Spectroscopic and antibacterial study of biochemically-derived silver nanoparticles

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ScienceDirect Materials Today: Proceedings 19 (2019) 86–93

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NANOTEXNOLOGY2018

Spectroscopic and antibacterial study of biochemically-derived silver nanoparticles Α. Ntoliaa, N. Matisioudisa,d, E. Triantafilloud, G. Evangelopouloud, V. Karachristoua, E. Rizosa, T. Karamanidouc, A. Tsouknidasc, D. Papadopoulosc, D. Tsipasc, N. Michailidisb,c & A. Aggeli a* b

a Laboratory of Biomedical Engineering, Department of Chemical Engineering, Department of Mechanical Engineering, Aristotle University, 54124 Thessaloniki, Greece c PLiN-Nanotechnology S.A., 57001 Thermi, Thessaloniki, Greece d Hellenic Army, 41334 Larissa, Greece

Abstract Silver nanoparticles (AgNPs) constitute a promising approach for the development of new antimicrobial systems. The major global concern about the emergence of bacterial strains resistant to widely used therapeutic agents for human infections has increased research for the discovery of new antimicrobial compounds as therapeutic alternatives. The aim of the present study was a preliminary investigation of the spectroscopic properties and inhibitory activity of silver nanoparticles on four reference gram negative and gram positive bacterial strains: Salmonella Typhimurium, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus, at two different concentrations (103 and 106 cfu/ml) using cultivation media. The results confirmed that AgNPs absorb light in the visible area of the electromagnetic spectrum, as a fairly dominant and sharp peak appears at around 425 nm wavelength. These nanomaterials are shown here to be very effective antibacterial agents in both pathogen (Listeria, Salmonella) and non-pathogens (E.coli, Staphylococcus) microorganisms; furthermore the present study proves their efficacy against both Gram positive (Staphylococcus, Listeria) and Gram negative (E.coli, Salmonella) bacteria. © 2019 Published by Elsevier Ltd. Selection and peer-review under responsibility of International Conferences & Exhibition on Nanosciences & Nanotechnologies and Flexible Organic Electronics 2018, June 30th - July 6th, 2018 Keywords: Silver nanoparticles, antibacterial, Escherichia coli, Salmonella, Staphylococcus, Listeria

* Corresponding author. Assoc. Professor Amalia Aggeli, , tel.: +30 2130 996218. E-mail address: [email protected] 2214-7853 © 2019 Published by Elsevier Ltd. Selection and peer-review under responsibility of International Conferences & Exhibition on Nanosciences & Nanotechnologies and Flexible Organic Electronics 2018, June 30th - July 6th, 2018

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1. Introduction In the last decade, nanoparticles’ science and technology have been widely developed due to their fascinating physical, chemical and optical properties. Silver and gold nanoparticles are widely used in various fields such as medicine, biology, chemistry, physics and electronics [1]. More specifically, silver nanoparticles (AgNPs) are employed as antibacterial in consumer products such as textiles, personal care, and food storage containers[2,6]. The largest group (55.4%) of all of the nanobased consumer products available on the market as of March, 2011 are based on silver nanoparticles [3]. Some of the proposed reasons for the special properties of silver nanoparticles are an increased relative surface area and the influence of quantum effects [4]. Many researchers have prepared and characterized silver nanoparticles (AgNPs) using a variety of methods such as chemical methods, gamma radiation, laser ablation and electrochemical approaches [7,8]. Silver ions have long been known to exert strong inhibitory and bacteriocidal effects as well as to possess a broad spectrum of antimicrobial activities [14]. Silver ions cause the release of K+ ions from bacteria, thus, the bacterial cytoplasmic membrane, which is associated with many important enzymes and DNA, is a target site of silver ions [15, 16, 17, 22]. When bacterial growth is inhibited, silver ions may be deposited into the vacuole and cell walls as granules [18]. They may also inhibit cell division and damage the cell envelope and cellular contents of the bacteria [26]. The sizes of the bacterial cells can also increase, and the cytoplasmic membrane, cytoplasmic contents, and outer cell layers can exhibit structural abnormalities. In addition, silver ions can interact with nucleic acids [24]; they preferentially interact with the bases in the DNA rather than with the phosphate groups, although the importance of this mechanism in terms of their lethal action remains unclear [14, 19, 23]. Many studies have proved that the efficiency of the silver ions against Gram-negative bacteria is better than Gram-positive bacteria due to their difference in the peptidoglycan layer thickness. [11]

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b) Figure 1: a) Schematic representation of different proposed mechanisms of antibacterial activity of silver nanoparticles (adapted from [9]). b) Transmission electron micrographs of E. coli bacterial cells in contact with silver nanoparticles (scale bar : 200 nm) (adapted from [10]).

In Fig. 1 below the possible ways that silver nanoparticles affect microorganisms, are depicted. More specifically, the large surface area of silver nanoparticles provides a better contact with bacteria that get attached to the cell membrane and penetrate inside bacteria. In addition, silver nanoparticles, react with sulfur-containing proteins of

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bacterial membranes and cause problems in their function. Furthermore, bacterial cell death can be caused by the interaction of silver ions or silver nanoparticles with the respiratory chain of the bacterial mitochondria. AgNPs can be released continually inside bacterial cells, increasing the possibility of free radicals to be formed and consequently enhance AgNPs antimicrobial activity. Although, AgNPs seem to have remarkable functional properties, a significant number of studies in the literature report that they are stable without precipitation only for a short period of time [12]. For this reason, it is necessary to define the factors that destabilize AgNPs, in order to find better ways to store them after synthesis and consequently maintain their specific properties. The long-term stability of nanoparticles depends firstly on the medium that are submerged in [27]. Silver is also known to be extremely photosensitive. It is proposed that electromagnetic irradiation provides energy that facilitates changes in AgNPs [28]. In addition, the surface of nanosilver is vulnerable to oxidation by O2 and other molecules of environmental and biological media. The proposed mechanism of AgNPs oxidation is shown in Figure 2. The dissolution of silver nanoparticles is assumed to be carried out by two irreversible surface reactions [26]:

1 2 Ag  O2  Ag2O( s ) (1) 2 Ag2O( s )  2H   2 Ag   H 2O (2)

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b) Figure 2: a) Proposed physicochemical changes and toxicity of nanosilver in environmental and biological media [13]. b) Oxidative dissolution of silver nanoparticles [24].

Figure 2 illustrates mechanisms that can result in the transformation of AgNPs in environmental or biological media. First of all, surface coating agents are usually in an equilibrium state with the ligand molecules of the liquid. The dispersion of AgNPs in a solution forces these surface-coating agents to re-establish equilibrium leading to a loss of these surface molecules that can be replaced by available biological macromolecules and become unstable. As a result AgNPs agglomerate. As mentioned above, silver atoms of the nanosilver surface, interact with molecular oxygen and produce silver oxide. In this work, a preliminary investigation into the stability and the antibacterial properties of silver nanoparticles is presented. Antibacterial activity was screened against pathogenic and non pathogenic Gram + and Gram - bacteria, in particular against S. aureus, E. coli, Listeria and Salmonella. The samples of AgNPs were analyzed spectroscopically due to their important optical properties. The strong interaction of the silver nanoparticles with light occurs because the electrons on the metal surface undergo a collective oscillation when excited by light at

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specific wavelengths (Figure 3). it is known as surface plasmon resonance (SPR) and this oscillation results in unusually strong scattering and absorption properties [5].

Figure 3. a): Extinction (scattering and absorption) spectra of silver nanoparticles with diameters ranging from 10-100 nm (adapted from [5]). b) Surface plasmon resonance where the free electrons in the metal nanoparticle are driven into oscillation due to a strong coupling with photons of specific wavelength of incident light (adapted from [5]).

Due to the unique optical properties of silver nanoparticles, a great deal of information about the physical state of the nanoparticles can be obtained by analyzing the spectral properties of silver nanoparticles in solution. The spectral response of silver nanoparticles as a function of diameter is shown in the above Figure 3a. 2. Materials and Methods 2.1. Materials Two different Aqueous Silver Nanoparticles solutions (Batch No.5, Batch No.6) 300ppm were obtained from PLiN Nanotechnologies S.A. Their synthesis involved a starting silver ion solution and the basic steps of the AgNPs synthesis have been presented previously. TSYEA (Tryptic Soy Yeast Exctract Agar, BIOLIFE), Escherichia coli (WDCM 00090), Staphylococcus aureus (WDCM 00032), Salmonella typhimurium (NCTC 12023) and Listeria monocytogenes (NCTC 11994) were used for the antibacterial experiments. A Shimadzu UV-Vis spectrophotometer was employed for spectroscopic analysis of AgNPs. 2.2. Characterization of silver nanoparticles First, the aq. solution of AgNPs 300ppm (Batch No.6) was stored in a plastic bottle impenetrable to sunlight. Then, a small amount of the solution was placed in a transparent sterile plastic bottle in order to be evaluated for its optical properties. The silver nanoparticles solutions were analyzed using a UV-Vis spectrophotometer in the range of 300-800nm using a glass cuvette with deionized water as reference. The first measurement was conducted immediately after the transfer of the solution in the transparent plastic bottle. In order to investigate the effect of the storage conditions on the AgNPs, periodical absorbance measurements were carried out for a period of 2 weeks. For the study of AgNPs stability in environmental conditions such as oxygen and sunlight the solution of AgNPs (Batch No.5) was further divided in three transparent sterile plastic bottles. Each one was stored in different environmental conditions. The first one was immediately measured after the storage in the bottle. The second one was measured after about 2 weeks at ambient environmental conditions. The third one, was firstly exposed for a week in bright sunlight and then measured.

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2.3. Viscosity of AgNPs solution In order to obtain the viscosity of AgNPs solution, a steady state flow step test was performed at room temperature (25 °C), using a TA Instruments AR-G2 Rheometer with a parallel plate geometry (40mm steel plate). The performing shear rates ranged from 0.1 to 1000 s-1 and the viscosity value was calculated taking the average of the values at every shear rate. 2.4. Filtration of AgNPs Microfiltration (MF) is a type of sterilization as it is a physical filtration process where a contaminated fluid is passed through a special pore-sized membrane to separate microorganisms from a liquid. In this study, preliminary experiments in sterilization of AgNPs solution were conducted, in order to sterilize the sample of 300 ppm silver nanoparticles prior to antibacterial studies. More specifically, AgNPs solution was passed through a 0.2 μm poresized membrane. 2.5. Micro-organism preparation Since an 18 hour incubation of the microorganisms in a nutrient broth would result in a concentration of 109 cfu / ml, overnight culture of the above strains was used in BHI (Brain Heart Infusion Broth, BIOLIFE). In order to achieve desirable populations of microorganisms, successive decimal dilutions were made in Maximum Recovery Medium (BIOLIFE). 2.6. Bacterial growth To examine bacterial growth or elimination in presence of silver nanoparticles different initial populations of E.coli, Streptococcus aureus, Listeria and Salmonella cells (103cfu/ml, 106cfu/ml) were grown. For this purpose, a stock solution of AgNPs of an initial concentration of 300ppm was used. Necessary dilutions were performed using sterile deionized water, double strength BHI and inoculating an appropriate amount of bacterial populations to achieve for these two microorganisms the desired initial dilutions of bacteria with the desired final concentration of nanoparticles (50ppm). Then, all the nutrient broths were incubated in an oven at 37 oC. To determine the remaining live cell population of the bacteria, counts were taken after 0, 6, 24 and 48 hours. The counting was performed on TSYEA (Tryptic Soy Yeast Extract Arar, BIOLIFE) by the incorporation method. In particular, 100 μl of bacterial suspension was placed in petri dishes and an appropriate amount of thermostated at 47 °C, nutrient medium was added. After solidification of the substrate, the plates were incubated at 37 °C for 24-48 hours. It should be noted that the same procedure was performed in the original solution of silver nanoparticles to confirm its sterility. At the end of the incubation, colonies were counted using a colony counter. 3. Results and Discussion 3.1. Spectroscopic characterisation The aq. solution of AgNPs was a thin Newtonian fluid with a viscosity of 0.881 Pa·s and a standard deviation of 0.022 Pa·s, as measured by viscosity experiments. In Figure 4a, it is shown that due to the surface plasmon resonance the silver nanoparticles solution (Batch No.6) produced a fairly sharp peak at around 425 nm wavelength, and taking into account previous studies [5], this may correspond to AgNPs with size <30 nm. The fairly stable nature of this particular AgNPs preparation over time (Batch No. 6), is demonstrated in figure 4b. For comparison a less stable previous, less optimized AgNPs preparation is also included (Batch 5, Figure 4c). It can be seen that the UV-Vis spectra of freshly prepared AgNPs is presenting a λmax at around 420nm. When the solution of AgNPs is exposed to environmental conditions the UVVis spectra changes and a decrease in the absorption peak can be observed. However the wavelength of the absorption peak does not change.

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This decrease in the absorption peak is accompanied by a slight alteration in the solution color that becomes darker. Moreover, we can assume that the AgNPs tend to agglomerate as it can be shown from the solution spectra. On the other hand, the solution of AgNPs that is exposed to intense sunlight seems to have a totally opposite behavior. The absorbance peak increases and a red shift of the λmax is observed. These results need further investigation in order to be clearly estimated. However, it can be easily speculated that the combination of sunlight irradiation and environmental oxygen led to these different results.

Figure 4. Absorption spectra : a) AgNPs measured fresh, b) AgNPs measured periodically over a period of 2 weeks of storage, c) AgNPs stability related to the environmental conditions of storage, d) AgNPs during microfiltration.

Some preliminary sterilization experiments were also carried out by using the method of microfiltration (Figure 4d). It was showed clearly that by microfiltaring the AgNPs solution, a large amount of silver nanoparticles were removed by the filter. More specifically, the optical density at 425 nm, which corresponds to the maximum absorbance before microfiltration was around 1.05 while after microfiltration was around 0.35. This difference in absorbance of 0.7, leads to the conclusion that microfiltration is not an appropriate method in order to sterilize the aqueous solution of silver nanoparticles.

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Figure 5. Bacterial growth in the presence of 50 ppm AgNPs as a function of time : A) E.coli, B) Salmonella, C) Staphylococcus, D) Listeria

3.2. Antibacterial experiments According to the results shown in Figure 5, by using silver nanoparticles at concentration of 50ppm, it is possible to kill pathogen, non-pathogen, gram positive and gram negative bacteria. The nanoparticles, were bactericidal after 24h of incubation, showing qualitatively similar final results for all the microorganisms. In the present study, the AgNPs showed extreme efficacy against the four bacterial species tested, regardless of their Gram positive or negative nature. This strong bactericidal property of the AgNPs against different bacterial species, including multi drug resistant (MDR) bacteria, is in agreement with previous studies, making AgNPs a promising tool for the manufacturing of novel antibacterial products for controlling pathogens [29-32]. 4. Conclusions With the rapid progress in the field of nanotechnology, inorganic nanoparticles such as AgNPs, become commonly used materials in a variety of application fields. The SPR phenomenon of AgNPs is shown in agreement with previous studies, to be a useful tool which allows to track the size and physicochemical state of the nanoparticles as a function of a variety of conditions. As shown in the present initial study, AgNPs have a high antimicrobial efficacy at low levels and may have potential as realistic substitutes for common chemical antimicrobial materials. These nanomaterials are shown here to be effective antibacterial agents against both pathogen (Lysteria, Salmonella) and non-pathogen (E.coli, Staphylococcus) microorganisms. Furthermore the present study shows their efficacy against both Gram positive (Staphylococcus, Listeria) and Gram negative (E.coli,

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