Antimicrobial properties of metallic nanoparticles: a qualitative analysis

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ScienceDirect Materials Today: Proceedings 17 (2019) 155–160

www.materialstoday.com/proceedings

ICAMEES 18

Antimicrobial properties of metallic nanoparticles: a qualitative analysis Nidhi Dhull1 *, Nidhi2, Vinay Gupta1, Monika Tomar3 ** 1

Department of Physics and Astrophysics, University of Delhi, Delhi-110007 Amity Institute of Applied Sciences, Amity University, Uttar Pradesh-201303 3 Department of Physics, Miranda House, University of Delhi, Delhi-110007 Email – *[email protected], **[email protected]

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Abstract Nanoparticles exhibit unique physiochemical properties leading to their potential applications in the field of biotechnology, electronics and drug delivery. The synthesis of uniform nanosized particles with specific properties related to size, shape, and physical and chemical properties is of great interest for different applications. The bacterial resistance towards conventional antibiotics has increased over time and development of new antibacterial agents is required to fight the pathogenic bacteria. Metallic nanoparticles are safe, durable and heat resistance compared to the conventional antibacterial agents. In the present work, silver (Ag), gold (Au) and zinc oxide (ZnO) nanoparticles have been synthesized via polyol and precipitation methods. The nanoparticles have been characterized by UV-Visible Spectroscopy and X-ray Diffraction studies. The size and morphology of the synthesized nanoparticles has been studied using FESEM analysis. The antibacterial activity of these nanoparticles was investigated qualitatively against Escherichia coli (E. coli) bacteria as a model for a pathogenic gram-negative bacterium. Bacteriological tests were performed in Luria–Bertani (LB) liquid medium and on solid agar plates using UV-Visible spectroscopy. The nanoparticles show enhanced antibacterial activities and potential application towards realization of bactericides. © 2019 Elsevier Ltd. All rights reserved. Peer-review under responsibility of the scientific committee of the International Conference on Advanced Materials for Technological Applications – ICAM 18. Keywords: Silver; Gold; Zinc Oxide; nanoparticles; antimicrobial

1. Introduction Several kinds of bacterial infections have emerged around the globe causing chronic diseases and large-scale mortality. The preferred method of treatment so far is the use of antibiotics. However, the penetration in the 2214-7853© 2019 Elsevier Ltd. All rights reserved. Peer-review under responsibility of the scientific committee of the International Conference on Advanced Materials for Technological Applications – ICAM 18.

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bacterial cell wall mode of action of the antibiotics is such that it has led to appearance of drug-resistant bacterial strains. The excessive use of antibiotics has resulted in the emergence of super-bacteria which is resistant to about all kind of antibiotics [1]. In such a scenario, the antimicrobial properties of nanoparticles provide hopes to fight against lethal bacterial infections as they act by being in direct contact with the cell wall instead of penetrating through it, reducing the chances of bacterial resistance to a great extent [2] Nanotechnology has been a well-known research arena since a century ago and has delivered several kinds of materials in the nanoscale range. Nanoparticles form a broad category of materials that combine particulate materials having at least one dimension in the range 1100 nm. Nanoparticles of different materials have demonstrated antimicrobial actions against both Gram-negative and Gram-positive strains of bacteria [3]. Being effective bactericides, nanoparticles have found application in implantable devices, wound dressings and dental materials apart from antibiotic delivery [4]. The present work focuses on the qualitative analysis of antimicrobial properties of silver (Ag), gold (Au) and zinc oxide (ZnO) nanoparticles. A pathogenic strain of E. coli, a Gram-negative bacterium, has been chosen to demonstrate the antimicrobial action of the nanoparticles. Nanoparticles of all the three materials have been produced via chemical routes and characterized using X-Ray diffraction (XRD) and UV-Vis spectroscopy. Also, their morphologies and sizes have been analyzed using FESEM technique. It has been well established in the literature that changes in the optical spectra of microorganisms during the period of their growth reveal quantitative information [5]. Thus, in the present work, the antimicrobial properties have been successfully presented in the liquid media and also on solid agar plates using UV-Vis spectroscopy. 2. Experimental 2.1. Materials and Apparatus E. coli O157:H7 (ATCC #700728) was purchased from BCCM/LMG (Belgium-Europe). The bacterial culturing and surface plating were performed at Defence Institute of Physiological and Allied Research (DIPAS), a laboratory of the Defence Research and Development Organization (DRDO), India. Fresh colonies of E. coli were inoculated in 10 mL of broth (Luria Bertani (LB)) media and also on solid agar plates. The crystallographic structure of the synthesized nanoparticles was studied using Rigaku Ultima XRD. The spectroscopic analysis of the nanoparticles and the antimicrobial studies were performed using PerkinElmer Lambda 35 UV-Vis spectrophotometer. Zeiss FESEM has been employed to study the size and morphology of the synthesized nanoparticles. All other reagents; Zinc acetate dihydrate ((CH3COO)2Zn.2H2O from Titan Biotech), silver nitrate (AgNO3 from SRL Pvt. Ltd.), Chloroauric acid (HAuCl4.xH2O from Molychem), ethanol (Spectrochem), lithium hydroxide monohydrate (LiOH.H2O from Spectrochem), n-heptane (Spectrochem), ethylene glycol (Titan Biotech) and polyvinyl pyrrolidone (PVP from SRL Pvt. Ltd.) were used without further purification. 2.2. Synthesis of Nanoparticles The Ag and Au nanoparticles were synthesized via polyol method as reported elsewhere [6]. Briefly, the precursors of respective metals (AgNO3 for Ag and HAuCl4.H2O for Au) were dissolved in 20 mL of ethylene glycol separately, amounting to the concentration of 0.05 mM each. 0.5 mM PVP was supplemented to both the solutions

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under rigorous magnetic stirring. The solutions were irradiated with microwave radiations for about 1 minute at 800 W after transferring to a quartz beaker. The resulting solutions of yellow and pink color indicated the formation of Ag and Au nanoparticles respectively. ZnO nanoparticles were synthesized by using a wet chemical route, as announced by Spanhel and Anderson [7]. The process consists of two steps, (1) preparation of the precursor by dissolving 0.01 M of zinc acetate into 75 mL of boiling ethanol and (2) refluxed at 75 oC for 30 minutes, followed by hydrolyzing the precursor using 0.014 M of lithium hydroxide. A transparent ZnO sol was obtained after constant magnetic stirring at room temperature. The ZnO nanoparticles in the powder form are recovered through the precipitation of prepared ZnO sol. n-heptane in the volume ratio of 3:1 with the ZnO sol was used as the precipitation agent. This was followed by repeated washing of the precipitate with ethanol. The supernatant after the precipitation process was expelled by decantation and centrifugation (3000 rpm for 10 min). The ZnO nanoparticles were at last recovered by heating the precipitate for 2 hours at 80 °C. 3. Results and discussion 3.1. Structural and morphological characterization Figure 1 (a), (b) and (c) present the XRD spectrum of Ag, Au and ZnO nanoparticles respectively. The XRD peaks at around 37.9o, 43.5o and 64.2o in figure 1(a) can be attributed to the diffraction from (111), (200), and (220) planes of silver respectively (JCPDS file #04–0783). The well-defined peaks with (111) having maximum intensity indicates dominating FCC structure [8]. The crystalline nature of the Au nanoparticles was confirmed from figure 1 (b). Prominent Bragg reflections at around 38.3o, 44.5o and 64.4o may be assigned to the (111), (200) and (220) planes of Au respectively (JCPDS file #04-0784). The XRD spectra of the ZnO nanoparticles prepared by the precipitation method is shown in figure 1 (c). All the diffraction peaks may be marked to the hexagonal wurtzite structure of ZnO (JCPDS file #36–1451). Absence of extra unidentified peaks in the XRD patterns confirm the high purity of the synthesized nanoparticles. (a)

(b)

(c)

Figure 1: XRD spectra of (a) Ag (b) Au and (c) ZnO nanoparticles. The UV-Visible absorption spectrum of the synthesized nanoparticles is shown in figure 2 (a), (b) and (c). Strong absorption peaks at around 410 nm and 590 nm are observed for Ag (Figure 2(a)) and Au (Figure 2(b)) respectively

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[6]. The onset of fundamental absorption edge around 350 nm can be observed in figure 2 (c). It can be attributed to the growth of nanosized ZnO particles in the solution [7]. Thus, the absorption spectrum confirms the synthesis of nanoparticles of the respective metals. The morphologies of the synthesized nanoparticles as depicted by FESEM analysis are shown in the inset of figure 2. Inset of figure 2 (a) reveals well dispersed typical silver nano cubes having uniform size of around 80 nm. Inset of figure 2 (b) indicates that spherical shaped gold nanoparticles have an average size of about 70 nm. Polymeric ligands such as PVP protect the particles prepared in solution form agglomeration. However, when a small amount of PVP is used, agglomeration occurs as a result of incomplete covering of the gold nanoparticles with PVP. This may be the reason of agglomeration of Au nanoparticles as evident in the image. The size of ZnO nanoparticles turned out to be varying between 20 to 50 nm (Inset of figure 2 (c)). Also, it can be observed that the nanoparticles have been agglomerated into cluster which depicts the drawback of the present methodology in which entire deagglomeration after precipitation of the nanoparticles is not possible. (a)

(b)

(c)

Figure 2: Absorbance spectra of (a) Ag (b) Au and (c) ZnO nanoparticles Inset in each figure shows the FESEM image of the respective nanoparticle. 3.2. Antimicrobial Studies The agar plates and the LB media containing E. coli were supplemented with nanoparticles for studying their bacterial activity. In addition, one agar plate and LB media sample of E. coli without nanoparticles was also prepared as control sample. The plates and the liquid sample were incubated for 12 hours at 37 oC and the growth of cells was studied using UV-Vis spectroscopy. A significant increase in the intensity of absorbance and an efficient growth of E. coli colonies on agar plates in the absence of nanoparticles after incubation for 12 hours at 37 oC can be observed from the figure 3 (a) and the corresponding inset. With addition of the nanoparticles, there was no significant increase in the intensity of absorbance spectrum and also no significant cell growth could be observed in both the agar plates and the LB media even after 12 hours of incubation at 37 oC (Figure 3 (b), (c) and (d)). This is due to the antimicrobial nature of the nanoparticles which inhibited the further growth of cells. Thus, the Ag, Au and ZnO nanoparticles prove to be effective bactericides. The effect of parameters such as the particle size, shape and the time of action is yet to be quantified.

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(a)

(c)

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(b)

(d)

Figure 3: Absorbance spectra of (a) E. coli cells (b) Ag nanoparticles dispersed in E. coli culture (c) Au nanoparticles dispersed in E. coli culture (d) ZnO nanoparticles dispersed in E. coli culture just after culture preparation (0 hour) and after incubation at 37 oC for 12 hours. Inset in each figure represents the bacterial growth in agar plates with corresponding combination of nanoparticles and E. coli culture. 4. Conclusion Silver, gold and zinc oxide (Ag, Au and ZnO) nanoparticles were successfully synthesized using polyol and precipitation methods. The UV-Vis Spectroscopy and the XRD pattern confirmed the formation of respective nanoparticles. The size in the nano regime and a uniform morphology was revealed in the FESEM images. The antibacterial activity of these nanoparticles was successfully demonstrated against E. coli bacteria. Thus, the nanoparticles were shown to be effective bactericides. Acknowledgment The authors are thankful to DIPAS, DRDO, India for providing the bacterial culture facilities. Technical support from the University of Delhi is deeply acknowledged. One of the authors (Nidhi Dhull) gratefully acknowledge the Department of Science and Technology (DST), Ministry of Science and Technology for financial support.

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