Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 416–420

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Facile synthesis of silver chloride nanoparticles using marine alga and its antibacterial efficacy T. Stalin Dhas, V. Ganesh Kumar ⇑, V. Karthick, K. Jini Angel, K. Govindaraju Nanoscience Division, Centre for Ocean Research, Sathyabama University, Chennai 600119, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Silver chloride nanoparticles

(AgClNPs) synthesized successfully using Sargassum plagiophyllum.  Powder diffraction analysis revealed the particle to have silver and silver chloride facets.  Synthesized particles exhibit excellent antibacterial activity revealed using SEM and fluorescence microscopic analysis.

SEM images of E. coli (A) Control and (B) Treated A

500 nm



500 nm


Silver Chloride nanoparticles

Fluorescence microscopic image of (A) Live and (B) Dead cells of E. coli

a r t i c l e

i n f o

Article history: Received 28 August 2013 Received in revised form 1 October 2013 Accepted 9 October 2013 Available online 19 October 2013 Keywords: Sargassum plagiophyllum Silver chloride nanoparticles Electron microscopy Antibacterial activity Fluorescence microscopy

a b s t r a c t Exploitation of advancements in antimicrobial agent synthesis assisted by nanomaterials has received considerable attention in the recent years. Based on this, an eco-friendly approach for the synthesis of silver chloride nanoparticles (AgClNPs) using aqueous extract of Sargassum plagiophyllum is emphasized. UV–vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), high resolution transmission electron microscopy (HR-TEM), field emission scanning electron microscopy (FESEM) were used to characterize the formation of AgClNPs. X-ray diffraction (XRD) patterns clearly illustrate the presence of AgClNPs. The synthesized AgClNPs were tested for its antibacterial activity and it was found to cause considerable amount of deterioration to bacterial cells, when examined using electron microscope and cell viability analysis. Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved.

Introduction Silver nanomaterials are antimicrobial agents and more effective [1], due to their large surface area and high reactivity compared to a bulk solid, nanosized metal particles which exhibit excellent physical, chemical and biological properties [2,3]. There are several physical, chemical methods for synthesis of noble metal nanomaterials [4], however the toxicity issues of the nanoparticles synthesized through these methods render them unsuitable for ⇑ Corresponding author. Tel./fax: +91 44 24500646. E-mail address: [email protected] (V. Ganesh Kumar).

biomedical applications, hence biosynthesis protocols are preferred for noble metals [5–12]. Silver in the form of nanoparticles could be more reactive, they may pass through cell membranes leading to the accumulation of intracellular nanoparticles which can cause malfunctioning of cells [13,14]. Silver ions have the capability to inhibit the bacterial multiplication, by binding and denaturing bacterial DNA, thus affecting the ribosomal subunit protein and some enzymes important for bacterial cell growth by penetrating the cells [15–17]. Among silver associated materials, silver chloride is more important due to its applications in wound healing products, antimicrobial agent in deodorants and a long term preservative of drinking water in

1386-1425/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.10.044


T. Stalin Dhas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 416–420

water tanks. Many protocols have been established for the synthesis of silver chloride nanoparticles [18–21], however utilization of marine resource in the synthesis is yet to be pursued. The present investigation involves Sargassum plagiophyllum mediated biosynthesis of silver chloride nanoparticles (AgClNPs) and its antibacterial activity against gram negative bacteria (Escherichia coli). S. plagiophyllum, an industrially important brown algae having anticancer and antioxidant properties [22] is used as a source for synthesis of AgClNPs. Materials and methods Preparation of seaweed extract S. plagiophyllum were collected along the coast of Mandapam (Lat. 09°17’N; Long. 79°08’E), Rameswaram, Tamilnadu, India. The freshly collected seaweed was washed with seawater followed by deionized water to remove other impurities. Collected samples were shade dried for 8–10 days and ground to powder and stored for further studies. The seaweed extract was prepared in a conical flask by taking 5 g of seaweed powder along with 50 mL of deionized water. It was placed in an orbital shaker for 24 h and filtered through whatman No. 1 filter paper to obtain the extract. Synthesis of silver chloride nanoparticles Silver nitrate (AgNO3) was purchased from Qualigens Fine Chemicals, India and used as received. For typical experiments, the seaweed extracts (50 lL, 100 lL, 150 lL, 200 lL, 250 lL, 300 lL, 350 lL, 400 lL, 450 lL and 500 lL) were mixed with 5 mL of 1 mM AgNO3 solution and it was placed in room temperature for 24 h.

Morphological changes of E. coli treated with silver chloride nanoparticles The antibacterial activity of the AgClNPs was analyzed in liquid medium, 20 lg/mL of AgClNPs was added into eppendorf tubes containing E. coli culture and incubated at 37 °C with shaking incubator at 150 rpm for 12 h. The morphological changes in the treated cells were examined using FESEM. Fluorescence microscopy analysis of E. coli treated with silver chloride nanoparticles The staining reagent was prepared by mixing of 0.05 mL of stock solution of 1% Acridine orange (HiMedia, India) and 5 mL of acetate buffer 0.2 M (pH 4.0) [23]. Slide with bacterial sample (E. coli) was dried at 50 °C, fixed in absolute methanol for 2 min and air dried. The slides were then covered with acridine orange (AO) staining reagent for 1 min, washed with tap water and air dried. The samples were then examined with epifluorescence microscope (Eclipse 80i, Nikon, Japan). Result and discussion The formation of AgClNPs was qualitatively confirmed by color change (yellowish brown) in 24 h. The color change is observed owing to collective oscillation of free conduction electrons induced by an interacting electromagnetic field [24]. Fig. 1a shows the UV– vis spectra of synthesized AgClNPs for varying concentration. A sharp intense peak at 417 nm was observed in 250 lL of extract with 5 mL AgNO3 solution. The chloride ions in the seaweed extracts were confirmed by EDAX (Fig 1b). The chloride ions convert into AgNO3 to form of AgClNPs. Thus, the chlorine content of



Characterization studies 50 µL

Antibacterial activity of silver chloride nanoparticles Agar diffusion test For antibacterial experiments, E. coli (ATCC 25922) were selected as the target organism. Luria Bertani (LB) broth and Muller Hinton Agar (MHA) were used as sources for culturing E. coli. Agar plates were prepared from autoclaved agar (MHA) solution. Three wells (6 mm diameter) were punched in the agar with a sterile cork borer and the bacterial culture was evenly spread using cotton swab over the agar. The test sample (10 lg and 20 lg of AgClNPs) and control sample (10 lL of seaweed extract) were dropped within the well and the zone of inhibition was measured after 18 h incubation at 37 °C.

100 µL

Absorbance (a.u.)

UV–vis spectral analysis was performed on a Shimadzu-1800 spectrophotometer. The biosynthesized AgClNPs solution was centrifuged at 10,000 rpm for 15 min and the suspension was redispersed in sterile distilled water. Finally, dried samples were palletized with KBr for FTIR measurements. The spectrum was recorded in the range of 4000–500 cm 1 using Bruker Optic GmbH Tensor 27. HR-TEM studies were prepared by drop coating AgClNPs onto carbon-coated HR-TEM grids performed by JEOL 3010 operated at an accelerating voltage of 300 kV. X-ray diffraction (XRD) measurement of the AuNPs was carried out using Rigaku smart lab instrument operated at a voltage of 40 kV and a current of 30 mA with Cu Ka1 radiations. Field emission scanning electron microscopy–energy dispersive X-ray analysis (FESEM–EDAX) was performed by SUPRA 55-CARL ZEISS, Germany.

150 µL


200 µL 250 µL 300 µL 350 µL 400 µL


450 µL 500 µL

0.0 300





Wavelength (nm)


Fig. 1. UV–vis spectra of S. plagiophyllum reduced silver chloride nanoparticles at various concentration (A) and EDAX profile of dried extract of S. plagiophyllum.


T. Stalin Dhas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 416–420

Fig. 2. FTIR spectra of silver chloride nanoparticles synthesized using S. plagiophyllum extracts.

extracts could be source for the formation of AgClNPs [25]. FTIR spectrum of the synthesized nanoparticles shows strong band at 3417 cm 1 attributes to the alcoholic/carboxylic groups present. The peak at 2927 cm 1 designates the asymmetric –CH bending thus indicating its role in reduction of silver ions. Further, the presence of peak at 1659 cm 1 confirms the amide I stretching group [26,27]. The role of [email protected] in the reduction of silver salt is evident by the peak at 1390 cm 1 (Fig. 2). HR-TEM image reveals that nanoparticles were spherical in shape and the size of the particles were around 18–42 nm (Supplementary data Fig. 1a–d) and the bio-components from the S. plagiophyllum extracts are seen in the pictures. Bio-organic materials bound to the nanoparticles surface which is responsible for the stability of nanoparticles [28]. FESEM micrograph shows AgClNPs which are spherical in shape and size of the nanoparticles in the range 21–48 nm (Supplementary data Fig. 2a), which are agrees with TEM results. In EDAX analysis two (silver and chloride) strong signal were observed (Supplementary data Fig. 2b). XRD of AgClNPs was analyzed with standard XRD files of AgClNPs which was published by Joint Committee on Powder



Intensity (a.u.)



AgCl (111)



100 Ag (111) (311) (222)



(420) (331)

0 20







2 theta (degree) Fig. 3. X-ray diffraction pattern showing peaks for silver and silver chloride facets.

Diffraction Standards (JCPDS file no. 85-1355). The intense peaks were raised at the 2h peak values of 27.68°, 32.13°, 44.34°, 46.02°, 54.58°, 57.23°, 67.15°, 74.11° and 76.38° which corresponds to (1 1 1), (2 0 0), (1 1 1), (2 2 0), (3 1 1), (2 2 2), (4 0 0), (3 3 1) and (4 2 0) respectively [19] confirming the formation of AgClNPs (Fig. 3). The antibacterial activity of AgClNPs investigated against E. coli and the inhibitory action of AgClNPs was found to be dose dependent. Increase in concentration of AgClNPs resulted in larger zone of inhibition. The diameter of inhibition was shown in Supplementary data Fig. 3. The improved antibacterial activity could be due to the amount of silver ions released from the nanoparticles which act as reservoirs for the same [29]. The antibacterial activity of AgClNPs was also investigated in culture grown in liquid medium. After 12 h incubation, the morphological changes of E. coli cells were examined using FESEM. Fig. 4a shows the surface of E. coli cells untreated with AgClNPs retained their smooth and original rod shape and appears to have regular morphology. In contrast, considerable morphological changes were observed in the bacterial cells treated with AgClNPs and distribution of AgClNPs on bacterial cell surfaces was observed (Fig. 4b and c). Silver has many possibilities to disturb biochemical processes, the formation of sparingly soluble silver salt (AgCl, Ag2S) adverse effect of silver ions due to interactions with thiol and amino groups of proteins, with nucleic acids and with cell membranes [30–32]. The possible mechanisms could be associated with interaction between nanoparticles and bacteria which lead to production of increased levels of reactive oxygen species (ROS), mostly hydroxyl radicals and singlet oxygen [33–36] and deposition of the nanoparticles on the surface of bacteria or the accumulation of periplasmic region which originates causing disruption of cellular function and disorganization of membranes [37,38]. Generally, acridine orange (AO) was used to determine the viability of bacterial cells [39,40] as AO has the tendency to distinguish viable and non-viable cells with its fluorescence. According to Strugger [41], viable cells fluorescence green whereas, nonviable cells fluorescence red [41]. Supplementary data Fig. 4a shows the fluorescence image of control cells indicating the viability of cells as evident by the green fluorescence. Fluorescence orange color indicates the dead cells as shows in Supplementary data Fig. 4b. Fluorescence shift to red-orange occurred in highly degraded cells, the shift was not obligatorily linked to viability when cells were killed by

T. Stalin Dhas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 416–420


Conclusion The present investigation emphasizes the role of AgClNPs in inhibiting the control bacterial pathogen E. coli. A novel protocol to synthesize AgClNPs using marine alga S. plagiophyllum has been successfully carried out and the antibacterial activity of AgClNPs was studied using fluorescence and electron microscopic analysis. Synthesis of AgClNPs and its antibacterial potential will be helpful in providing an insight in the development of viable antimicrobial agents in near future. Acknowledgments We thank DST – Nanomission, Government of India for its financial support for the project (SR/NM/NS-06/2009) and management of Sathyabama University, Chennai for its stanch support in research activities. SAIF, IIT Madras and Centre for Nanoscience and Nanotechnology, Sathyabama University is gratefully acknowledged for characterization studies. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.10.044. References

Fig. 4. Morphological analysis of E. coli using FESEM before (A) and after (B and C) treatment with silver chloride nanoparticles (20 lg/mL). Insert: figure shows individual bacterial cells before and after treatment.

various physical treatments [40]. To the best of our knowledge, this is the first report involving marine alga the synthesis of AgClNPs.

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Facile synthesis of silver chloride nanoparticles using marine alga and its antibacterial efficacy.

Exploitation of advancements in antimicrobial agent synthesis assisted by nanomaterials has received considerable attention in the recent years. Based...
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