Acta Polytechnica doi:10.14311/AP.2018.58.0365 Acta Polytechnica 58(6):365–369, 2018 © Czech Technical University in Prague, 2018 available online at http://ojs.cvut.cz/ojs/index.php/ap THE INFLUENCE OF SILVER NANOPARTICLES SYNTHESIS ON THEIR PROPERTIES Anna Mražíkováa, ∗, Oksana Velgosováa, Jana Kavuličováb, Stanislav Krumc, Jaroslav Málekc a Institute of Materials and Quality Engineering, Faculty of Materials, Metallurgy and Recycling, Technical University of Kosice, Slovakia b Institute of Metallurgy, Faculty of Materials, Metallurgy and Recycling, Technical University of Kosice, Slovakia c Department of Materials Engineering, Faculty of Mechanical Engineering, Czech Technical University in Prague, Czech Republic ∗ corresponding author: anna.mrazikova@tuke.sk Abstract. Application of green methods to replace physical and chemical methods for synthesis of silver nanoparticles (AgNPs) has become necessary not only from economic aspect but especially due to its significant impact on ecosystem. The properties of biologically synthesized AgNPs using green algae Parachlorella kessleri (P. kessleri) and chemically prepared were investigated and compared. The UV- vis analysis confirmed a high stability of biosynthesized AgNPs as well as chemically synthesized gelatin modified citrate-AgNPs. Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) revealed different sizes and shapes of AgNPs synthesized in different ways. Biosynthesized AgNPs have similar inhibitory antimicrobial activity as gelatin/sodium citrate-AgNPs. Keywords: silver nanoparticles; biosynthesis; chemical reduction; gelatin; anti-microbial activity. 1. Introduction In recent years the use of noble metal nanomaterials in many industrial applications including physics, chem- istry, electronics, optics, material science has rapidly increased. Furthemore, silver containing materials have gained a great attention especially due to their antimicrobial properties. Therefore, AgNPs are now being used to reduce infections, to prevent biofilm formation on protheses, catheters, dental materials and also on stainless steel materials [1–5]. Physical and chemical methods generally used for AgNPs synthesis very often involved toxic chemicals that can contaminate the nanoparticles [6]. Such nanoparticles are released into environment in differ- ent stages of their production, apllication and even disposal of nanowastes what can consequently lead to contamination of the whole ecosystem. Finally, the majority of nanoparticles accumulates in fresh and marine ecosystems [7]. Oukarroum et al. [8] in their reports outlined a negativelly effect of such Ag- NPs on both freshwater and marine algae by strong decrease in viable algal cells. Therefore there is a need to replace physical and chemical techniques of AgNPs preparation by green alternatives, which are cost-effective, safe, environment-friendly and easily scaled up for large syntheses of NPs. The use of biomolecules like proteins and lipids present on NPs surfaces has a great potential in AgNPs synthesis due to their non-toxic nature and also gentle synthetic procedures [9, 10]. Therefore there is a growing con- cern to apply biomimetic which use plants, bacteria, fungi, yeast, actinomycetes and algae for synthesis of nanostructures of biocompatible metals and semicon- ductors [5]. The common problem of AgNPs application typ- ically prepared via reduction of a silver precursor using chemical or physical means is dispersion in- stability against aggregation. One of the possibili- ties to enhance stabilization of nanoparticles is ad- dition of surface-protecting agents such as organic ligands namely chitosan, polysacharides and gelatin or inorganic capping materials [11–13]. Lee [12] and Sivera [13] reported the gelatin-modified AgNPs ex- hibited long-term stability against aggregation and maintained unchanged optical and physical properties and a high antibacterial activity for several months at ambient temperature. The similar properties were also observed in biologically synthesized AgNPs. Func- tional groups of the biological materials are responsible for the reduction of the Ag+ and subsequent stabi- lization of nanoparticles [9, 14, 15]. Green algae are well-known by biomass containing different organic biologically active compounds such as chlorophylls, carotenoids, flavonoids, proteins, vitamins and miner- als [5]. Natural polymer on algae is considered to be suitable for stabilization of inorganic silver nanoparti- cles. We have recently reported that AgNPs biosynthe- sized using P. kessleri showed long-term stability at the higher pH values [16]. The novelty of this work is investigation and comparison of properties biologically synthesized AgNPs using the algae P. kessleri and chemically synthesized AgNPs. Chemically synthe- sized AgNPs were performed by two different ways: using sodium citrate and both sodium citrate and gelatin as a reducing and capping agent. This study 365 http://dx.doi.org/10.14311/AP.2018.58.0365 http://ojs.cvut.cz/ojs/index.php/ap Anna Mražíková, Oksana Velgosová, Jana Kavuličová et al. Acta Polytechnica Figure 1. UV-vis spectra of (a) biosynthesized AgNPs (b) gelatin/sodium citrate-AgNPs (c) sodium citrate-AgNPs. also compares the antimicrobial activities of AgNPs against the algae P. kessleri. 2. Materials and methods 2.1. Synthesis of silver nanoparticles The green algae P. kessleri were cultivated on agar plates in Petri dishes for 3 weeks at the ambient tem- perature and light mode (12 : 12). The extract was filtered and the filtrate was centrifuged at 9000 rpm for 15 min and supernatant was added into Erlenmeyer flasks containing 250 ml of AgNO3 solution (0.29 mM) and used for biosynthesis of AgNPs. The Erlenmayer flasks were stored under lighting condition at the am- bient temperature to allow reducing the silver ions into AgNPs. Chemically synthesized AgNPs were prepared us- ing chemical reduction method [7, 17]. The 15 ml of sodium citrate (0.5 wt.%) solution as a reducing agent was added drop by drop to the 250 ml of aque- ous AgNO3 (0.29 mM). In the case of chemically syn- thesized AgNPs using mixed gelatin/sodium citrate, first of all, to prepare stock solution of AgNO3, the gelatin (0.01 wt.%) was dispersed in 250 ml of 0.29 mM of AgNO3 to prevent particle agglomeration. Silver nanoparticles were prepared by adding drop-wise of 15 ml of (0.5 wt.%) sodium citrate solution into AgNO3 solution. Both solutions of chemically prepared Ag- NPs were stirred at 700 rpm with a magnetic stirring bar at 70 °C for 30 minutes. Erlenmayer flasks were stored in lighting condition at the ambient tempera- ture and the end point of the reaction was the appear- ance of pale yellow-brown and dark brown colour. 2.2. Antimicrobial assay The ability of AgNPs to inhibit the formation of algae biofilm was performed by standard disk- diffusion method [18]. The 1 ml of algal suspension (105 CFU/ml) was used to seed agar plates consisted of 2 % agar and culture medium (Milieu Bristol). The 25 µl of colloidal sollutions AgNPs were added to ster- ile swabs (6 mm) placed on agar plates seeded with microorganisms. The minimum inhibitory concentra- tion (MIC) was read after 7 days of incubation at the ambient temperature and light mode (12 : 12). 2.3. Characterizations The nanoparticle colloidal solutions were stirred with magnetic stirrer with heating (IKA C-MAG HS4). The absorbance of the AgNPs dispersions was an- alyzed using an UV–vis spectra from 300 to 800 nm with UNICAM UV/vis Spectrometer UV4. The ab- sorbance was recorded on days 7 and 120. Transmission Electron Microscope (TEM; JEM- 2000FX, JEOL) at 200 kV was used to determine the size and morphology of AgNPs on day 120. Scanning electron Microscopy (SEM) analysis was done using JEOL JSM-7600F and used to determine the surface morphology properties on day 120. EVETM-NanoEnTek was used to automate cell counting by the standard trypan blue technique. The observation of algae cells eradication on agar plates was done by macroscope LEICA WILD M32. 3. Results and discussion The formation of silver nanoparticles synthesized ei- ther chemically and biologically was clearly observed after 3 and 24 hours reaction time by solution colour changes and confirmed by UV-vis spectroscopy as depicted in Fig. 1. In the case of P. kessleri the solu- tion colour changed from pale yellow to yellow-brown. In the presence of citrate and gelatin/sodium citrate the solution colour changed to pale yellow-brown and dark brown, respectively. The UV-vis measurements showed the increase of the absorption maximum within 120 days in all three nanoparticle samples. This in- dicated the formation of AgNPs in the solution at the time. The biologically synthesized nanoparticles exhibited an increase of the broad absorption band on day 120 and only minor shift of UV-visible spec- trum (Fig. 1a), what indicated a long-term stability of the silver nanoparticles [19]. Kadukova [15] reported that AgNPs produced by P. kessleri can be stable even more than 6 months. The AgNPs chemically produced using gelatine as a capping agent exhibited the most significant deviation at increase of the ab- sorption maximum at 433 nm on day 120 (Fig. 1b). As reaction time increased more amine residues of gelatine were being released into the reaction system and consequently, reduction of silver ions slowly pro- ceeded [12]. Shift of UV-visible spectrum (Fig. 1b) 366 vol. 58 no. 6/2018 The Influence of Silver Nanoparticles Synthesis on Their Properties Figure 2. SEM and TEM images and the antimicrobial effect of (a,top) biosynthesized AgNPs (b,middle) gelatin/sodium citrate-AgNPs (c,bottom) sodium citrate-AgNPs. from 416 to 433 nm points to the creation of larger average particle sizes as it was reported by many au- thors [19, 20]. For AgNPs obtained and stabilized only using sodium citrate the UV-vis increase of the absorption maximum on day 120 was not as strong as in the case of biologically synthesized AgNPs and gelatin modified sodium citrate-AgNPs as well. The most significant shift of UV-vis absorption maximum from day 7 to 120 (from 360 to 450 nm) was observed in the solution with citrate-AgNPs without addition of capping agents. Such broadening and shift of SPR indicate the presence of larger nanoparticle sizes than above mentioned and also showed their short-term stability [20]. The occurrence of symmetrical sharp UV-vis absorp- tion peaks typically located around 400 nm observed on day 120 in the solutions with biologically synthe- sized AgNPs as well as gelatin/sodium citrate-AgNPs (Fig. 1ab) indicated the presence of stable nanoparti- cles. The broad absorption band observed in citrate- AgNPs, (Fig. 1c) indicated much less uniform and stabilized nanoparticles. The SEM and TEM micro- graphs (Fig. 2c) obtained after 120 days revealed the presence of large particle sizes (from 7 to 85 nm) and also small agglomerates and some dispersed AgNPs. It is very likely that agglomeration was caused by diminishing electrostatic repulsion [21]. Based on our results the addition of gelatin to Ag+ solution in the process of AgNPs formation improved stability and dispersibility of nanoparticles. Biomolecules like pep- tides and proteins present in gelatin are able readily interact with metals and hydrophilic ligands protect gelatin coated AgNPs in aqueous solution. The coat- ing serves to provide proper gap between the silver core [12, 13]. The silver nanoparticles, which were reached us- ing biological approaches, showed on SEM and TEM micrographs (Fig. 2a) spherical particles with aver- age particle size of 15 nm. The role of active com- pounds of biomass responsible for the process of Ag- NPs formation and stabilization was confirmed by the work of several authors [6, 15, 23]. Their re- sults indicated that the various functional groups, especially amine, carboxyl, sulphydril and hydroxyl moieties present in the proteins, primarily cause the reduction of nanoparticles. Formation of smaller sizes 367 Anna Mražíková, Oksana Velgosová, Jana Kavuličová et al. Acta Polytechnica gelatin/sodium citrate-AgNPs (from 4 to 55 nm) might be caused by presence of a higher gelatin concentra- tion as demonstrated [14, 22]. SEM and TEM images (Fig. 2b) of gelatin-protected particles also revealed the nanoparticles of different spherical and pyramidal shapes. Antimicrobial effects of biosynthesized and chem- ically synthesized AgNPs against the green algae P. kessleri were observed in all three cases. The results revealed that chemically synthesized sodium citrate- AgNPs caused only particular inhibition (Fig. 2c) against algae. Double zone of inhibition observed around the swabs impreganated with sodium citrate- AgNPs was attributable to their bigger sizes, where AgNPs were not able to pass through the pores on the cell wall. Such aggregate formation might act as a binding agent between cells and inhibited algal cells growth [8]. Stronger extent of algae cells eradi- cation and clear circular inhibition zone was observed around the swabs impregnated with gelatin/sodium citrate-AgNPs and biosynhesized AgNPs (Fig. 2ab). According to literature [3, 14], the nanoparticles of smaller sizes have a higher antibiofilm activity due to the largest surface/volume ratio what is most eas- ily to reach cellular proximity. Such AgNPs cause structural changes and damages of cellular membrane that lead to cell death [3]. Our results indicated that biosynthesized AgNPs have similar inhibitory an- timicrobial activity as gelatin/sodium citrate-AgNPs against biofilm formation and owing to their easy and inexpansive synthesis appear to be good alternative to chemically prepared AgNPs. 4. Conclusion Silver nanoparticles were synthesized bio- and chemi- cal reduction of Ag+ ions. The UV-vis spectroscopy revealed that the addition of gelatin positively affected size and long-term stability of chemically synthesized citrate-AgNPs. Gelatin coated citrate-AgNPs also dis- played enhanced antialgal effects in comparison with citrate-AgNPs. The UV-vis, SEM and TEM analy- ses revealed that biosynthesized AgNPs using algae extract exhibited long-term stability and also good antimicrobial activity against the green algae which could be attributed to the smallest sizes of the Ag- NPs. The extract from the green algae P. kessleri can adequately act as both reducing and capping agents. The results implied that biosynthesized AgNPs can be good alternative for preparation of materials which inhibit the biofilm formation. Acknowledgements This work was financially supported by Slovak Grant Agency (VEGA 1/0134/19). References [1] Zhang X., Wang H., Li J., He X., Hang R., Yang Y., Tang B.: The fabrication of Ag-containing hierarchical micro/nano-structure on titanium and its antibacterial activity. Mater. Lett. 193, 2017, p. 97-100. doi:10.1016/j.matlet.2017.01.094 [2] Guzmán M. G., Dille J., Godet S.: Synthesis of silver nanoparticles by chemical reduction method and their antibacterial activity. Int. J. of Chemical and Biomolecular Eng., 2:3, 2009, p. 104-111. doi:10.1016/j.nano.2011.05.007 [3] Inbakandan D., Kumar C., Abraham L. S., Kirubagaran R., Venkatesan R., Khan S.A.: Silver nanoparticles with anti microfouling effect: A study against marine biofilm forming bacteria. Colloids and Surfaces B: Biointerfaces 111, 2013, p. 636-643. doi:10.1016/j.colsurfb.2013.06.048 [4] Oluwafemi O. S., Vuyelwa N., Scriba M., Songca S. P.: Green controlled synthesis of monodispersed, stable and smaller sized starch-capped silver nanoparticles. Mater. Lett. 106, 2013, p. 332-336. doi:10.1016/j.matlet.2013.05.001 [5] Shankar P.D., Shobana S., Karuppusamy I., Pugazhendhi A., Ramkumar V.S., Arvindnarayan S., Kumar G.: A review on the biosynthesis of metallic nanoparticles (gold and silver) using bio-components of microalgae: Formation mechanism and applications. Enz. and Microb. Technol. 95, 2016, p. 28-44. doi:10.1016/j.enzmictec.2016.10.015 [6] Bogireddy N. K. R., Kumar H. A. K., Mandal B. K.: Biofabricated silver nanoparticles as green catalyst in the degradation of different textile dyes J. of Environ. Chem. Eng. 4, 2016, p. 56-64. doi:10.1016/j.jece.2015.11.004 [7] Girilal M., Krishnakumar V., Poornima P., Fayaz A.M., Kalaichelvan P.T.: A comparative study on biologically and chemically synthesized silver nanoparticles induced Heat Shock Proteins on fresh water fish Oreochromis niloticus. Chemosphere, 139, 2015, p.461-468. doi:10.1016/j.chemosphere.2015.08.005 [8] Oukarroum A., Bras S., Perreault F., Popovic R.: Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Ecotoxicology and Environ. Safety, 78, 2012, p. 80-85. doi:10.1016/j.ecoenv.2011.11.012 [9] Sharma D., Kanchi S., Bisetty K.: Biogenic synthesis of nanoparticles: A review. Arabian J. of Chemistry. 2015. doi:10.1016/j.arabjc.2015.11.002 [10] Srinithia B., Kumar V.V., Vadivel V., Pemaiah B., Anthony S. P., Mathuraman M. S.: Synthesis of biofunctionalized AgNPs using medicinally important Sida cordifolia leaf extract for enhanced antioxidant and anticancer activities. Mater. Lett. 170, 2016, p. 101-104. doi:10.1016/j.matlet.2016.02.019 [11] Abdulla-Al-Mamun M., Kusumoto Y., Muruganandham M.: Simple new synthesis of copper nanoparticles in water/acetonitrile mixed solvent and their characterization. Mater. Lett. 63, 2009, p. 2007-2009. doi:10.1016/j.matlet.2009.06.037 [12] Lee Ch. and Zhang P.: Facile synthesis of gelatin-protected silver nanoparticles for SERS applications. J. Raman Spectrosc. 44, 2013, p. 823-826. doi:10.1002/jrs.4304 368 http://dx.doi.org/10.1016/j.matlet.2017.01.094 http://dx.doi.org/10.1016/j.nano.2011.05.007 http://dx.doi.org/10.1016/j.colsurfb.2013.06.048 http://dx.doi.org/10.1016/j.matlet.2013.05.001 http://dx.doi.org/10.1016/j.enzmictec.2016.10.015 http://dx.doi.org/10.1016/j.jece.2015.11.004 http://dx.doi.org/10.1016/j.chemosphere.2015.08.005 http://dx.doi.org/10.1016/j.ecoenv.2011.11.012 http://dx.doi.org/10.1016/j.arabjc.2015.11.002 http://dx.doi.org/10.1016/j.matlet.2016.02.019 http://dx.doi.org/10.1016/j.matlet.2009.06.037 http://dx.doi.org/10.1002/jrs.4304 vol. 58 no. 6/2018 The Influence of Silver Nanoparticles Synthesis on Their Properties [13] Sivera M., Kvitek L., Soukupova J., Panacek A., Prucek R., Vecerova R., Zboril R.: Silver Nanoparticles Modified by Gelatin with Extraordinary pH Stability and Long-Term Antibacterial Activity. PLOS ONE, 9(8) e103675, 2014. doi:10.1371/journal.pone.0103675 [14] Ethiraj A. S., Jayanthi S., Ramalingam Ch., Benerjee Ch.: Control of size and antimicrobial activity of green synthesized silver nanoparticles. Mater. Lett. 185, 2016, p. 526-529. doi:10.1016/j.matlet.2016.07.114 [15] Kadukova J.: Surface sorption and nanoparticle production as a silver detoxification mechanism of the freshwater alga Parachlorella kessleri Biores. Tech. 216, 2016, p. 406-413. doi:10.1016/j.biortech.2016.05.104 [16] Velgosová O., Mražíková A., Marcinčáková R.: Influence of pH on green synthesis of Ag nanoparticles. Mater. Lett. 180, pp. 336-339 (2016). doi:10.1016/j.matlet.2016.04.045 [17] Sileikaite A., Prosycevas I., Puiso J., Juraitis A., Guobiens A.: Analysis of Silver Nanoparticles Produced by Chemical Reduction of Silver Salt Solution. Materials Science (Medziagotyra) 12(4) 2006, p. 287–291. [18] Kavita K., Singh V.K., Jha B.: 24-Branched ∆5 sterols from Laurencia papillosa red seaweed with antibacterial activity against human pathogenic bacteria. Microbial Research. 169 (4), 2014, p. 301-306. doi:10.1016/j.micres.2013.07.002 [19] Villanueva-Ibáñez M., Yañez-Cruz M.G., R. Álvarez- García R., Hernández-Pérez M.A., Flores-González M.A.: Aqueous corn husk extract – mediated green synthesis of AgCl and Ag nanoparticles Mater. Lett. 152, 2015, p. 166-169. doi:10.1016/j.matlet.2015.03.097 [20] Rashid M.U., Bhuiyan M.K.H., Quayum M.E.: Synthesis of Silver Nano Particles (Ag-NPs) and their uses for Quantitative Analysis of Vitamin C Tablets. J. Pharm. Sci. 12, 2013, p. 29-33. doi:10.3329/dujps.v12i1.16297 [21] Roh J., Umh H. N., Sim J., Park S., Yi J., Kim Y.: Dispersion stability of citrate- and PVP-AgNPs in biological media for cytotoxicity test Korean. J. Chem. Eng. 30 (3), 2013, p.671-674. doi:10.1007/s11814-012-0172-3 [22] Pootawang P., Saito N., Takai O.: Ag nanoparticle incorporation in mesoporous silica synthesized by solution plasma and their catalysis for oleic acid hydrogenation. Mater. Lett. 65, 2011, p. 1037-1040. doi:10.1016/j.matlet.2011.01.009 [23] Patel V., Berthold D., Puranik P., Gantar M.: Screening of cyanobacteria and microalgae for their ability to synthesize silver nanoparticles with antibacterial activity. Biotechnol. Reports. 5, 2015, p. 112-119. doi:10.1016/j.btre.2014.12.001 369 http://dx.doi.org/10.1371/journal.pone.0103675 http://dx.doi.org/10.1016/j.matlet.2016.07.114 http://dx.doi.org/10.1016/j.biortech.2016.05.104 http://dx.doi.org/10.1016/j.matlet.2016.04.045 http://dx.doi.org/10.1016/j.micres.2013.07.002 http://dx.doi.org/10.1016/j.matlet.2015.03.097 http://dx.doi.org/10.3329/dujps.v12i1.16297 http://dx.doi.org/10.1007/s11814-012-0172-3 http://dx.doi.org/10.1016/j.matlet.2011.01.009 http://dx.doi.org/10.1016/j.btre.2014.12.001 Acta Polytechnica 58(6):365–369, 2018 1 Introduction 2 Materials and methods 2.1 Synthesis of silver nanoparticles 2.2 Antimicrobial assay 2.3 Characterizations 3 Results and discussion 4 Conclusion Acknowledgements References