Comparative study of antifungal activity of silver and gold nanoparticles synthesized by facile chemical approach

Accepted Manuscript Title: Comparative study of Antifungal Activity of Silver and Gold Nanoparticles Synthesized by Facile Chemical Approach Authors: Umme Thahira Khatoon, G.V.S. Nageswara Rao, Mantravadi Krishna Mohan, Almira Ramanaviciene, Arunas Ramanavicius PII: DOI: Reference:

S2213-3437(18)30441-X https://doi.org/10.1016/j.jece.2018.08.009 JECE 2556

To appear in: Received date: Revised date: Accepted date:

13-4-2018 22-7-2018 4-8-2018

Please cite this article as: Khatoon UT, Rao GVSN, Mohan MK, Ramanaviciene A, Ramanavicius A, Comparative study of Antifungal Activity of Silver and Gold Nanoparticles Synthesized by Facile Chemical Approach, Journal of Environmental Chemical Engineering (2018), https://doi.org/10.1016/j.jece.2018.08.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Full Title: Comparative study of Antifungal Activity of Silver and Gold Nanoparticles Synthesized by Facile Chemical Approach Short Title: Silver- and Gold-Nanoparticle Anti-fungal Activity Umme Thahira Khatoon1, 2, G.V.S. Nageswara Rao1, Mantravadi Krishna Mohan1, Almira Ramanaviciene3, Arunas Ramanavicius2,* Metallurgical and Materials Engineering Department, National Institute of Technology,

Warangal-506004, Telangana state, India; 2

Department of Physical Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko 24,

03225 Vilnius 6, Lithuania; 3

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NanoTechnas –Centre of Nanotechnology and Materials Science, Faculty of Chemistry, Vilnius

University, Naugarduko 24, 03225 Vilnius 6, Lithuania.

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First author email address: [email protected]

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Corresponding author: Tel.: +370-5-2336310; Tel.: +370-600-32332; Fax.: +370-5-2330987

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E-mail address: [email protected] (ArūnasRamanavičius)

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Graphical abstract

Highlights

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Synthesis of pure silver nanoparticles (Ag-NPs) and gold nanoparticles (Au-NPs) via facile chemical approach with new protocol. Pure phases of Ag and Au visible in XRD, TEM, SAED, EDAX. The Comparative analytic analysis compliments each other with respect to particle size and presence of element. Candida albicans and Sacharomyces cerevesiae fungal strains have shown more sensitivity towards Ag-NPs than Au-NPs.

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ABSTRACT

Colloidal solutions of silver nanoparticles (Ag-NPs) and gold nanoparticles (Au-NPs) have been proved to be suitable antimicrobial agents. In this study, Ag-NPs (yellow color) and Au-NPs (wine red color) have been produced by chemical reduction method using silver nitrate and chloroauric acid as metal precursors with sodium borohydride as reducing agent for Ag-NPs and tannic acid

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as reducing agent for Au-NPs and trisodium citrate as a stabilizing agent in both cases. UV-Visible spectroscopy graph shows the adsorption for Ag-NPs and Au-NPs to be 440 nm and 520 nm

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respectively. X-ray diffraction (XRD) analysis showed the highly crystalline nature of Ag-NPs

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and Au-NPs with (111), (200), (220), (311) orientations. TEM and SEM reveal the uniform

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spherical morphology of Ag-NPs and Au-NPs with the presence of elemental Ag and Au metal analyzed by EDAX. SAD confirms the orientation of Ag-NPs and Au-NPs with XRD analysis. The average particle size of Ag-NPs and Au-NPs was found to be 25 nm and 34 nm respectively

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investigated by DLS. The antifungal activity of Ag-NPs and Au-NPs on fungal cells, i.e., Candida

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Albicans and S.Cerevesiae showed diminished fungal growth by developing inhibition zones. The comparative studies demonstrate that Ag-NPs are more active than Au-NPs in diminishing fungus.

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Keywords: Silver nanoparticles; Gold nanoparticles; Chemical reduction; Fungal/Microbial inhibitory concentration; Zone of inhibition

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1. Introduction

Nanoparticles have great scientific interest because of their unique physical, chemical and mechanical properties, which are different from that of bulk metals. The physical-, chemical- and ‘bio'-properties of nanosized particles are strongly dependent on particle size and shape. When the size decreases from bulk scale to nanoscale, the properties of nanoparticles vary, and they behave

completely different from bulk-sized aliquots of their primary metals [1-2]. The large surface area to volume ratio of nanoparticles changes their color, appearance, reduces their incipient melting temperature [3], etc. Hence, the interest in the synthesis and application of nanoparticles with wellcontrolled size and shape has continuously grown over the years [4-5]. Among the metal-based nanoparticles, the Ag-NPs and Au-NPs synthesized by using chemical reduction methods have

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exhibited best monodispersity in size [2, 6]. In the recent past, the synthesis and characterization of Ag-NPs and Au-NPs have been received increasing attention [2, 6]. Due to their bio-affinity properties [2, 7], Ag-NPs and Au-NPs are being used in some biotechnology related applications [8-9]. Because of their potential resources, Ag-NPs and Au-NPs have become attractive in biomedical applications, microelectronics and also in optical, electronic and magnetic devices [10, 11].

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So far, there have been many studies on the synthesis of Ag-NPs and Au-NPs by biological,

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physical and chemical reduction methods [12]. Agnihotri et al., [13] have reported chemical reduction synthesis of Au-NPs over a wide size range (3.5 nm - 13 nm) with varying reaction

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conditions. Recently few studies have evaluated the size-dependent antimicrobial activity of Ag-

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NPs and Au-NPs [3, 7, 8, 13]. The surface plasmon bands of Ag-NPs and Au-NPs have been reported in the range of 350 nm - 800 nm [14] and 500 nm - 800 nm [15] respectively.

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A very high surface area to volume ratio of nanoparticles is attractive for advanced

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reactivity and catalytic action. This property also provides a tremendous driving force for antimicrobial studies. The antimicrobial activity of the Ag-NPs and Au-NPs play a crucial role in the inhibition of microbial growth in aqueous and robust media [16, 17]. For example, in medicine,

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a silver colloidal solution is applied to reduce infections as well as to prevent microbial colonization on prostheses, catheters, vascular grafts, dental materials, stainless steel materials and

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human skin. Antimicrobial susceptibility testing method is most often performed using the ‘disc diffusion method’ [15, 18]. Ag-NPs and Au-NPs are also employed in medicine as antimicrobial

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agents and drug or DNA delivery systems, carriers for various drugs, viz. anti-cancer drug like paclitaxel and as nano-sensors for sensing devices [19]. Au-NPs have been successfully used in rheumatoid arthritis therapy [20]. In cancer research, colloidal gold is used to target tumours and provide their detection using surface-enhanced raman spectroscopy (SERS), also used in chemistry, catalysis, sensors, nano-reactors, etc. [21]. The color of gold colloid is purple, orange,

red, violet, blue, brown and black and that of silver colloid is green and yellow with a change in the size of the particle [22].

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Currently, many methods have been reported for the synthesis of metal NPs by using chemical, physical, photochemical and biological routes. Each method has advantages and disadvantages with common problems being costs, scalability, particle sizes and size distribution. Among the existing methods, the chemical methods have been mostly used for production of NPs. Chemical methods provide an easy way to synthesize Ag-NPs and Au-NPs in solution [23]. The main drawback with the chemical and physical methods of silver nanoparticle formation is that they are extremely costly and also involve the use of toxic, hazardous chemicals and they contain potential environmental and biological stakes [24]. The way in which the nanoparticles blended must be taken care of by human and must be accessible at low evaluated rates for their compelling usage; hence, there is a requirement for an ecologically and financially doable approach to incorporate these nanoparticles [25]. In the present work, the preparation, characterization and size-specific antifungal efficacy

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of Ag-NPs and Au-NPs were studied. Synthesis of pure silver nanoparticles (Ag-NPs) and gold

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nanoparticles (Au-NPs) via facile chemical approach with new protocol has been presented, where;

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pure phases of Ag and Au elements are visible in XRD, TEM, SAED, EDAX. The antifungal characteristics of Ag-NPs and Au-NPs have been evaluated by directly exposing fungal cells to

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colloidal silver and gold solutions by using disc diffusion method [17]. The comparative analytic analysis compliments each other with respect to particle size and presence of element are observed.

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Furthermore, Candida albicans and Sacharomyces cerevesiae fungal strains have shown more

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sensitivity towards Ag-NPs than Au-NPs. 2. Materials and Methods

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2.1 Chemicals

Silver nitrate (AgNO3), chloroauric acid (HAuCl4), sodium borohydride (NaBH4), tri-sodium

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citrate (C6H5Na3O7.2H2O) and tannic acid (C76H52O46) are the chemicals used in the present investigation. Merck, Germany supplied chemicals. All the chemicals were used as received. Double distilled water was used for dilution of chemicals. Silver nitrate and chloroauric acid were

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used as metal precursors, and sodium borohydride, trisodium citrate, and tannic acid were used as reducing agents. Tri-sodium citrate also has an additional action as a stabilizing agent. 2.2 Preparation of Ag-NPs Silver nitrate (AgNO3), sodium borohydride (NaBH4) and tri-sodium citrate (Na3C6H5O7) solutions were prepared with respective weights in distilled water. NaBH4 (0.015% ~ 0.004 M) and Na3C6H5O7 (0.1032% ~ 0.004 M) were mixed in 100 ml water with magnetic stirrer. AgNO3

(0.0153% ~ 0.0009 M) was added through burette, and the color change was noticed. Initially, the color changes from colorless to yellow in first few drops. The color changes from yellow to light brown to light algae green and final color of the sample observed was yellow cum light algae green once the reaction was complete. The solution was under stirring for one hour with a magnetic stirrer at room temperature for ensuring a complete response. The perfect colloidal dispersed

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solution was prepared. The prepared silver colloid sample is shown in figure 1(a). 2.3 Preparation of Au-NPs

Chloroauric acid (HAuCl4), tannic acid (C76H52O46) and tri-sodium citrate (C6H5Na3O7.2H2O) solutions were prepared with respective weights in distilled water. Two solutions, viz. Solution A (1% HAuCl4.3H2O ~ 0.0294 M) and solution B (1% trisodium citrate ~ 0.0388 M + 1% tannic acid ~ 0.0059 M) were prepared in two different erlenmeyer flasks using distilled water. Initially,

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solutions A and B were heated till 65 C. Solution A was added slowly to solution B while stirring.

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Color changes to red while the process of heating and stirring was continued until the temperature reached to 95 C. The solution was cooled to room temperature, and the final color of the solution

3. Characterization techniques

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colloid is shown in figure 1(b).

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was observed to be wine red. The solution was then filtered and stored at 4 C. The prepared gold

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Surface Plasmon Resonance (SPR) of the sample was determined by using UV-Visible

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spectroscopy (Lambda25–PerkinElmer-USA). The morphology of Ag-NPs and Au-NPs was determined by using Scanning Electron Microscope (SEM–SU–78-Hitachi, Japan). Samples for

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SEM studies were prepared by placing a sol-drop of Ag-NPs and Au-NPs solution on a silicon chip and dried under atmospheric conditions. The average particle size of Ag-NPs and Au-NPs was investigated by Dynamic Light Scattering (DLS) (Malvern, Zetasizer Nano ZS-Germany).

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The crystal structure and phases were explored by X-ray diffraction (XRD) (Bruker D8 Advanced, Germany. Surface morphology was analyzed by using Transmission Electron Microscopy (TEM)

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(D2083, FAITECNAI G2, Netherlands). The chemical composition studied by Fourier transform infrared spectrum (FTIR, Perkin Elmer Spectrum 100, United Kingdom). 3.1 Antifungal activity of Ag and Au nanoparticles The antimicrobial susceptibility of synthesized Ag-NPs and Au-NPs were investigated by KirbyBauer diffusion method [16 - 18]. It is also known as radial/disc diffusion method. Disc diffusion sample sensitivity testing is a test which uses particular sample placed in holes within the nutrient

agar media of petri dish to test whether the particular fungus is susceptible to specific sample/material. The antifungal activity of Ag-NPs and Au-NPs were investigated against fungal strains, i.e., fungi (yeast) Saccharomyces cerevisiae, Candida Albicans using well diffusion technique and disc diffusion technique. 50 microliters each of fungi strain are spread with wells containing 134 microliters of Ag-NPs and Au-NPs. Simultaneously, the circular discs with 5 mm

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diameter made of filter papers were soaked in 50 microliters of Ag-NPs and Au-NPs for 10 minutes and also kept on fungus spread nutrient media petri dish. If the fungus is susceptible to a particular sample, a clean area free from fungus surrounds the small hole, where microbes are not capable of growing (called as Zone of Inhibition, ZoI). Depending on size and shape, the antifungal properties were determined; the plates were then incubated further at 37 C. The ZoIs were measured after 24 hours.

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4. Results and discussion

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The Ag-NPs exhibited yellow color (Fig. 1(a)), which is characteristic of the chemical reduction

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and formation of Ag-NPs that has been reported in the literature [11]. Whereas the prepared AuNPs exhibited wine red color (Fig. 1(b)), similar to that reported earlier by Jones et al., [26].

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4.1 X-ray diffraction studies (XRD)

The XRD pattern (Fig. 2(a)) of the Ag-NPs synthesized after reduction using sodium borohydride

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displayed four sharp peaks at the 2theta angles of 38.10, 44.40, 64.47 and 77.35 respectively. The

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patterns are in good agreement with four different JCPDS cards # 87-0718; 87-0719; 87-0717 and 03-0921 respectively [24 - 30, 42, 43]. Table 1 represents the details of the 2theta, d-spacing and

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hkl values obtained in Ag-NPs. According to the JCPDS card details, the Ag-NPs produced exhibit an FCC lattice structure with the cubic system. The XRD pattern (Fig. 2(b)) of the Au-NPs synthesized after reduction using tannic acid,

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and sodium borohydride displayed four sharp peaks at different 2theta angles of 38.91, 44.51, 64.75, and 77.72 respectively. The patterns are in good agreement with four different JCPDS card

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# 04-0784 [31, 44]. Table 1 represents the details of the 2theta, d-spacing and hkl values obtained in Au-NPs. According to the JCPDS card details, the Au-NPs produced exhibit an FCC lattice structure with the crystalline system. 4.2 Scanning Electron Microscopy (SEM) Studies The synthesized Ag-NPs and Au-NPs (figure 3(a) and figure 3 (b)) show SEM images of the dispersed Ag-NPs and Au-NPs along with the size of the particles. Figure 3(a) shows the spherical

morphology of Ag-NPs with a magnitude range between 9.97 nm - 12.3 nm. From figure 3 (b), it has been observed that the morphology of Au-NPs is spherical with a diameter of 13 nm - 19 nm, which is in agreement with that reported previously by Khademi et al., [32]. 4.3 Transmission electron microscopy (TEM) and selected area diffraction (SAD) pattern Studies

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TEM images of Ag-NPs (Fig. 4 (a-c)) and of Au-NPs (Fig. 5 (a-c)) exhibit a typical spherical morphology. The image also shows loosely bound particles created due to the effect of sonication treatment. The approximate particle diameter was found to be in the range of 10 nm - 20 nm for Ag-NPs and 12 nm - 23 nm Au-NPs. The SAD patterns (Fig. 4(d) & Fig. 5(d)) were provided with the diffraction rings along with the spots and the d-spacings indexed as an FCC crystalline structure of Ag-NPs and Au-NPs according to the JCPDS cards just below their respective TEM images.

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Based on the analysis, it is evident that the Ag-NPs and Au-NPs appear to be of excellent

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crystalline, as the SAD patterns show a strong presence of bright spots with their crystal orientations appearing within the diffraction rings. The diameter measurement of the pattern from

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the center towards the rings was also consistent with the d-spacing and coincided with the FCC

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phase of Ag-NPs and Au-NPs respectively [30, 33] as investigated using XRD. 4.4 UV-Vis spectroscopy based evaluation

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Ag-NPs and Au-NPs at nano-range exhibit an unusual optical phenomenon called Surface Plasmon

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Resonance (SPR), due to the cumulative oscillation of the conducting metal surface electrons in resonance with the non-particulate radiation. This property is largely governed and dependent upon

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the particle type, size, shape, and the local chemical ambiance. The UV-Vis spectroscopy revealed the formation of Ag-NPs and Au-NPs by exhibiting a

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typical surface plasmon absorption maxima at wavelengths of 410 nm for Ag-NPs and 520 nm for Au-NPs which are presented in Figures 6(a) and 6(b), are in agreement with that reported previously [34 - 41]. Based on the color transitions and UV-vis spectroscopy analysis, it has been

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observed that with the increase in particle size, the corresponding spectra display a redshift [27, 34, 35]. A spectrum of silver colloids using silver nitrate as silver precursor contains a strong plasmon band close to 396 nm, which confirms that silver ions (Ag+) were reduced to Ag° in the aqueous phase. It is also reported that the silver precursor had a substantial effect on the crystallinity of the silver nanoparticles [34]. 4.5 Dynamic Light Scattering (DLS) based investigations

The size distribution analyses of the Ag-NPs and Au-NPs were carried out using a DLS instrument. The study was conducted at 25 oC in a standard monodispersed medium maintained at a viscosity of 0.892 mPa.s. The graphs of the samples are shown in Fig. 7(a) & 7(b). The particle size analysis (PSA) shows that the average particle sizes observed around 24 nm and 35 nm for Ag-NPs and Au-NPs respectively. The remarkable results of current experimental

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investigations are shown in Table 2.

The obtained nanoparticle size distribution of Ag-NPs and Au-NPs using DLS is also in good agreement with the TEM results. 5. Antifungal study of Ag-NPs and Au-NPs

The zone of inhibition and rate of sample diffusion are used to estimate the microbe's sensitivity to the particular material which is present in the sample. In general, zones at which the microbes

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are not able to grow to correlate with Minimal Inhibitory Concentration (MIC) in the sample for

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investigated micro-organism [34]. Such information can be used to choose appropriate material or antibiotics to combat or kill microorganisms, which are inducing particular infection. The results

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of MIC (in mm) for Ag-NPs and Au-NPs against fun lower reactivity against fungus and lower

activity is observed as expected.

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concentration, compared to silver, the MIC of gold nanoparticles is not seen clearly, or much less

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Simultaneously, the circular discs with 5 mm diameter made of filter papers which are

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soaked in 50 microliters of Au-NPs and Ag-NPs for 10 minutes are also kept on fungus spread nutrient media petri dish. The final inoculum of fungal strain, i.e., Saccharomyces cerevisiae with

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optical density (OD) 0.1456 at 600 nm, and Candida albicans with OD 1.8927 at 600 nm was read. From the study/observation, the highest antifungal activity was observed with Ag-NPs

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when compared with the same 1 mM concentration Au-NPs. The measured radius of inhibition zones around each diffusion well with Ag-NPs and Au-NPs are shown in Table 3. The study shows

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that Ag-NPs exhibits better antifungal activity than Au-NPs with the presented concentrations. Also due to less particle size as declared by researchers, lower the particle size, higher is the zone of inhibition. Ag-NPs (24 nm) showed high Zone of Inhibition (ZoI) than Au-NPs (35 nm) which complements the property of nanoparticle with less size has more antifungal activity than micro size [36, 37]. ZoI presented in graphical form in Fig. 8(a) & 8(b) which show the domination of Ag-NPs over Au-NPs due to size and shape of the particle. In comparison, Jayabrata das et al., has

performed antifungal effect against candida albicans at 50 mg/l, showing 7 mm ZoI [38]. Also Suganthy et al. could able to gain ZoI 7 mm at 60 mg/l concentration of nanoparticles against candida albicans [39]. Hence, Ag-NPs and Au-NPs obtained from the presented experiments are more approachable.

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The extent of inhibition depends on the concentration of Ag-NPs as well as on the initial microbial population. This finding is supported by the research, which was conducted by Mlalila et al., [40] who reported that the interaction of these particles with intracellular substances from lysed cells caused their coagulation and the particles were thrown out of the liquid system. The mechanism of inhibitory action of gold ions and silver ions on micro-organism shows that the treatment of microbe by some metal ions induces DNA loses, its replication ability and expression

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of ribosomal sub-unit proteins, as well as other cellular proteins and enzymes essential for ATP (Adenosine Tri-Phosphate, coenzyme used as an energy carrier in the cells of all known organisms)

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production resulting in inactivation of living cell [34]. It has also been hypothesized that metal

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ions primarily affects the function of membrane-bound enzymes, in the respiratory chain.

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6. Conclusions

The simple and efficient method of synthesis of Au-NPs and Ag-NPs with well-defined size and antimicrobial activity was demonstrated. (i) It is an easy, deficient energy based and economic

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process. (ii) This process doesn't require extra chemicals besides needed from the protocol for

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reduction and processing. (iii) There is no need of using other sophisticated machines for synthesis. The XRD graph plotted on Ag-NPs and Au-NPs are in good agreement with standard

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JCPDS file reported by many researchers. The XRD pattern of the samples revealed the crystalline (FCC) lattice structure of elemental silver and gold.

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The 3D structure of spherical and uniform arrangement of Ag-NPs and Au-NPs are showed in TEM and SEM micrographs. A formal crystalline ring structure which is in good agreement with the miller indices of polydisperse silver and gold nanoparticles shown in the SAD pattern.

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Moreover, no elements are observed during EDAX scan, witnessing the purity of the synthesized sample. UV-Vis spectroscopy confirmed the presence of Ag-NPs and Au-NPs due to a high surface plasmon resonance (SPR) value observed at wavelengths of 400 nm - 460 nm and 500 nm - 550 nm respectively. DLS histogram of Ag and Au nanoparticles shows the average particle size to be

25 nm and 34 nm respectively. The smaller the size (Ag-NPs, 25 nm) the larger the ZoI which is proved and observed by the antifungal studies. Hence DLS and ZOI complement each other. Gold and silver have always been an excellent choice for antibacterial and antifungal activity and has been used for this purpose. Finally, this study shows that silver nanoparticles have good antifungal activity against Candida albicans and Saccharomyces cerevisiae than gold. This

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work, integrates nanotechnology and microbiology, leading to possible advances in the formulation of new types of fungicides. However, future studies on the biocidal influence of this nanomaterial on Candida albicans, Saccharomyces cerevisiae are necessary to fully evaluate its possible use as a new fungicidal material.

Conflict of Interest: The authors declare no conflict of Interest.

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Acknowledgements

The authors would like to acknowledge institute of nanotechnology, Vilnius University for

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providing microbiology lab for experiments and use of sophisticated equipment like XRD, SEM-

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EDX, and DLS. We would also like to acknowledge center for nanotechnology, HCU, Hyderabad

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for providing the use of TEM facility. AR and AR acknowledge support by Lithuanian Research council project No (SEN-21/2015).References:

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29. Dhanapal, K., Revathy, T. A., Dhanavel, S., Narayanan, V., & Stephen, A. (2017). Phosphorus role on the enhancement in catalytic activity of magnetic Ni-P alloy. Surfaces and Interfaces, 7, 58-68.

30. Enrichi, F., Armellini, C., Battaglin, G., Belluomo, F., Belmokhtar, S., Bouajaj, A., &

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Mardegan, M. (2016). Silver doping of silica-hafnia waveguides containing Tb 3+/Yb 3+

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rare earths for downconversion in PV solar cells. Optical Materials, 60, 264-269. 31. Konował, E., Modrzejewska-Sikorska, A., Motylenko, M., Klapiszewski, Ł., Wysokowski,

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M., Bazhenov, V. V., & Jesionowski, T. (2016). Functionalization of organically modified

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silica with gold nanoparticles in the presence of lignosulfonate. International journal of biological macromolecules, 85, 74-81.

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32. Srivastava, S. K., Guix, M., & Schmidt, O. G. (2015). Wastewater mediated activation of

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micromotors for efficient water cleaning. Nano letters, 16(1), 817-821. 33. Thong, N. M., Quang, D. T., Bui, N. H. T., Dao, D. Q., & Nam, P. C. (2015). Antioxidant properties of xanthones extracted from the pericarp of Garcinia mangostana (Mangosteen):

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a theoretical study. Chemical Physics Letters, 625, 30-35. 34. Jogee, P. S., Ingle, A. P., & Rai, M. (2017). Isolation and identification of toxigenic fungi

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from infected peanuts and efficacy of silver nanoparticles against them. Food Control, 71, 143-151.

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35. Zhang, D., Gökce, B., & Barcikowski, S. (2017). Laser synthesis and processing of colloids: fundamentals and applications. Chemical Reviews, 117(5), 3990-4103.

36. Nayak, D., Ashe, S., Rauta, P. R., Kumari, M., & Nayak, B. (2016). Bark extract mediated green synthesis of silver nanoparticles: evaluation of antimicrobial activity and antiproliferative response against osteosarcoma. Materials Science and Engineering: C, 58, 44-52.

37. Syafiuddin, A., Salim, M. R., Beng Hong Kueh, A., Hadibarata, T., & Nur, H. (2017). A review of silver nanoparticles: Research trends, global consumption, synthesis, properties, and future challenges. Journal of the Chinese Chemical Society, 64(7), 732-756. 38. Das, J., & Velusamy, P. (2013). Biogenic synthesis of antifungal silver nanoparticles using aqueous stem extract of banana. Nano Biomed Eng, 5(1), 34-38.

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39. Suganthy, N., Ramkumar, V. S., Pugazhendhi, A., Benelli, G., & Archunan, G. (2017). Biogenic synthesis of gold nanoparticles from Terminalia arjuna bark extract: assessment of safety aspects and neuroprotective potential via antioxidant, anticholinesterase, and antiamyloidogenic effects. Environmental Science and Pollution Research, 1-16.

40. Levard, C., Hotze, E. M., Lowry, G. V., & Brown Jr, G. E. (2012). Environmental transformations of silver nanoparticles: impact on stability and toxicity. Environ. Sci.

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Technol, 46(13), 6900-6914.

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41. Yu, Y., Chen, Z., He, S., Zhang, B., Li, X., & Yao, M. (2014). Direct electron transfer of glucose oxidase and biosensing for glucose based on PDDA-capped gold nanoparticle

Bioelectronics, 52, 147-152.

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graphene/multi-walled

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modified

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photooxidation in the gas phase. Catalysis Today, 230, 104-111.

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43. Zhu, C., Du, D., Eychmüller, A., & Lin, Y. (2015). Engineering ordered and nonordered porous noble metal nanostructures: synthesis, assembly, and their applications in electrochemistry. Chemical reviews, 115(16), 8896-8943.

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44. Singh, S., Ashfaq, M., Singh, R. K., Joshi, H. C., Srivastava, A., Sharma, A., & Verma, N. (2013). Preparation of surfactant-mediated silver and copper nanoparticles dispersed in

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hierarchical

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biotechnology, 30(6), 656-665.

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List of Figures:

Figure 1. (a) Silver colloid (Ag-NPs) prepared using sodium borohydride as a reducing agent, (b) Gold colloid (Au-NPs) prepared using tannic acid as a reducing agent. Figure 2. (a) X-ray diffractogram of Ag-NPs synthesized using sodium borohydride, (b) Xray diffractogram of Au-NPs synthesized using tannic acid and sodium borohydride.

Figure 3. (a) SEM monograph of Ag-NPs (size < 15nm) synthesized using sodium borohydride as reducing agent, (b) SEM monograph of Au-NPs (size < 20nm) synthesized using tannic acid and sodium borohydride as reducing agent. (c) Elemental composition of AgNPs using energy dispersive X-ray analysis (EDAX), (d) Elemental composition of Au-NPs using energy dispersive X-ray analysis (EDAX).

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Figure 4. (a-d) Transmission electron microscope and SAD patterns images of Ag-NPs (a - 50 nm magnification), (b - 20 nm magnification), (c – 5 nm magnification).

Figure 5. (a-d) Transmission electron microscope and SAD patterns images of Au-NPs (a - 50 nm magnification), (b - 20 nm magnification), (c – 5 nm magnification).

Figure 6. (a) UV-Visible spectroscopy of Ag-NPs with a peak at 440 nm, (b) UV-Visible spectroscopy of Au-NPs with a peak at 521 nm.

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Figure 7. (a) Size distribution of Ag-NPs with average size 25 nm as analyzed using DLS, (b)

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Size distribution of Au-NPs with average size 34 nm as analyzed using DLS. Figure 8. Antifungal tests carried out on Candida albicans, S.cerevesiaein a nutrient agar

A

medium. (a) Ag-NPs and Au-NPs inhibiting the growth of Candida albicans(disc diffusion

M

technique), (b) Ag-NPs and Au-NPs inhibiting the growth of S. cerevisiae(disc diffusion technique), (c) Ag-NPs and Au-NPs inhibiting the growth of Candida albicans(well diffusion

D

technique), (d) Ag-NPs and Au-NPs inhibiting the growth of S. cerevisiae(well diffusion

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technique), (e) control plate after inoculation, (d) graphical representation of ZOI using well diffusion technique of Ag-NPs and Au-NPs against Candida albicans and S.cerevesiae, (e) graphical representation of ZOI using disc diffusion technique of Ag-NPs and Au-NPs against

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Candida albicans and S.cerevesiae. List of Tables:

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Table 1. XRD details with 2theta and hkl values of the obtained Ag-NPs and Au-NPs. Table 2. The salient observations of experimental investigations in this study.

A

Table 3. MIC study of Ag-NPs and Au-NPs against fungal strains.

(b)

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(a)

Figure 1: (a) Silver colloid (Ag-NPs) prepared using sodium borohydride as a reducing agent, (b) Gold colloid (Au-NPs) prepared using tannic acid as a reducing agent.

N

2000

500

500

Au (311)

1000

Au (200)

M

1000

Ag (311)

1500

Ag (220)

Ag (200)

2000

Au-NPs

1500

A

2500

Intensity (a.u.)

3000

(b)

Au (220)

Ag (111)

(a)

Au (111)

Ag-NPs

3500

Intensity (a.u.)

U

2500

4000

20

30

40

50

2 Theta (Degrees)

60

70

80

20

30

TE

10

D

0

0

40

50

60

2 Theta (Degrees)

70

80

Figure 2: (a) X-ray diffractogram of Ag-NPs synthesized using sodium borohydride, (b) X-ray

A

CC

(a)

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diffractogram of Au-NPs synthesized using tannic acid and sodium borohydride.

(b)

(d)

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(c)

Figure 3: (a) SEM monograph of Ag-NPs (size < 15nm) synthesized using sodium borohydride as reducing agent, (b) SEM monograph of Au-NPs (size < 20nm) synthesised using tannic acid and sodium borohydride as reducing agent. (c) Elemental composition of Ag-NPs using energy dispersive X-ray analysis (EDAX), (d) Elemental composition of Au-NPs using energy dispersive

A

N

U

X-ray analysis (EDAX).

A

CC

EP

TE

D

M

(a)

(b)

(d)

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(c)

Figure 4: (a - d) Transmission electron microscope and SAD patterns images of Ag-NPs (a - 50

N

(b)

CC

EP

TE

D

M

A

(a)

A

U

nm magnification), (b - 20 nm magnification), (c – 5 nm magnification).

(d)

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(c)

Figure 5: (a - d) Transmission electron microscope and SAD patterns images of Au-NPs (a - 50

N

U

nm magnification), (b - 20 nm magnification), (c – 5 nm magnification).

0.9

A

(a)

0.35

M D

0.6

0.5

0.4

0.3 400

500 Wavelength (nm)

EP

300

TE

Absorbance (a.u.)

0.7

600

Au-Nps

(b)

0.30

Absorbance (a.u.)

0.8

0.40

Ag-NPs

440nm

521 nm

0.25 0.20 0.15 0.10 0.05

700

300

400

500

Wavelength (nm)

600

700

Figure 6: (a) UV-Visible spectroscopy of Ag-NPs with peak at 440 nm, (b) UV-Visible

A

CC

spectroscopy of Au-NPs with peak at 521 nm.

(b)

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(a)

Figure 7: (a) Size distribution of Ag-NPs with average size 25 nm as analysed using DLS, (b) Size

A

(b)

A

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CC

(c)

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D

M

(a)

N

U

distribution of Au-NPs with average size 34 nm as analysed using DLS.

(d)

(e)

10

(f)

7

7 6 5 4 3 2

6 5 4 3 2 1

1

0

0 S. Cerevesiae

Ag-NPs (filter) Au-NPs (filter)

(g)

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8

8

Ag-NPs (wells) Au-NPs (wells)

Zone of Inhibition (mm)

Zone of Inhibition (mm)

9

S. Cerevesiae

Candida albicans

Candida albicans

Figure 8: Antifungal tests carried out on Candida albicans, S.cerevesiae in a nutrient agar medium. (a) Ag-NPs and Au-NPs inhibiting the growth of Candida albicans (disc diffusion technique), (b) Ag-NPs and Au-NPs inhibiting the growth of S. cerevesiae (disc diffusion technique), (c) Ag-NPs

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and Au-NPs inhibiting the growth of Candida albicans (well diffusion technique), (d) Ag-NPs and

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Au-NPs inhibiting the growth of S. cerevesiae (well diffusion technique), (e) control plate after inoculation, (f) graphical representation of ZoI using well diffusion technique of Ag-NPs and Au-

A

NPs against Candida albicans and S.cerevesiae, (g) graphical representation of ZoI using disc

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diffusion technique of Ag-NPs and Au-NPs against Candida albicans and S.cerevesiae. Table 1: XRD details with 2theta and hkl values of the obtained Ag-NPs and Au-NPs. Sample

2 theta (degree) d-Spacing

D

S.

Ag-NPs

CC

A

(b)

Au-NPs

Particle size (nm)

38.20

2.35

111

30.53

44.37

2.05

200

23.59

64.53

1.44

220

25.84

77.48

1.23

311

22.18

EP

(a)

TE

no.

hkl values

25.53 (Average)

38.22

2.31

111

14.89

44.54

2.02

200

5.07

64.68

1.44

220

10.45

77.94

1.23

311

10.59 10.25 (Average)

Table 2: The salient observations of experimental investigations in this study.

Ag-NPs

Au-NPs

Color of final sample

Yellow

Wine red

Nature of final Sample

Suspension

Clear solution

SEM (shape)

Spherical +deformed

Spherical

SEM (particle size)

9.97±3.2 nm

13±5.2 nm

TEM (shape)

Spherical

TEM (particle size)

8±1.2 nm

SAD patterns

111, 200, 220, 311

XRD 2 theta (degree)

38.10, 44.40, 64.47, 77.35

38.91, 44.51, 64.75, 77.72

XRD-Average particle size (nm)

25.53

10.25

UV-Visible spectroscopy (peak)

440 nm

DLS (Particle size)

25 nm

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Analysis

Spherical 12±1 nm

111, 200, 220, 311

34 nm

M

A

N

U

521 nm

D

Table 3: MIC study of Ag-NPs and Au-NPs against fungal strains.

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Test organism

Zone of inhibition (mm)

Well diffusion technique

Disc diffusion technique

Sterilized

EP

water

Ag-

Au-

Standard

Ag-

Au-

Standar

NPs

NPs

errors

NPs

NPs

d errors

5

4.5

4.75 ± 0.25

4.6

4

4.3 ± 0.3 0

4

4.405 ± 0.5

5

4

4.5 ± 0.5 0

CC

A

Candida

Control

(de-ionized)

Albicans Sacharomyces 4.9 cerevesiae

D

TE

EP

CC

A

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U

N

A

M