectra indicates the formation of spherical shaped silver nanoparticles (Mie’s theory)7. Broad peaks for silver nanoparticles were observed when the concentration of the reducing agent was increased from 1% to 5% (V/V) with respect to silver nitrate solution. This might be due to formation of the anisotropic silver nanoparticles which always exhibit a broad SPR band depending on their shape compared with single SPR for small spherical nanocrystals23.

Synthesis of Silver nanoparticles: Silver nanoparticles were synthesized using biological reduction method described by Kumar and Yadav20 with minor modifications. We used colocasia as reducing agent for the synthesis of Ag-Nps. Freshly collected colocasia stalks (50 gm) were thoroughly washed and ground in a mortar pestle. The paste obtained was then re-suspended in 50 mL of distilled water and then filtered with clean muslin cloth at ambient temperature and centrifugation was carried out at 10,000 rpm for 5 minutes in a desktop centrifuge (Hermle Laboratechnik GmbH, Germany) to obtain clear solution of colocasia extract.

The maximum peak of the absorption spectra was shifted from 419 nm to 438 nm and the broad absorption peaks with decreasing intensity were observed when the concentration of colocasia extract was increased. The shifting absorption peaks are probably due to the increase in the size of the nanoparticles26. Broadening of the absorption peaks for silver nanoparticles and a red shift of metallic nanoparticles with increasing particle size have been reported26. TEM images of silver nanoparticles have a good agreement with this approximation.

The extract was used immediately for synthesis of silver nanoparticles and the remaining extract was stored at 40C for future use. For the synthesis of Ag-NPs, 0.5 mL (1%) of colocasia extract was added drop by drop to 50 mL of 1mM aqueous silver nitrate solution. The reaction mixture was stirred with a magnetic stirrer at 200 rpm until (~30 min) the reaction mixture became yellowish brown. Formation of yellowish brown color indicates the synthesis of silver nanoparticles21. The pH of the reaction was maintained at basic condition throughout the experiment22 and the concentration of the colocasia extract was increased sequentially from 1% to 5% (V/V) to observe the effect of concentration of reducing agent on the characteristics of Ag-NPs.

TEM images of the silver nanoparticles proved the formation of silver nanoparticles of nearly spherical shape and an increment in the diameter of prepared silver nanoparticles was observed as the concentration of the colocasia extract was increased from 1% to 5% (V/V) [Fig. 2(a)-(f)]. The average diameters of silver nanoparticles were measured using Image J (1.46r) software. The variations in average size of silver nanoparticles with different concentration of reducing agent are represented in fig. 2(g). The average size of the nanoparticles at 1%, 2%, 3%, 4% and 5% colocasia extract was found as 10 nm, 13 nm, 18nm, 20nm and 30nm respectively. This increase in sizes of silver nanoparticles may be due to the increasing concentration of reducing agent.

Characterization: UV–visible spectra of the samples were taken in UV spectrophotometer (Thermo Scientific, Evolution 201) within the wavelength range of 300 to 750 nm. JEOL JEM 100 CX II transmission electron microscope (TEM) at operating voltage of 100 kV was used to determine the size and distribution of nanoparticles.

Reducing agents have great influence in the size and shape of nanoparticles, as the pressure of the reaction, directly depends on the decomposition of reducing agents. At high concentrations of reducing agents the reaction rate is greatly accelerated and the process of nucleation and growth of the nanoparticles will occur at a higher rate24. The selected area electron diffraction (SAED) pattern recorded from the silver nanoparticles indicates the crystalline nature of silver nanoparticles25. These data support our hypothesis that colocasia extract can be used as a reducing agent for biological synthesis of Ag-NPs.

Antibacterial activity studies: The disc diffusion method was used to detect the antimicrobial activity of silver nanoparticles against B. flexus, E. coli, S. marcescens and S. aureus. Nutrient agar plates were spreaded with 100μl of each bacterial culture and sterile paper discs of about 5mm impregnated with 20 μl of well dispersed silver nanoparticles having 100μg/ml concentration were placed on each plate. Plates were then incubated for 24h at 37 0C.

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Vol. 10 (2) February (2015) Res. J. Biotech 2. Pacios R., Marcilla R., Pozo-Gonzalo C., Pomposo J. A., Grande H., Aizpurua J. andMecerreyes D., Combined electrochromic and plasmonic optical responses in conducting polymer/metal nanoparticle films, J. Nanosci. Nanotechnol., 7(8), 2938-41 (2007)

In our present study, it was observed that Ag-NPs synthesized by biological method showed reasonable antibacterial activity. Antibacterial activity was observed against the all tested bacteria. Among them S. aureus (Fig. 3d) was most susceptible to Ag-NPs and S. mercenscens (Fig. 3c) was least susceptible. Rest of the two bacteria showed moderate range of susceptibility.

3. BoroR. C., KaushalJ., NangiaY., WangooN.,Bhasin A. and Suri C. R., Gold nanoparticles catalyzed chemiluminescence immunoassay for detection of herbicide 2,4-dichlorophenoxyacetic acid, Analyst, 136(10), 2125-30 (2011)

Conclusion Silver nanoparticles were synthesized by simple biological method using colocasia extract as reducing agent. Spherical shape of synthesized silver nanoparticles was confirmed by the UV-spectroscopy and Transmission Electron Microscopy studies. TEM micrograph reveals that the size of the synthesized silver nanoparticles was increased with the increasing concentration of colocasia extract. Antibacterial action of the prepared silver nanoparticles was observed against four bacterial strains and was found effective against all the four test organisms. We conclude that colocasia can be a good source of reducing agent required for biological synthesis of nanoparticles and it eliminates the use of hazardous chemical that makes synthesis eco-friendly as well as cost effective due to the wide availability of colocasia plants.

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Wavelength (nm) Fig. 1: UV-Visible absorption spectra of silver nanoparticles at different concentration of reducing agent.

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(g) Average Diameter of nanoparticles (nm)

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Fig. 2: TEM images of Silver nanoparticles at different concentrations of (a) 1% colocasia extract, (b) 2% colocasia extract, (c) 3% colocasia extract, (d) 4% colocasia extract, (e) 5% colocasia extract, (f) SAED pattern recorded from the silver nanoparticles and (g) graphical representation of average sizes of the nanoparticles with respect to the different concentration of colocasia extract.

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(Received 01st August 2014, accepted 30th September 2014)

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