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/lp/springer-journals/antimicrobial-activities-of-silver-nanoparticles-synthesized-from-BTNpSmS0ru
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- Springer Journals
- Copyright
- 2014 Maiti et al.; licensee Springer.
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- 2093-3371
- DOI
- 10.1186/s40543-014-0040-3
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Abstract
Background: It has been known for quite some time now that silver nanoparticles (AgNP) can inhibit microbial growth and even kill microbes. Our investigation reports the antimicrobial activity of AgNP against a model bacterium, Escherichia coli. Methods: The aqueous extract of Lycopersicon esculentum (red tomato) was used for the rapid synthesis of AgNP, which is very simple and eco-friendly in nature. The UV-visible spectroscopy technique was employed to establish the formation of AgNP. Results: The transmission electron microscopic images showed that the particles were of mostly spherical shape. For the bacteriological tests, the microorganism E. coli was inoculated on Luria broth (LB) agar plate in the presence of varied amounts of AgNP. The antibacterial activity was obvious from the zone of inhibition. At concentration 20 μg/ml and above, the AgNP showed a clear zone of inhibition and the minimum inhibitory concentration of AgNP to E. coli was 50 μg/ml. Growth rates and bacterial concentrations were determined by measuring optical density at 600 nm at different time points. Conclusions: From the slope of the bacterial growth curve, it has been concluded that the nanoparticles are bacteriostatic at low concentration and bactericidal at high concentration. So these nanoparticles are believed to act as preventive for bacterial contamination. Keywords: Silver nanoparticle; Green synthesis; Lycopersicon esculentum; Antibacterial activity; Escherichia coli Background prevent HIV from binding to host cells (Sun et al. 2005), Disease-causing microbes that have become resistant to but the effects of silver nanoparticles (AgNP) on microor- drug therapy are an increasing public health problem. ganisms have not been developed fully. Nanosilver, being Many researchers are now engaged in developing new ef- less reactive than silver ions, is expected to be more suitable fective antimicrobial reagents with the emergence and for medical applications. Reducing the particle size of increase of microbial organisms resistant to multiple antibi- metals is also an efficient and reliable tool for improving otics, which will increase the cost of health care. Therefore, their biocompatibility, which facilitates their applications in there is an urgent need to develop new bactericides. Silver different fields such as bioscience and medicine. The mech- has been used for years in the medical field for antimicro- anism of the bacterial effect of AgNP as proposed is due to bial applications such as burn treatment (Parikh et al. 2005; the attachment of AgNP to the surface of the cell mem- Ulkur et al 2005), elimination of microorganisms on textile brane, thus disrupting permeability and respiration func- fabrics (Jeong et al. 2005; Lee et al. 2007; Yuranova et al. tions of the cell (Kevitec et al. 2008). It is also proposed 2003), disinfection in water treatment (Russell and Hugo that AgNP not only interact with the surface of a mem- 1994; Chou et al. 2005), prevention of bacteria colonization brane but can also penetrate inside the bacteria (Morones on catheters (Samuel and Guggenbichler 2004; Alt et al. et al. 2005). The antibacterial activity of AgNP is signifi- 2004; Rupp et al. 2004), etc. It has also been found to cantly enhanced when it is modified with sodium dodecyl sulfate (SDS) (Kevitec et al. 2005; Carpenter 1972). * Correspondence: j.laha@yahoo.co.in Midnapore College, Midnapore, West Bengal, India Full list of author information is available at the end of the article © 2014 Maiti et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maiti et al. Journal of Analytical Science and Technology 2014, 5:40 Page 2 of 7 http://www.jast-journal.com/content/5/1/40 In this study, we have investigated the antimicrobial nanoparticles (Barman et al. 2013), a smooth and narrow effects of silver nanoparticles prepared by a biosynthesis absorption band of AgNP at 410 nm was observed only in −3 method. The chemical reduction method is widely used the presence of SDS of 3 × 10 M (Figure 1A). So we pre- to synthesize AgNP because AgNP could be synthesized ferred to synthesize AgNP using SDS as the stabilizing under a mild as well as on a large scale (Cao et al. 2010). agent. However, the use of environmentally benign materials An absorption band was observed at 410 nm for 1:1 like plant leaf extract, bacteria, and fungi for the synthe- extract composition. The plasmon band shifted to higher sis of silver nanoparticles is more acceptable as they values with the increase of the concentrations of tomato offer several benefits over chemical methods like condi- in aqueous extracts and reached to 415 nm for 3:2 com- tions of high temperature, pressure, and toxic chemicals position (Figure 1B). At concentrations higher than 3:2 which are not required in the synthesis protocol (Singh composition, the plasmon band shifted to higher values et al. 2010). Therefore, preparation of AgNP by a green and the extinction coefficient of the band decreased ap- synthesis approach has compatibility for pharmaceutical preciably. However, tomato extract of 1:1 composition and biomedical applications. was used throughout the work. In the present work, the synthesis of silver nanoparti- A bathochromic shift of the SPR bands from 388 to 445 cles has been carried out using the aqueous extract of nm was observed while the concentration of AgNO varied −3 −2 Lycopersicon esculentum (red tomato). The water extract from 3 × 10 to 5 × 10 M keeping the extract compos- −3 of tomato juice mostly contains proteins and water- ition constant at 1:1 using SDS of 3 × 10 M(Figure 1C). soluble organic acids (Gould 1983) which are believed to When the particle size increased, the absorption peak act as stabilizing and reducing agents, respectively. With shifted towards the red wavelength, which indicated the for- these nanoparticles, a preliminary test for antibacterial mation of larger sized nanoparticles (Peng et al. 2010). activity was carried out by cup diffusion method and the The shape and size distribution of the synthesized effects of AgNP on bacterial growth has been studied by AgNP were characterized by TEM study. The TEM im- employing minimum inhibitory concentration (MIC) ages were taken using JEOL JEM-2100 high-resolution method. Results obtained by us prove that AgNP pre- transmission electron microscope (HR-TEM; JEOL Ltd., pared by the green method is suitable for the formula- Akishima-shi, Japan). Samples for the TEM studies were tion of new types of bactericidal materials. prepared by placing a drop of the aqueous suspension of particles on carbon-coated copper grids followed by Results and discussion solvent evaporation under vacuum. The TEM images of Results AgNP produced from 1:1 composition of tomato extract Characterization and optimization of AgNP preparation showed that the particles were mostly spherical and their The absorbance spectra of the AgNP were analyzed by sizes varied from 10 to 40 nm. Selected area electron dif- using a ‘SHIMADZU’ UV 1800 spectrophotometer fraction (SAED) pattern illustrated the crystalline nature (Shimadzu Corporation, Kyoto, Japan). AgNP exhibited of AgNP (Figure 2). a reddish yellow color in water due to the excitation of the localized surface plasmon vibrations of the metal Antibacterial activity of AgNP against the microorganism nanoparticles. Generally, the surface plasmon reson- Preliminary test for antibacterial activity The anti- ance (SPR) bands are influenced by the size, shape, microbial activity of AgNP was evaluated against Escheri- morphology, composition, and dielectric environment chia coli by cup diffusion method. Approximately 10 of the synthesized nanoparticles (Kelly et al. 2003; colony-forming units (CFU) of the microorganism E. coli Stepanov 1997). Previous studies showed that spherical were inoculated on Luria broth (LB) agar plate, and then AgNP contribute to the absorption bands at around 400 different concentrations of AgNP (1, 2, 5, 10, 20, 50, 100, nm in the UV-visible spectra (Maiti et al. 2013; Barman and 200 μg/ml) were added to the well present in the LB et al. 2014). The SPR band due to AgNP was observed in agar plate. A reaction mixture containing no AgNP was −3 our case at around 410 nm (Figure 1) when 3 × 10 M put in the well in the LB plate and cultured under the silver nitrate solution was used. This strongly suggested same condition as the control test. All the LB plates were that AgNP were nearly spherical in shape and it was con- incubated at 37°C overnight. After incubation, the plates firmed by the transmission electron microscopy (TEM) were observed for the presence of a zone of inhibition. results. The antibacterial activity of AgNP was proved from the In this study, AgNP have been synthesized both in the zone of inhibition (Figure 3). At concentration 20 μg/ml presence and in the absence of a stabilizer and both an- and above, the AgNP showed a clear zone of inhibition. ionic and neutral surfactants were used one at a time. No zone of inhibition was found in the vehicle control Though soluble proteins and amino acids present in well (spot in the middle of the plate) which suggested that L. esculentum extract were expected to act as stabilizer for the antimicrobial activity was specifically due to AgNP. Maiti et al. Journal of Analytical Science and Technology 2014, 5:40 Page 3 of 7 http://www.jast-journal.com/content/5/1/40 Figure 1 UV-Vis spectra and digital photographic images of AgNP. (A) UV-Vis spectra of AgNP: spectrum 1A-A with surfactant SDS, spectrum 1A-B with surfactant TX-100, spectrum 1A-C without any surfactant, and spectrum 1A-D is for pure Lycopersicon esculentum extract. (B) UV-Vis spectra of AgNP at different compositions of Lycopersicon esculentum extract. (C) UV-Vis spectra of AgNP with varying concentrations of silver −3 −2 −2 −3 nitrate (a) at 3 × 10 M, (b) at 1 × 10 M, and (c) at 5 × 10 M using 1:1 extract composition and 3 × 10 M SDS solution in each case. (D) Digital photographic images of AgNP produced from different concentrations of silver nitrate. Evaluation of antibacterial effectiveness using minimum concentrations of AgNP (0.2,0.5,1,2,5,10,20,50, inhibitory concentration method The antimicrobial and 100 μg/ml). The cultures were incubated at 37°C activity of AgNP was evaluated using the MIC method. at 250 rpm. Bacterial concentrations were determined The antimicrobial effectiveness was determined against by measuring optical density (OD) at 600 nm (0.1 OD600 6 8 the bacterial concentration of 10 CFU/ml with different corresponding to 10 cells per milliliter). With the in- crease of concentration of nanoparticles, the final bacterial concentration decreased. When the concentration of AgNP was 50 μg/ml, growth of E. coli was completely inhibited, which indicated that the MIC of AgNP to E. coli was 50 μg/ml (Figure 4). Effect of AgNP on bacterial growth To determine the growth curve in the presence of silver nanoparticles, E. coli bacteria were grown in liquid LB medium till they Figure 2 TEM micrographs and SAED pattern of AgNP. TEM reached the log phase. Then they were diluted in fresh micrographs of AgNP synthesized from Lycopersicon esculentum LB liquid medium to optical density (OD600) 0.05, 0.1, extract (A, B). SAED pattern of AgNP synthesized from Lycopersicon and 0.2. AgNP solution was added into the cell culture esculentum extract (C). medium at different concentrations, and the culture was Maiti et al. Journal of Analytical Science and Technology 2014, 5:40 Page 4 of 7 http://www.jast-journal.com/content/5/1/40 AgNP concentration necessary to completely inhibit bacterial growth was also low. So silver nanoparticles produced by us will be suitable for preventing bacterial contamination. Discussion Chemical antimicrobial agents are increasingly becoming resistant to a wide spectrum of antibiotics. An alternative way to overcome the drug resistance of various microor- ganismsistherefore urgently needed.Agionsand silver salts have been used for decades (Silver and Phung 1996) as antimicrobial agents in various fields due to their growth- Figure 3 Antibacterial activity of AgNP. Antibacterial activity of inhibitory abilities against microorganisms. However, there AgNP having different concentrations: (A) 1, 2, 5, and 10 μg/ml and are some limitations in using Ag ions or Ag salts as anti- (B) 20, 50, 100, and 200 μg/ml, with 10 CFU of E. coli inoculated on microbial agents. Probable reasons include the interfering Luria broth agar plate. The ‘B’ spot in the middle of the agar plate is effects of salts. This type of limitation can be removed by for the blank test, having no AgNP. using silver in nano form. Due to the increase of the surface area in nano state, the contact area between Ag(0) and that incubated at 37°C and 250 rpm. Growth rates and bac- of the microorganism increases. To use AgNP against mi- terial concentrations were determined by measuring OD crobes in various fields, it is important and necessary to at 600 nm at different time points (Figure 5A,B,C). prepare AgNP in a green environment. In this study, we re- The slope of the bacterial growth curve continuously de- port a green method for the preparation of AgNP which is creased with increasing nanoparticle concentration. This environmentally benign and cost-effective. means that at low concentration of nanoparticles, the For the assessment of the antimicrobial effects of AgNP, growth of bacteria was delayed and at higher concentra- E. coli was used in our study. The effect was investigated tion, growth was completely inhibited. So it can be con- by growing E. coli on agar plates and in liquid LB medium, cluded that the nanoparticles are bacteriostatic at low supplemented with AgNP. The bacterial growth was com- concentration and bactericidal at high concentration. It pletely inhibited in the presence of AgNP on the LB agar was also clear from the graphs that the bacterial growth plate. The inhibition solely depended upon the AgNP con- was dependent on the initial number of cells present in centration. It showed a clear zone of inhibition at and the medium. It was observed that at lower initial OD, the above the concentration 20 μg/ml. Figure 4 Optical density vs concentration of AgNP. MIC assay 50 μg/ml. Maiti et al. Journal of Analytical Science and Technology 2014, 5:40 Page 5 of 7 http://www.jast-journal.com/content/5/1/40 Figure 5 Growth curves with initial OD 0.05 (A), 0.10 (B), and 0.20 (C). To study the antimicrobial effectiveness of AgNP, we nanoparticles, bacterial growth was delayed and growth treated a bacterial concentration at high CFU (10 /ml) was completely inhibited at higher concentrations. So it ap- with varying concentrations of AgNP from 0.2 to 100 pears that these particles are bacteriostatic at low concen- μg/ml. When the concentration of AgNP was increased, tration and bactericidal at high concentration. It is also the bacterial concentration was found to decrease. At clear from the graphs that the bacterial growth is concentration 50 μg/ml of AgNP, the growth of E. coli dependent on the initial number of cells present in the was completely inhibited, which indicated that the mini- medium. It was observed that at lower initial OD, the mum inhibitory concentration was 50 μg/ml (Figure 4). AgNP concentration necessary to completely inhibit bacter- Since high CFU are seldom found in real-life systems, it ial growth was also low. So it is confirmed that these nano- may be concluded that these AgNP have a biocidal effect particles may be used to prevent bacterial contamination. and effectiveness in delaying bacterial growth, findings The mechanism of the inhibitory effects of Ag ions on which may lead to valuable inventions in the future in microorganisms is partially known. It is reported that various fields like in antimicrobial systems as well as the positive charge on the silver ion is the reason for medical devices. antimicrobial activity as it can attract the negatively The slope of the bacterial growth curves (Figure 5A,B,C) charged cell membrane of microorganisms through the continuously decreased with increasing nanoparticle con- electrostatic interaction (Dibrov et al. 2002; Hamouda centration. This indicated that at low concentration of et al. 2000). Due to their unique size and greater surface Maiti et al. Journal of Analytical Science and Technology 2014, 5:40 Page 6 of 7 http://www.jast-journal.com/content/5/1/40 area, silver nanoparticles can easily reach the nuclear apparent from the zone of inhibition. At concentrations 20 content of bacteria (Chen et al. 2010; Chudasama et al. μg/ml and above, the AgNP showed a clear zone of inhib- 2009). A survey of the literature showed that the electro- ition and the MIC of AgNP to E. coli was 50 μg/ml. Growth static attraction between negatively charged bacterial rates and bacterial concentrations were determined by cells and positively charged nanoparticles was crucial for measuring OD at 600 nm at different time points. From the antibacterial activity (Stoimenov et al. 2000). The the slope of the bacterial growth curve, it has been con- AgNP used in this study, however, received negative cluded that the nanoparticles are bacteriostatic at low con- charge from SDS, an anionic surfactant, used during centration and bactericidal at high concentration. So these synthesis. The bacterium E. coli being gram-negative, the nanoparticles are believed to act as preventive for bacterial interaction with the negatively charged nanoparticles contamination. might have occurred through ‘pit’ formation in the cell wall of the bacteria (Sondi and Salopek-Sondi 2004) Competing interests The authors declare that they have no competing interests. which helped the permeability and resulted in cell death. Authors' contributions Experimental SM and DK carried out the experiments. SM, GB, and DK drafted the Materials manuscript. JKL and SKG guided the research and modified the manuscript. Silver nitrate and SDS, both of AR grade, were purchased All authors read and approved the final manuscript. from Sigma-Aldrich Chemical Ltd. (St Louis, MO, USA) Sodium hydroxide was purchased from Merck (Darmstadt, Acknowledgements We are thankful to Central Research Facility at IIT Kharagpur, India, for the Germany). Double-distilled de-ionized water was used in all HR-TEM measurements. experiments. Author details 1 2 Midnapore College, Midnapore, West Bengal, India. Department of Green synthesis of silver nanoparticles by L. esculentum Biotechnology, Indian Institute of Technology, Kharagpur, India. extract Silver nanoparticles were made according to the recipe de- Received: 26 July 2014 Accepted: 21 October 2014 scribed below. For this purpose, red tomato (L. esculentum) was collected from the local market and washed with References double-distilled de-ionized water. 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Photochem Photobiol A 161:27–34 doi:10.1186/s40543-014-0040-3 Cite this article as: Maiti et al.: Antimicrobial activities of silver nanoparticles synthesized from Lycopersicon esculentum extract. Journal of Analytical Science and Technology 2014 5:40. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com
Journal
"Journal of Analytical Science and Technology" – Springer Journals
Published: Dec 1, 2014
Keywords: Analytical Chemistry; Characterization and Evaluation of Materials; Monitoring/Environmental Analysis