Green synthesis of silver nanoparticles: characterization and determination of antibacterial potency
Silver ions (Ag+) and its compounds are highly toxic to microorganisms, exhibiting strong biocidal effects on many species of bacteria but have a low toxicity toward animal cells. In the present study, silver nanoparticles (SNPs) were biosynthesized using aqueous extract of Chlorella vulgaris as reducing agent and size of SNPs synthesized ranged between 15 and 47 nm. SNPs were characterized by UV–visible spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction and Fourier infrared spectroscopy, and analyzed for its antibacterial property against human pathogens. This approach of SNPs synthesis involving green chemistry process can be considered for the large-scale production of SNPs and in the development of biomedicines.
KeywordsSilver ions Green chemistry Human pathogens Biomedicines
Nanotechnology is an exploitation of strange properties of materials smaller than 100 nm (nm) to create new useful objects. Nanomaterials display unique, superior and indispensable properties and have attracted much attention for their distinct characteristics that are unavailable in conventional macroscopic material. Their uniqueness arises specifically from higher surface-to-volume ratio and increased percentage of atoms at the grain boundaries. They represent an important class of materials in the development of novel devices that can be used in various physical, biological, biomedical and pharmaceutical applications.
Silver nanoparticles are nontoxic, safe inorganic antibacterial agent used for centuries and are capable of killing about 650 types of diseases causing microorganisms. Silver has been described as oligodynamic because of its ability to exert a bactericidal effect at minute concentrations. The first scientific papers describing the medical use of silver report the prevention of eye infection in neonates in 1881 and internal antisepsis in 1901 (Russell and Hugo 1994). After this, silver nitrate and silver sulfadiazine have been widely used for the treatment of superficial and deep dermal burns of wounds and for the removal of warts (Rai et al. 2009).
SNP mode of action is presumed to be dependent on Ag+ ions that strongly inhibit bacterial growth through suppression of respiratory enzymes and electron transport components and through interference with DNA functions (Li et al. 2006). Silver in a nanometric scale (less than 100 nm) has strong toxicity to a wide range of microorganisms (Elechiguerra et al. 2005). Morones et al. (2005) defined the antibacterial activity of SNPs in four types of Gram-negative bacteria Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa and Salmonella typhi, and suggested that SNPs attach to the surface of the cell membrane and disturb its function by penetrating into bacterial cell and release of silver ions. Silver nanoparticles have also been found to exert antibacterial activity against some drug-resistant bacteria (Birla et al. 2009; Inoue et al. 2010).
Chlorella vulgaris is a medicinal unicellular green microalgae, which contains more than 20 different vitamins and minerals, 19 of 22 amino acids and high content of protein. The high profile in phytochemistry with carboxyl, hydroxyl and amino groups serves as an effective reducing agent and capping agent to robust coating on SNPs. Economical- and environmental-friendly approach to synthesize metal nanoparticles terms to be initiative in developing nanotechnology and nanoparticle-based products. In this study, SNPs are biosynthesized in the aqueous extract of C. vulgaris using aqueous solution of silver nitrate and characterized for their average particle size, morphology, surface capping, crystal structure and bactericidal properties by UV/Vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and antimicrobial assay.
Materials and methods
The fresh water green algal strain of Chlorellavulgaris was collected from Algal Culture Collection, Center for Advanced Studies in Botany, University of Madras, India, and was inoculated in Bold Basal medium. The culture was maintained at 24 ± 1 °C in a thermostatically controlled room and illuminated with cool fluorescence lamps (Phillips 40 W, cool day light 6500 K) at an intensity of 2000 lux in a 16:8 h light/dark regime. In the exponential log phase, when the pigment, protein and carbohydrate measured were maximum, the cells were harvested. The collected cells were washed with double distilled water and sonicated using ultrasonic vibration at 30 % amplitude for 20 min to release the water-soluble biomolecules. The homogenate was subjected to centrifugation (3–4 times), the supernatant was diluted through a series of dilutions with 1 mM AgNO3, and the reaction mixtures (10 mL) were incubated at 37 °C.
Characterization of silver nanoparticles
UV–visible spectroscopy analysis
Color changes in reaction mixture were visually observed from white to pale brown then to dark brown. Bioreduction of the silver ions was monitored by sampling 1 mL aliquots at different time intervals (0, 24, 48 and 72 h). The absorption measurements were carried out by Beckman DU 64 UV–visible spectrophotometer.
Scanning electron microscopy analysis
Scanning electron microscopic (SEM) analysis of synthesized SNPs was performed using Hitachi S-4500 SEM machine. Thin films of the SNPs were prepared on a carbon-coated copper grid by dropping a very small amount of the aqueous SNPs on the grid, and excess were removed using blotting paper. These films on the SEM grid were then allowed to dry under mercury lamp for 5 min.
Transmission electron microscopy analysis
Synthesized SNPs by the biological reduction were prepared for TEM analysis by placing a drop over carbon-coated copper grids and allowing the solvent to evaporate. TEM measurements were performed on a JEOL model 1200EX instrument operated at an accelerating voltage at 80 kV.
X-ray diffraction (XRD) measurement
Bio-reduced aqueous silver nitrate solution drop was coated on glass substrate and subjected to XRD measurement using XRD (Model D/Max-2500). Scanning was executed in a 2θ region from 30 to 80º. The pattern was recorded using Cu-Kα radiation with a wavelength (λ) of 1.5406 Å at a tube voltage of 40 kV and a tube current of 30 mA. Drop-coated on glass were done on a Phillips PW 1830 instrument operating at a voltage of 40 kV and current of 20 mA with Cu-Kα radiation.
Fourier transform infrared (FTIR) spectroscopy measurements
After complete reduction in AgNO3− ions by aqueous extract of C.vulgaris, the reaction mixture was centrifuged at 10,000 rpm for 10 min to isolate the SNPs from free proteins or other compounds present in the solution. The SNPs pellets obtained were freeze-dried and diluted with potassium bromide at 1:100 ratio. FTIR spectrum of samples was recorded on Shimazdu IR Prestige-21 FTIR instrument; all measurements were carried out in the range of 400–4000 cm−1.
The antimicrobial activity of synthesized SNPs was performed by using agar well diffusion method. About 20 mL of sterile molten Mueller–Hinton agar (HiMedia Laboratories Pvt. Limited, Mumbai, India) was poured into the sterile petriplates. Triplicate plates were swabbed with the overnight culture (108 cells/mL) of human pathogens: Escherichia coli, Proteus vulgaries, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. The solid medium was gently punctured with cork borer to make a well. Finally, the aqueous SNPs (20 µL) were added into each well and incubated for 24 h at (37° C). After incubation, the zone of inhibition was measured and expressed as millimeter (mm) in diameter.
Results and discussion
Synthesis of silver nanoparticles
Characterization of silver nanoparticles
UV–visible spectroscopy is one of the most widely used techniques for structural characterization of nanoparticles (Sun et al. 2001). The biosynthesized SNPs were measured by UV–visible spectroscopy at different time intervals to study the change in light absorption and increase in intensity. The absorption spectra of nanoparticles showed highly symmetric single band absorption with peak maximum at 421 nm with steadily increase in intensity as a function of time of reaction without any shift in intensity (Fig. 1b). This indicates the presence of SNPs, which is due to the excitation of surface plasmons (Jae and Beom 2009). After 72 h, no further increase in intensity was recorded, indicating the complete reduction in silver ions.
The biosynthesized SNPs from algal aqueous cell-free extract were moderately susceptible to fungal pathogen C. albicans, suggesting that the increase in dose may tend to be efficiently toxic. In a study a Kim et al. (2008), SNPs showed potent activity against Trichophyton mentagrophytes, Trichosporon beigelii and C. albicans which was compared with the commercially available antifungal agents (amphotericin and fluconazole). Stable solutions containing up to 35 ppm SNPs were found to have effective antifungal property against Aspergillus, Penicillium and Trichoderma sps (Petica et al. 2008). SNPs at 100 ppm totally inhibited the bacterial growth, but the activity against mold and dermatophytes was low; bacteria and molds exhibited resistance against SNPs at 50 ppm concentration (Falkiewicz-Dulik and macura et al. 2008). In case of C.albicans also, SNPs are found to be cytotoxic disrupting the cell membrane and formation of pits and pores on membrane surface, subsequently leads to cell death (Kim et al. 2008, 2009). SNPs have found its application against fungal infection and in biostabilization of footwear materials.
A critical need in the field of nanotechnology is the development of reliable and eco-friendly process for the synthesis of metallic nanoparticles. SNPs are toxic to human beings at high concentration, while nontoxic at low concentration. The growing interest in metallic nanoparticles is due to their exclusive catalytic, optical, electronic, magnetic and antimicrobial properties. In our study, we have investigated the synthesis of silver nanoparticles in a cost-effective approach using extract of Chlorella vulgaris. Synthesized SNPs were been characterized to be stable without any impurities and of average size 27 nm. Thus, biosynthesized SNPs can find immense application in the field of biomedical appliances, formulation of antimicrobial agents and in combination with antibiotics.
- Cullity BD (1956) Elements of X-ray Diffraction. Addison-Wesley Company, USAGoogle Scholar
- Falkiewicz-Dulik M, Macura AB (2008) Nanosilver as substance biostabilising footwear materials in the foot mycosis prophylaxis. Mikol Lek 15:145–150Google Scholar
- Inoue Y, Uota M, Torikai T, Watari T, Noda I, Hotokebuchi T (2010) Antibacterial properties of nanostructured silver titanate thin films formed on a titanium plate. J Biomed Mater Res 92A(3):1171–1180Google Scholar
- John R, Florence SS (2009) Structural and optical properties of ZnS nanoparticles synthesized by solid state reaction method. Chalcogenide Lett 6:535–539Google Scholar
- Kim KJ, Sung WS, Moon SK, Choi JS, Kim JG, Dong GL (2008) Antifungal effect of silver nanoparticles on dermatophytes. J Microbiol Biotechnol 18:1482–1484Google Scholar
- Mohan N, Hanumantha rao P, Ranjith kumar R, Sivasankaran S, Sivasubramanian V (2009) Studies on mass cultivation of Chlorella vulgaris and effective harvesting of bio-mass by low-cost methods. J Algal biomass utln 1(1):29–39Google Scholar
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