Materials Science and Engineering C 47 (2015) 351–356
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Blue green alga mediated synthesis of gold nanoparticles and its antibacterial efficacy against Gram positive organisms K.S. Uma Suganya a, K. Govindaraju a,⁎, V. Ganesh Kumar a, T. Stalin Dhas a, V. Karthick a, G. Singaravelu b, M. Elanchezhiyan c a b c
Centre for Ocean Research, Sathyabama University, Chennai 600 119, India Nanoscience Division, Department of Zoology, Thiruvalluvar University, Vellore 632115, India Department of Microbiology, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai 600113, India
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Article history: Received 3 July 2014 Received in revised form 9 September 2014 Accepted 11 November 2014 Available online 13 November 2014 Keywords: Spirulina platensis Biofunctionalization Gold nanoparticles Gram positive organisms Antibacterial activity
a b s t r a c t Biofunctionalized gold nanoparticles (AuNPs) play an important role in design and development of nanomedicine. Synthesis of AuNPs from biogenic materials is environmentally benign and possesses high bacterial inhibition and bactericidal properties. In the present study, blue green alga Spirulina platensis protein mediated synthesis of AuNPs and its antibacterial activity against Gram positive bacteria is discussed. AuNPs were characterized using Ultraviolet–visible (UV–vis) spectroscopy, Fluorescence spectroscopy, Fourier Transform-Infrared (FTIR) spectroscopy, Raman spectroscopy, High Resolution-Transmission Electron Microscopy (HR-TEM) and Energy Dispersive X-ray analysis (EDAX). Stable, well defined AuNPs of smaller and uniform shape with an average size of ~ 5 nm were obtained. The antibacterial efficacy of protein functionalized AuNPs were tested against Gram positive organisms Bacillus subtilis and Staphylococcus aureus. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nanoscience offers unique approaches to influence a wide variety of biological and medical processes and is believed to have a great impact on biology and medicine. Interestingly, nanoparticles possess size and shape dependent properties [1] and its preparation remains a major obstruction in research even after the establishment of different protocols. On tuning the size and shape of nanoparticles, its intrinsic properties can be controlled for potential applications in different fields of science [2]. Recently, nanomaterials have gained more importance due to their potential applications in diagnostic imaging, biosensing, cancer therapeutics and targeted drug delivery [3]. The properties exhibited by gold nanoparticles (AuNPs) depend on their morphology and dimensions [4,5] and hence, controlling their size and shape remains the foremost need. In general, size and shape controlled AuNPs are synthesized chemically in the presence of reducing agents like trisodium citrate, sodium borohydride and some thiol functionalized agents which may hamper the surface modifications and functionalization of agents for particular applications [6,7]. Interaction of AuNPs with microorganisms to carry out a broad range of antibacterial activities is enhanced by their small size and high surface to volume ratio [8]. Nanoparticles alter the permeability of
⁎ Corresponding author. E-mail address: [email protected] (K. Govindaraju).
http://dx.doi.org/10.1016/j.msec.2014.11.043 0928-4931/© 2014 Elsevier B.V. All rights reserved.
bacterial cell membrane causing pits and gaps; suppress the activity of respiratory chain enzymes and finally leads to cell death [9,10]. Functionalized AuNPs with doxorubicin drug was efficient to facilitate better transport and delivery of loaded drug in human system [11]. Herein, S. platensis protein functionalized AuNPs serves as a potential antibacterial agent against Gram positive bacteria. Freshwater blue green alga S. platensis was selected for the study due to its various properties such as antioxidant, antiviral, and anticancer effects [12–14]. Reports suggest that the aqueous extract of S. platensis has inhibitory action on viral replication in human T-cells, peripheral blood mononuclear cells and Langerhan cells [15]. Synthesis of AuNPs with stringent control over size and shape is distinct from earlier studies on biological synthesis of Au, Ag and Au/Ag bimetallic nanoparticles from the blue green algae S. platensis [16]. Emerging Multi Drug Resistance (MDR) in bacteria has raised a necessity to develop novel antibacterial agents against Gram positive and Gram negative organisms. Gram negative bacteria possess a thin bacterial cell wall which is more susceptible to the antibacterial action of nanoparticles when compared to Gram positive organisms [17]. In contrast, Gram positive organisms possess a thick mesh like peptidoglycan layer of cell wall which shows greater resistance to various antibacterial agents. Gram positive organisms in particular, Bacillus subtilis and Staphylococcus aureus responsible for various pathogenic diseases require strong inhibitory agents for its control. In the present study, synthesis of protein protected AuNPs and its antibacterial efficacy against Gram positive organisms B. subtilis and S. aureus have been discussed.
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2. Experimental 2.1. Materials S. platensis was collected from a culture pond at Thandalam (Latitude — 12.99° N, Longitude — 80.117° E) near Sriperumbudur, Tamilnadu, India. Chloroauric acid (HAuCl4·3H2O) was purchased from Loba Chemie and sodium hydroxide (NaOH) was purchased from Sd-Fine Chemicals, India. Mueller Hinton agar (MHA) and Luria Bertani (LB) broth were obtained from Himedia Laboratories, Mumbai. All other chemicals were of analytical grade and used as received. Antibacterial efficacy of AuNPs was studied in B. subtilis (ATCC 6633) and S. aureus (ATCC 29213). 2.2. Extraction of protein For protein extraction, 1 g of dry S. platensis powder was mixed with 10 mL of millipore water and ground using mortar and pestle. The mixture was centrifuged at 5000 rpm for 10 min. The supernatant was used for synthesis of AuNPs and the amount of protein present was quantitatively estimated by Lowry's method [18]. 2.3. Synthesis of AuNPs For synthesis of AuNPs, S. platensis protein extract was added to 10 mM HAuCl4·3H2O solution in a ratio of 1:1 (10 mL:10 mL) followed by the addition of 1 N NaOH under stirring condition. On addition of 1 N NaOH, there was an immediate color change from green to greenish yellow and was kept under constant stirring for 3 h and incubated at room temperature for 48 h. The appearance of ruby red color indicated the formation of AuNPs. 2.4. Physico-chemical characterization of AuNPs Formation of AuNPs was confirmed by measuring the Ultraviolet– visible spectra (UV–vis) of the solution after diluting a small aliquot (0.2 mL) of the sample to 10 fold. UV–vis spectra were recorded using UV–vis spectrophotometer (Shimadzu UV-1800) in the range of 300–700 nm. Further, stability studies were performed by incubating AuNPs at different temperatures (4 °C, 15 °C, 25 °C, 60 °C and 80 °C) and the identity was measured by recording the absorbance spectrum using UV–vis spectrophotometer. Fluorescence spectroscopy of AuNPs was recorded using Jobin Yvon Fluorimeter to investigate the emission properties of AuNPs. The nature of ligand protection of synthesized AuNPs was studied using Fourier Transform Infrared spectroscopy (FTIR). The AuNPs were centrifuged at 2000 rpm for 15 min and the resulting pellet was washed with millipore water to remove free proteins. The purified pellet was lyophilized to obtain dry powder and they were pelletized further using Potassium Bromide (KBr). FTIR spectra were recorded usingThermo Nicolet Avatar 300 spectrometer in the range of 4000–500 cm−1. Fourier Transform-Raman (FT-Raman) spectroscopy was performed to investigate the molecules involved in the reduction process. Sample preparation was similar to FTIR. AuNPs were analyzed using a Witech GmbH confocal Raman spectrometer equipped with a 514.5 nm Argon ion laser in the range 5000–500 cm−1. Energy Dispersive X-ray analysis (EDAX) of AuNPs was carried out using HITACHI-S3400N. High Resolution-Transmission Electron Microcopy (HR-TEM) was performed using JEOL 3010 equipment operating at an accelerating voltage of 300 kV with an ultra high resolution pole piece. 2.5. Antibacterial studies 2.5.1. Well diffusion assay Mueller Hinton agar (MHA) plates were prepared to evaluate the zone of inhibition (ZOI) against two bacterial species B. subtilis and S. aureus. Wells of 5 mm diameter were made on culture plates using
gel puncture and different concentrations of protein protected AuNPs (50, 100, 150 and 200 μg/mL) were added and incubated at 37 °C for 24 h. After incubation, the zone was measured using a zone reader. The antibiotic (Vancomycin—30 μg/mL) was kept as positive control and S. platensis protein extract (200 μg/mL) was kept as negative control. The tests were performed in triplicate to avoid error. 2.5.2. Bactericidal studies of AuNPs To estimate the bactericidal activity of AuNPs by Colony Forming Units (CFU), serial dilution was performed for the two bacterial species to bring it to a concentration of 106 cells/mL. The number of cells was confirmed with 0.5 McFarland standards [19]. The bactericidal activity of AuNPs was carried out by standard CFU protocol [20]. Approximately, 106 bacterial cells/mL of the individual species were treated with different concentrations of AuNPs (10, 50, 100, 150 and 200 μg/mL) on LB agar plates. LB agar plates having 106 bacterial cells/mL and S. platensis protein (200 μg/mL) cultured under similar conditions were used as control. All plates were incubated at 37 °C for 24 h. After 24 h, the colonies were counted in a colony counter and the assays were performed in triplicate. 2.5.3. Growth kinetics study The bacterial growth kinetics was studied to determine the growth curve in the presence of AuNPs. Approximately, 1 mL of 106 cells of individual bacterial species was treated with different concentrations (10, 50, 100, 150 and 200 μg/mL) of AuNPs along with a protein treated (200 μg/mL) group. Control plates were free of protein and protein protected AuNPs and growth rates were determined by measuring the optical density (OD) at 600 nm at an interval of 2 h. 2.5.4. Determination of morphology changes using TEM studies Approximately, 1 mL of 106 cells of each bacterial species was mixed with 200 μg of protein protected AuNPs in a separate sterilized vial, shaken well and incubated for 1 h at 37 °C. The suspension was then centrifuged at 12,000 rpm and the pellet was washed thrice with millipore water to remove excess and unbound particles. TEM samples were prepared by standard procedures for fixing and embedding biological samples [21]. Ultrathin sections (b 100 nm) of the processed sample were cut with a diamond knife of ultramicrotome and placed on a copper grid and stained with uranyl acetate and Reynold's solution. Finally, the sections in the grid were washed with millipore water and the samples were dried in a dessicator overnight and examined using TEM (Philips 201C). 3. Results and discussion 3.1. Physico-chemical characterization of AuNPs The formation of AuNPs was evident with the appearance of ruby red color after 48 h. The formation of AuNPs is due to the reduction of Au3+ ions of chloroauric acid to Au0 by S. platensis protein. The appearance of ruby red color of AuNPs can be attributed to the collective oscillation of electrons induced by the interacting electromagnetic field in metallic nanoparticles [22] and formation of AuNPs was confirmed using UV–vis absorption spectra (Fig. 1). Three distinct peaks were observed at 685 nm, 524 nm and 385 nm for AuNPs synthesized using S. platensis protein which showed excitation maximum at 620 nm. These excitations are attributed to the electronic transitions and the peaks in visible region at 685 nm and 620 nm may be due to HOMO (High Occupied Molecular Orbital)–LUMO (Low Unoccupied Molecular Orbital) intraband transitions of the nanoparticles, the peak at 524 nm is attributed to intraband transition (HOMO-sp) and the peak at 385 nm is due to interband transition between LUMO and d-orbitals [23,24]. Intraband transition of the nanoparticles which comes at 680 nm is in good agreement with the gold nanoclusters synthesized so far [25].
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Fig. 1. UV–vis spectra of (A) S. platensis protein and (B) S. platensis protein protected AuNPs.
Stability of AuNPs is important for application in pharmacological and biomedical studies. Hence, the stability of protein protected AuNPs was evaluated by monitoring the plasmon wavelength (λmax) at different temperatures (4, 15, 25, 60 and 80 °C). From UV–vis spectra, it is evident that AuNPs subjected to different temperature ranges from 4 to 60 °C were stable and the stability of AuNPs is affected at 80 °C as shown in the spectrum (Supplementary data Fig. 1). Green synthesis of AuNPs using S. platensis protein offers stability in aqueous media for over two months. Based on the result, it can be confirmed that S. platensis has the ability to stabilize AuNPs under eco-friendly conditions without the intervention of any other chemicals. From fluorescence spectra (Supplementary data Fig. 2), it is evident that there is emission for all the absorption peaks observed and although there are three excitation peaks, emission maximum was found at 763 nm. The emission maximum around 763 nm for AuNPs exhibits a red shift of 78 nm in the NIR region from the excitation maximum at 685 nm [26].
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FTIR spectra recorded for both S. platensis protein and S. platensis protein mediated AuNPs (Fig. 2A & B) show absorption bands at 3317 cm−1, 3199 cm−1, 1664 cm−1, 1401 cm−1, and 1117 cm−1 and 3425 cm−1, 2942 cm− 1, 2350 cm− 1, 1651 cm−1, 1405 cm−1, and 1112 cm−1 respectively. On comparing, the band observed at 3317 cm−1 and 3199 cm−1 are attributed to the –NH stretching vibration of secondary amines (protein) and –OH (hydroxyl) functional groups present in protein. The bands in the S. platensis protein can be assigned as 1664 cm−1 (O = C–NH amide of protein), 1401 cm−1 and 1117 cm−1 (–COO−, carboxylate group) respectively. From both the spectra, it is found that the peak shift from higher wavelength (1664 cm−1) to lower wavelength (1651 cm−1) shows the reduction of Au3+ to Au0 ions and the peaks at 1405 and 1112 cm−1 shows the contribution of carboxylate group in the reduction [10]. The peaks at 3317 cm− 1 and 3199 cm− 1 that have merged to 3425 cm−1 as seen in AuNPs spectra indicates the reduction and capping of AuNPs [27] from which it can be concluded that the functional groups –COOH, – OH, and –NH would have caused the stabilization of AuNPs. Further, it was confirmed by Raman spectrum (Fig. 3A & B) that the –NH functional group of protein molecules are responsible for the stabilization of AuNPs. In S. platensis, there is a prominent peak centered at 3188 cm− 1 which has been split into two bands at 3233 cm− 1 and 3174 cm−1 that confirms the involvement of –NH functional group in the formation and stabilization of AuNPs. HR-TEM results gave a clear indication on size and shape of AuNPs. The nanoparticles were monodispersed and spherical shaped with size ranging from 2 to 8 nm having an average particle size of ~ 5 nm (Fig. 4A & B). The EDAX spectrum (Fig. 4C) showed strong signals from the gold metal atoms in the nanoparticles along with certain other elements like carbon, oxygen, sodium and potassium quantitatively [28]. From our earlier report on the direct addition of chloroauric acid to S. platensis, the AuNPs formed were spherical with a different size range (2–8 nm) and the reaction was completed in 5 days [16]. Recently, synthesis of protein encapsulated luminescent atomically precise AuNPs (clusters) having a very small sub-nanometer level core size [29] has also been reported. Similarly, in the present work, the synthesis of size controlled AuNPs (~5 nm) whose size also plays a key role in enhancing the antibacterial activity of protein protected AuNPs
Fig. 2. FT-IR spectra of (A) S. platensis protein and (B) S. platensis protein protected AuNPs.
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Fig. 3. Raman spectra of (A) S. platensis protein and (B) S. platensis protein protected AuNPs.
against Gram positive organisms by easily penetrating into the bacterial cell wall has been achieved. 3.2. Antibacterial efficacy of synthesized AuNPs Antibacterial activity of protein protected AuNPs has been investigated against Gram positive organisms B. subtilis and S. aureus. In the present study, Vancomycin was used as positive control and the diameter of ZOI for the test bacteria B. subtilis and S. aureus was measured. From Supplementary data Fig. 3, it is evident that the activity increases considerably with increase in concentration of AuNPs. However, no antibacterial activity was recorded for the protein extract. Further, the bactericidal activity of AuNPs was investigated by CFU method. Supplementary data Figs. 4 & 5 show the number of bacterial colonies formed in the presence of different concentrations of AuNPs treated when approximately 106 CFU were applied to each plate. The bactericidal activity of AuNPs against B. subtilis and S. aureus showed a maximum of 97.4% and 80.5% reduction in colonies with 150 μg/mL respectively. Almost 99% reduction in colonies was seen when supplemented with 200 μg/mL of AuNPs. The growth rate of Gram positive organisms was checked at different intervals (0 h, 2 h, 4 h, 6 h, 8 h, 10 h and 12 h) of time by treating with different concentrations (10, 50, 100, 150, 200 μg/mL) of AuNPs
(Fig. 5A & B). There was an increase in bacterial cell number in the control with time. When subjected to different concentrations of AuNPs, there was a substantial reduction in the bacterial cell number when compared with control. Interaction of AuNPs with Gram positive organisms (B. subtilis and S. aureus) was investigated using TEM studies. From TEM micrograph of B. subtilis, it is evident that AuNPs have caused damage to cells by distorting the bacterial cell wall causing pits at the exterior of cell and deformation of cell membrane thereby disturbing the bacterial functions like respiration, and permeability [30]. The antibacterial activity of Gram negative bacteria is highly feasible due to thin peptidoglycan layer which is easily penetrable [30,31]. It was also reported that easier permeability of nanoparticles can be achieved in Gram negative bacteria rather than Gram positive bacteria due to the cell wall nature [32]. Recently, biological synthesis of AuNPs using brown alga showed good activity against Gram negative bacteria [33]. Though, with thick mesh like peptidoglycan cell wall in Gram positive bacteria, the TEM images show a pronounced destruction of the bacterial cell membrane when treated with AuNPs (Fig. 6A & B). The TEM image of S. aureus show better antibacterial activity of AuNPs which is evident from irregular shaped cells and the visible damage caused to the cytoplasmic membrane. There are few studies which reported the synergistic effect of antibiotic coated AuNPs against Gram positive and Gram negative organisms [30].
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A
Bacterial cell number x 108
0.12 0.1 0.08 0.06 0.04 0.02 0 0h
2h
4h 6h 8h Time (hours)
10h
12h
Bacterial cell number x 108
0.16
B
0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0h
2h
4h
6h
8h
10h
12h
Time (hours) Fig. 5. Growth rate of 106 CFU bacterial species with different concentrations of AuNPs (A) B. subtilis and (B) S. aureus.
hinder the DNA replication by inhibiting its uncoiling and transcription finally leading to the lysis of cell [36–38].
4. Conclusions
Fig. 4. (A & B) HR-TEM and (C) EDAX images of protein protected AuNPs.
Similarly, the plant extract of Mentha piperita mediated synthesis of AuNPs has shown antibacterial activity against clinically isolated pathogens [32]. Many antibacterial studies using AuNPs were carried out on Gram negative bacteria [31] as they possess a thinner peptidoglycan layer (thickness 5–10 nm) surrounded by an outer phospholipidic membrane whereas Gram positive bacteria has a relatively thick and continuous cell wall (thickness 20–80 nm) consisting mainly of peptidoglycans [34]. Due to this, most of the Gram positive bacteria show greater resistance to drugs. The binding of AuNPs having large surface area for interaction could lead to enhanced antibacterial activity by penetrating the cell membrane of organisms [34]. AuNPs may initially get anchored to bacterial cell wall, interacts with the peptidoglycan layer thereby causing the breakage of bonds and enters inside the cell causing perforations at the exterior [35]. Nanoparticles, on its entry into the cytoplasmic matter of cell, move to the nucleus degrading the nuclear membrane and
In the present study, green synthesis of gold nanoparticles using S. platensis protein and its antibacterial activity against B. subtilis and S. aureus were discussed. The synthesized AuNPs were stable and spherical in shape having an average size of ~5 nm. The AuNPs were found to possess inhibitory action against B. subtilis and S. aureus. AuNPs managed to penetrate the thick peptidoglycan layer and caused damage to the cells as seen in electron microscopy. The results suggest that the protein functionalized AuNPs could be tuned for advanced medical applications in the treatment of infectious diseases caused due to Gram positive organisms.
Acknowledgments We thank Department of Science and Technology (DST-SERB), Government of India, for the financial support to carry out the work. We thank the management of Sathyabama University for its firm support to carry out research activities. We also thank SAIF and DST Nanoscience Unit, IITM, Chennai for characterization facilities.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.11.043.
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A
B
Fig. 6. Transmission electron micrograph of (A) B. subtilis (B) S. aureus cells treated with S. platensis protein protected AuNPs in agar medium for 1 h.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Blue green alga mediated synthesis of gold nanoparticles and its antibacterial efficacy against Gram positive organisms K.S. Uma Suganya a, K. Govindaraju a,⁎, V. Ganesh Kumar a, T. Stalin Dhas a, V. Karthick a, G. Singaravelu b, M. Elanchezhiyan c a b c
Centre for Ocean Research, Sathyabama University, Chennai 600 119, India Nanoscience Division, Department of Zoology, Thiruvalluvar University, Vellore 632115, India Department of Microbiology, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai 600113, India
a r t i c l e
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Article history: Received 3 July 2014 Received in revised form 9 September 2014 Accepted 11 November 2014 Available online 13 November 2014 Keywords: Spirulina platensis Biofunctionalization Gold nanoparticles Gram positive organisms Antibacterial activity
a b s t r a c t Biofunctionalized gold nanoparticles (AuNPs) play an important role in design and development of nanomedicine. Synthesis of AuNPs from biogenic materials is environmentally benign and possesses high bacterial inhibition and bactericidal properties. In the present study, blue green alga Spirulina platensis protein mediated synthesis of AuNPs and its antibacterial activity against Gram positive bacteria is discussed. AuNPs were characterized using Ultraviolet–visible (UV–vis) spectroscopy, Fluorescence spectroscopy, Fourier Transform-Infrared (FTIR) spectroscopy, Raman spectroscopy, High Resolution-Transmission Electron Microscopy (HR-TEM) and Energy Dispersive X-ray analysis (EDAX). Stable, well defined AuNPs of smaller and uniform shape with an average size of ~ 5 nm were obtained. The antibacterial efficacy of protein functionalized AuNPs were tested against Gram positive organisms Bacillus subtilis and Staphylococcus aureus. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nanoscience offers unique approaches to influence a wide variety of biological and medical processes and is believed to have a great impact on biology and medicine. Interestingly, nanoparticles possess size and shape dependent properties [1] and its preparation remains a major obstruction in research even after the establishment of different protocols. On tuning the size and shape of nanoparticles, its intrinsic properties can be controlled for potential applications in different fields of science [2]. Recently, nanomaterials have gained more importance due to their potential applications in diagnostic imaging, biosensing, cancer therapeutics and targeted drug delivery [3]. The properties exhibited by gold nanoparticles (AuNPs) depend on their morphology and dimensions [4,5] and hence, controlling their size and shape remains the foremost need. In general, size and shape controlled AuNPs are synthesized chemically in the presence of reducing agents like trisodium citrate, sodium borohydride and some thiol functionalized agents which may hamper the surface modifications and functionalization of agents for particular applications [6,7]. Interaction of AuNPs with microorganisms to carry out a broad range of antibacterial activities is enhanced by their small size and high surface to volume ratio [8]. Nanoparticles alter the permeability of
⁎ Corresponding author. E-mail address: [email protected] (K. Govindaraju).
http://dx.doi.org/10.1016/j.msec.2014.11.043 0928-4931/© 2014 Elsevier B.V. All rights reserved.
bacterial cell membrane causing pits and gaps; suppress the activity of respiratory chain enzymes and finally leads to cell death [9,10]. Functionalized AuNPs with doxorubicin drug was efficient to facilitate better transport and delivery of loaded drug in human system [11]. Herein, S. platensis protein functionalized AuNPs serves as a potential antibacterial agent against Gram positive bacteria. Freshwater blue green alga S. platensis was selected for the study due to its various properties such as antioxidant, antiviral, and anticancer effects [12–14]. Reports suggest that the aqueous extract of S. platensis has inhibitory action on viral replication in human T-cells, peripheral blood mononuclear cells and Langerhan cells [15]. Synthesis of AuNPs with stringent control over size and shape is distinct from earlier studies on biological synthesis of Au, Ag and Au/Ag bimetallic nanoparticles from the blue green algae S. platensis [16]. Emerging Multi Drug Resistance (MDR) in bacteria has raised a necessity to develop novel antibacterial agents against Gram positive and Gram negative organisms. Gram negative bacteria possess a thin bacterial cell wall which is more susceptible to the antibacterial action of nanoparticles when compared to Gram positive organisms [17]. In contrast, Gram positive organisms possess a thick mesh like peptidoglycan layer of cell wall which shows greater resistance to various antibacterial agents. Gram positive organisms in particular, Bacillus subtilis and Staphylococcus aureus responsible for various pathogenic diseases require strong inhibitory agents for its control. In the present study, synthesis of protein protected AuNPs and its antibacterial efficacy against Gram positive organisms B. subtilis and S. aureus have been discussed.
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2. Experimental 2.1. Materials S. platensis was collected from a culture pond at Thandalam (Latitude — 12.99° N, Longitude — 80.117° E) near Sriperumbudur, Tamilnadu, India. Chloroauric acid (HAuCl4·3H2O) was purchased from Loba Chemie and sodium hydroxide (NaOH) was purchased from Sd-Fine Chemicals, India. Mueller Hinton agar (MHA) and Luria Bertani (LB) broth were obtained from Himedia Laboratories, Mumbai. All other chemicals were of analytical grade and used as received. Antibacterial efficacy of AuNPs was studied in B. subtilis (ATCC 6633) and S. aureus (ATCC 29213). 2.2. Extraction of protein For protein extraction, 1 g of dry S. platensis powder was mixed with 10 mL of millipore water and ground using mortar and pestle. The mixture was centrifuged at 5000 rpm for 10 min. The supernatant was used for synthesis of AuNPs and the amount of protein present was quantitatively estimated by Lowry's method [18]. 2.3. Synthesis of AuNPs For synthesis of AuNPs, S. platensis protein extract was added to 10 mM HAuCl4·3H2O solution in a ratio of 1:1 (10 mL:10 mL) followed by the addition of 1 N NaOH under stirring condition. On addition of 1 N NaOH, there was an immediate color change from green to greenish yellow and was kept under constant stirring for 3 h and incubated at room temperature for 48 h. The appearance of ruby red color indicated the formation of AuNPs. 2.4. Physico-chemical characterization of AuNPs Formation of AuNPs was confirmed by measuring the Ultraviolet– visible spectra (UV–vis) of the solution after diluting a small aliquot (0.2 mL) of the sample to 10 fold. UV–vis spectra were recorded using UV–vis spectrophotometer (Shimadzu UV-1800) in the range of 300–700 nm. Further, stability studies were performed by incubating AuNPs at different temperatures (4 °C, 15 °C, 25 °C, 60 °C and 80 °C) and the identity was measured by recording the absorbance spectrum using UV–vis spectrophotometer. Fluorescence spectroscopy of AuNPs was recorded using Jobin Yvon Fluorimeter to investigate the emission properties of AuNPs. The nature of ligand protection of synthesized AuNPs was studied using Fourier Transform Infrared spectroscopy (FTIR). The AuNPs were centrifuged at 2000 rpm for 15 min and the resulting pellet was washed with millipore water to remove free proteins. The purified pellet was lyophilized to obtain dry powder and they were pelletized further using Potassium Bromide (KBr). FTIR spectra were recorded usingThermo Nicolet Avatar 300 spectrometer in the range of 4000–500 cm−1. Fourier Transform-Raman (FT-Raman) spectroscopy was performed to investigate the molecules involved in the reduction process. Sample preparation was similar to FTIR. AuNPs were analyzed using a Witech GmbH confocal Raman spectrometer equipped with a 514.5 nm Argon ion laser in the range 5000–500 cm−1. Energy Dispersive X-ray analysis (EDAX) of AuNPs was carried out using HITACHI-S3400N. High Resolution-Transmission Electron Microcopy (HR-TEM) was performed using JEOL 3010 equipment operating at an accelerating voltage of 300 kV with an ultra high resolution pole piece. 2.5. Antibacterial studies 2.5.1. Well diffusion assay Mueller Hinton agar (MHA) plates were prepared to evaluate the zone of inhibition (ZOI) against two bacterial species B. subtilis and S. aureus. Wells of 5 mm diameter were made on culture plates using
gel puncture and different concentrations of protein protected AuNPs (50, 100, 150 and 200 μg/mL) were added and incubated at 37 °C for 24 h. After incubation, the zone was measured using a zone reader. The antibiotic (Vancomycin—30 μg/mL) was kept as positive control and S. platensis protein extract (200 μg/mL) was kept as negative control. The tests were performed in triplicate to avoid error. 2.5.2. Bactericidal studies of AuNPs To estimate the bactericidal activity of AuNPs by Colony Forming Units (CFU), serial dilution was performed for the two bacterial species to bring it to a concentration of 106 cells/mL. The number of cells was confirmed with 0.5 McFarland standards [19]. The bactericidal activity of AuNPs was carried out by standard CFU protocol [20]. Approximately, 106 bacterial cells/mL of the individual species were treated with different concentrations of AuNPs (10, 50, 100, 150 and 200 μg/mL) on LB agar plates. LB agar plates having 106 bacterial cells/mL and S. platensis protein (200 μg/mL) cultured under similar conditions were used as control. All plates were incubated at 37 °C for 24 h. After 24 h, the colonies were counted in a colony counter and the assays were performed in triplicate. 2.5.3. Growth kinetics study The bacterial growth kinetics was studied to determine the growth curve in the presence of AuNPs. Approximately, 1 mL of 106 cells of individual bacterial species was treated with different concentrations (10, 50, 100, 150 and 200 μg/mL) of AuNPs along with a protein treated (200 μg/mL) group. Control plates were free of protein and protein protected AuNPs and growth rates were determined by measuring the optical density (OD) at 600 nm at an interval of 2 h. 2.5.4. Determination of morphology changes using TEM studies Approximately, 1 mL of 106 cells of each bacterial species was mixed with 200 μg of protein protected AuNPs in a separate sterilized vial, shaken well and incubated for 1 h at 37 °C. The suspension was then centrifuged at 12,000 rpm and the pellet was washed thrice with millipore water to remove excess and unbound particles. TEM samples were prepared by standard procedures for fixing and embedding biological samples [21]. Ultrathin sections (b 100 nm) of the processed sample were cut with a diamond knife of ultramicrotome and placed on a copper grid and stained with uranyl acetate and Reynold's solution. Finally, the sections in the grid were washed with millipore water and the samples were dried in a dessicator overnight and examined using TEM (Philips 201C). 3. Results and discussion 3.1. Physico-chemical characterization of AuNPs The formation of AuNPs was evident with the appearance of ruby red color after 48 h. The formation of AuNPs is due to the reduction of Au3+ ions of chloroauric acid to Au0 by S. platensis protein. The appearance of ruby red color of AuNPs can be attributed to the collective oscillation of electrons induced by the interacting electromagnetic field in metallic nanoparticles [22] and formation of AuNPs was confirmed using UV–vis absorption spectra (Fig. 1). Three distinct peaks were observed at 685 nm, 524 nm and 385 nm for AuNPs synthesized using S. platensis protein which showed excitation maximum at 620 nm. These excitations are attributed to the electronic transitions and the peaks in visible region at 685 nm and 620 nm may be due to HOMO (High Occupied Molecular Orbital)–LUMO (Low Unoccupied Molecular Orbital) intraband transitions of the nanoparticles, the peak at 524 nm is attributed to intraband transition (HOMO-sp) and the peak at 385 nm is due to interband transition between LUMO and d-orbitals [23,24]. Intraband transition of the nanoparticles which comes at 680 nm is in good agreement with the gold nanoclusters synthesized so far [25].
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Fig. 1. UV–vis spectra of (A) S. platensis protein and (B) S. platensis protein protected AuNPs.
Stability of AuNPs is important for application in pharmacological and biomedical studies. Hence, the stability of protein protected AuNPs was evaluated by monitoring the plasmon wavelength (λmax) at different temperatures (4, 15, 25, 60 and 80 °C). From UV–vis spectra, it is evident that AuNPs subjected to different temperature ranges from 4 to 60 °C were stable and the stability of AuNPs is affected at 80 °C as shown in the spectrum (Supplementary data Fig. 1). Green synthesis of AuNPs using S. platensis protein offers stability in aqueous media for over two months. Based on the result, it can be confirmed that S. platensis has the ability to stabilize AuNPs under eco-friendly conditions without the intervention of any other chemicals. From fluorescence spectra (Supplementary data Fig. 2), it is evident that there is emission for all the absorption peaks observed and although there are three excitation peaks, emission maximum was found at 763 nm. The emission maximum around 763 nm for AuNPs exhibits a red shift of 78 nm in the NIR region from the excitation maximum at 685 nm [26].
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FTIR spectra recorded for both S. platensis protein and S. platensis protein mediated AuNPs (Fig. 2A & B) show absorption bands at 3317 cm−1, 3199 cm−1, 1664 cm−1, 1401 cm−1, and 1117 cm−1 and 3425 cm−1, 2942 cm− 1, 2350 cm− 1, 1651 cm−1, 1405 cm−1, and 1112 cm−1 respectively. On comparing, the band observed at 3317 cm−1 and 3199 cm−1 are attributed to the –NH stretching vibration of secondary amines (protein) and –OH (hydroxyl) functional groups present in protein. The bands in the S. platensis protein can be assigned as 1664 cm−1 (O = C–NH amide of protein), 1401 cm−1 and 1117 cm−1 (–COO−, carboxylate group) respectively. From both the spectra, it is found that the peak shift from higher wavelength (1664 cm−1) to lower wavelength (1651 cm−1) shows the reduction of Au3+ to Au0 ions and the peaks at 1405 and 1112 cm−1 shows the contribution of carboxylate group in the reduction [10]. The peaks at 3317 cm− 1 and 3199 cm− 1 that have merged to 3425 cm−1 as seen in AuNPs spectra indicates the reduction and capping of AuNPs [27] from which it can be concluded that the functional groups –COOH, – OH, and –NH would have caused the stabilization of AuNPs. Further, it was confirmed by Raman spectrum (Fig. 3A & B) that the –NH functional group of protein molecules are responsible for the stabilization of AuNPs. In S. platensis, there is a prominent peak centered at 3188 cm− 1 which has been split into two bands at 3233 cm− 1 and 3174 cm−1 that confirms the involvement of –NH functional group in the formation and stabilization of AuNPs. HR-TEM results gave a clear indication on size and shape of AuNPs. The nanoparticles were monodispersed and spherical shaped with size ranging from 2 to 8 nm having an average particle size of ~ 5 nm (Fig. 4A & B). The EDAX spectrum (Fig. 4C) showed strong signals from the gold metal atoms in the nanoparticles along with certain other elements like carbon, oxygen, sodium and potassium quantitatively [28]. From our earlier report on the direct addition of chloroauric acid to S. platensis, the AuNPs formed were spherical with a different size range (2–8 nm) and the reaction was completed in 5 days [16]. Recently, synthesis of protein encapsulated luminescent atomically precise AuNPs (clusters) having a very small sub-nanometer level core size [29] has also been reported. Similarly, in the present work, the synthesis of size controlled AuNPs (~5 nm) whose size also plays a key role in enhancing the antibacterial activity of protein protected AuNPs
Fig. 2. FT-IR spectra of (A) S. platensis protein and (B) S. platensis protein protected AuNPs.
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Fig. 3. Raman spectra of (A) S. platensis protein and (B) S. platensis protein protected AuNPs.
against Gram positive organisms by easily penetrating into the bacterial cell wall has been achieved. 3.2. Antibacterial efficacy of synthesized AuNPs Antibacterial activity of protein protected AuNPs has been investigated against Gram positive organisms B. subtilis and S. aureus. In the present study, Vancomycin was used as positive control and the diameter of ZOI for the test bacteria B. subtilis and S. aureus was measured. From Supplementary data Fig. 3, it is evident that the activity increases considerably with increase in concentration of AuNPs. However, no antibacterial activity was recorded for the protein extract. Further, the bactericidal activity of AuNPs was investigated by CFU method. Supplementary data Figs. 4 & 5 show the number of bacterial colonies formed in the presence of different concentrations of AuNPs treated when approximately 106 CFU were applied to each plate. The bactericidal activity of AuNPs against B. subtilis and S. aureus showed a maximum of 97.4% and 80.5% reduction in colonies with 150 μg/mL respectively. Almost 99% reduction in colonies was seen when supplemented with 200 μg/mL of AuNPs. The growth rate of Gram positive organisms was checked at different intervals (0 h, 2 h, 4 h, 6 h, 8 h, 10 h and 12 h) of time by treating with different concentrations (10, 50, 100, 150, 200 μg/mL) of AuNPs
(Fig. 5A & B). There was an increase in bacterial cell number in the control with time. When subjected to different concentrations of AuNPs, there was a substantial reduction in the bacterial cell number when compared with control. Interaction of AuNPs with Gram positive organisms (B. subtilis and S. aureus) was investigated using TEM studies. From TEM micrograph of B. subtilis, it is evident that AuNPs have caused damage to cells by distorting the bacterial cell wall causing pits at the exterior of cell and deformation of cell membrane thereby disturbing the bacterial functions like respiration, and permeability [30]. The antibacterial activity of Gram negative bacteria is highly feasible due to thin peptidoglycan layer which is easily penetrable [30,31]. It was also reported that easier permeability of nanoparticles can be achieved in Gram negative bacteria rather than Gram positive bacteria due to the cell wall nature [32]. Recently, biological synthesis of AuNPs using brown alga showed good activity against Gram negative bacteria [33]. Though, with thick mesh like peptidoglycan cell wall in Gram positive bacteria, the TEM images show a pronounced destruction of the bacterial cell membrane when treated with AuNPs (Fig. 6A & B). The TEM image of S. aureus show better antibacterial activity of AuNPs which is evident from irregular shaped cells and the visible damage caused to the cytoplasmic membrane. There are few studies which reported the synergistic effect of antibiotic coated AuNPs against Gram positive and Gram negative organisms [30].
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A
Bacterial cell number x 108
0.12 0.1 0.08 0.06 0.04 0.02 0 0h
2h
4h 6h 8h Time (hours)
10h
12h
Bacterial cell number x 108
0.16
B
0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0h
2h
4h
6h
8h
10h
12h
Time (hours) Fig. 5. Growth rate of 106 CFU bacterial species with different concentrations of AuNPs (A) B. subtilis and (B) S. aureus.
hinder the DNA replication by inhibiting its uncoiling and transcription finally leading to the lysis of cell [36–38].
4. Conclusions
Fig. 4. (A & B) HR-TEM and (C) EDAX images of protein protected AuNPs.
Similarly, the plant extract of Mentha piperita mediated synthesis of AuNPs has shown antibacterial activity against clinically isolated pathogens [32]. Many antibacterial studies using AuNPs were carried out on Gram negative bacteria [31] as they possess a thinner peptidoglycan layer (thickness 5–10 nm) surrounded by an outer phospholipidic membrane whereas Gram positive bacteria has a relatively thick and continuous cell wall (thickness 20–80 nm) consisting mainly of peptidoglycans [34]. Due to this, most of the Gram positive bacteria show greater resistance to drugs. The binding of AuNPs having large surface area for interaction could lead to enhanced antibacterial activity by penetrating the cell membrane of organisms [34]. AuNPs may initially get anchored to bacterial cell wall, interacts with the peptidoglycan layer thereby causing the breakage of bonds and enters inside the cell causing perforations at the exterior [35]. Nanoparticles, on its entry into the cytoplasmic matter of cell, move to the nucleus degrading the nuclear membrane and
In the present study, green synthesis of gold nanoparticles using S. platensis protein and its antibacterial activity against B. subtilis and S. aureus were discussed. The synthesized AuNPs were stable and spherical in shape having an average size of ~5 nm. The AuNPs were found to possess inhibitory action against B. subtilis and S. aureus. AuNPs managed to penetrate the thick peptidoglycan layer and caused damage to the cells as seen in electron microscopy. The results suggest that the protein functionalized AuNPs could be tuned for advanced medical applications in the treatment of infectious diseases caused due to Gram positive organisms.
Acknowledgments We thank Department of Science and Technology (DST-SERB), Government of India, for the financial support to carry out the work. We thank the management of Sathyabama University for its firm support to carry out research activities. We also thank SAIF and DST Nanoscience Unit, IITM, Chennai for characterization facilities.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.11.043.
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A
B
Fig. 6. Transmission electron micrograph of (A) B. subtilis (B) S. aureus cells treated with S. platensis protein protected AuNPs in agar medium for 1 h.
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