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Green silver nanoparticles: Synthesis, characterization and uses


Environmentally sustainable production of silver nanoparticles

Rosa Cadena

Aislinn Morales

Ana Herrera

Raquel Villamizar

Microbiology Department, Universidad de Pamplona. Km 1, vía Bucaramanga, Ciudadela Universitaria. Building Eduardo Cote 202. Laboratory NANOSOST. Pamplona, Colombia

*E-mail: raqvillamizar@unipamplona.edu.co

Given the current problem of pollution of the environment and in search of more sustainable approaches to ensure natural resources for present and future generations, it is necessary to find out cleaner strategies for obtaining nanomaterials. This is how green synthesis has been emerging for several years as an effective and clean strategy for large-scale production of different types of metal nanoparticles.

The nanoparticles are defined as clusters of atoms with at least one of their dimensions on the nanometric scale (1 to 100 nm), which exhibit a very high surface area/volume ratio with respect to the bulk material. Among different metallic nanoparticles, silver nanoparticles (AgNP) display optical, catalytic and antimicrobial properties of great interest in different sectors such as clinical, textile or industrial [1]-[2]. These nanomaterials are usually produced through physical processes with “Top Down” approach where there is control over size and morphology. However, they also consume energy and are highly expensive. It is also common to use chemical methods with a “Bottom up” approach that are effective, but are usually contaminants due to the by-products generated after the synthesis process [3].

Given the current problem of pollution of the environment and in search of more sustainable approaches to ensure natural resources for present and future generations, it is necessary to find out cleaner strategies for obtaining nanomaterials. This is how green synthesis has been emerging for several years as an effective and clean strategy for large-scale production of different types of metal nanoparticles. Various biological materials are used in the green processes, including extracts of plants and algae, from which an arsenal of protein, polysaccharide and phytochemical compounds that act as reducing and stabilizing agents for nanoparticles can be obtained [4]-[5].

For this reason, in this study, the catalytic activity of green extracts obtained from Crescentia cujete (totumo), Solanum tuberosum (potato) and Actinidia deliciosa (Kiwi) in the production of silver nanoparticles was explored with the final aim of using these nanomaterials in different applications in clinical, food and environmental sectors.

Materials and methods

The nanoparticle biosynthesis process was  carried out following some of the recommendations of Rahj S and collegues 6 with some modifications (see figure 1). The amount of proteins and polysaccharide was quantified in each extract by using the Bradford and Phenol-Sulfuric methods respectively. Subsequently, AgNPs were characterized by the UV-vis spectrophotometry technique. The antimicrobial effect of the nanoparticles was performed using the diffusion method in solid medium. Sensidisks with 40 µL of the colloidal solution of nanoparticles was deposited on a Petri dish containing 0.5 Mcfarland concentration (1.5×108 bacteria/mL) of the strains of wild E.coli and methicillin resistant S. aureus.

Figure 1. Schematic representation of the biosynthesis of silver nanoparticles (AgNP) using different plant extracts.

Results and discussion

Extracts obtained from kiwi, potato and totumo presented protein contents of 0.0008 mg / mL, 0.0882 mg / mL and 0.23 mg / mL, while the polysaccharide content was 1.1 mg / mL, 1.98 mg / mL and 0.9 mg / mL, respectively. The results allowed concluding that the reducing agent obtained from totumo had a higher protein content, thereby it would follow a biosynthetic route very similar to that reported in Figure 2A[8], while the Polysaccharide residues would act as stabilizers. In contrast, reducing agents obtained from the potato, the kiwi, had a higher content of polysaccharides, and therefore, the biosynthesis route would be more adapted to those presented in Figure 2B ]9]. In the extracts of plants are also found phytochemical compounds such as flavonoids. Although its content was not determined, its presence cannot be deleted and therefore, the biosynthesis path would be very similar to those presented in Figure 2C [10].

Figure 2. Chemical representation of the biosynthesis process of silver nanoparticles using nitrate reductase-type green reducing agents (a-protein), Glucose molecule (b-polysaccharide) and flavonoids (c-phytochemicals) [8-10].

The bioreduction process from protein agents is usually carried out by nitrate reductase NADH-dependent enzymes. This process begins obtaining electrons from NADH. The nitrate reductase enzyme reduces nitrate (NO3-) to nitrite (NO2-) and the electron released during this process is transferred to the silver ion (Ag+) which is subsequently reduced to metallic silver (Ag0), obtained from this way the nanoparticles [8].  The study carried out by Seetharaman P and collagues9 reported that in the totumo´s extracts there are proteins, saponins, flavonoids, cardenolides, tannins, phenols and hydrogen cyanide, which act as reducing agents of silver nitrate salt, leading to to the formation of silver nanoparticles. In the case of the biosynthesis of nanoparticles using tubers such as Solanum tuberosum, the literature indicates that the glucose molecule through its hydroxyl group is linked to the Ag+ ion to perform substitution interaction forming bonds with the oxygen present in the hydroxyl groups (OH). In this way, it becomes O-Ag. The glucose molecule is a polyasociated forms of starch, which acts as a stabilizer of the synthesized nanoparticles [9].

When the reduction is mediated by a phytochemical compound, a route mediated by compounds such as flavonoids is used, which have a high redox potential because they are constituted by carboxyl groups (–C = O) and hydroxyls (-OH) that contribute to the reduction of Ag+ to Agº. The reactions of Ag+ silver ions with hydroxyl groups generate AgOH that initially appear as white colloidal particles that change to brown color.10 This can be seen in Figure 3 where the solution of the reducing agent and the product obtained are shown.

 Figure 3. Colloidal solution of nanoparticles obtained from green reducing agentsfrom A) Totumo B) Potato C) Kiwi. The clear solution corresponds to the reducing agent while the colored to the biosynthetized nanoparticles.

Characterization by UV-VIS spec-trophotometry showed an absorbance peak between 420-440 nm, given by the plasmon resonance effect of the AgNP (Figure 4).

Figure 4. Biosynthesized UV-Vis spectra using green reducing agents (Coffee-Totumo, Green-Kiwi, Purple-Potato).

The antimicrobial effect of the AgNP was evaluated against wild E. coli strains and a methicillin resistant S. aureus strain. Inhibition assays allowed determining that nanoparticles obtained from Totumo had a greater biocidal spectrum compared to those exhibited potatoes and Kiwi (Figure 5). Similarly, it was determined that the bactericidal effect of AgNP was greater on the wild E.coli strain than on the antibiotic resistant strain of S. aureus.Figure 5. Inhibitory effect of synthesized silver nanoparticles using TT (Totumo), TB (Papa), Kw (kiwi), AF (Control) on A) E. coli B) S. aureus (in this case the figure only shows the test performed with the AgNP obtained from totumo since with the other matrices the effect was undetected).

Through this assay it was possible determine that the E. coli strain is sensitive to AgNP synthesized from totumo and kiwi extracts with inhibition halos > 20 mm. In the case of methicillin resistant S. aureus, this bacterium presented an intermediate sensitivity to the AgNPS since the diameter of the inhibition halo was slightly> 10 mm (the sensitivity data were taken based on commonly used antibiotics to treat this type of pathogen according to the NCCLS) [11].

The inhibitory action of the nanoparticles depends mainly on the size, shape, surface area, concentration, pathogens (genus-species) and the type of phytoconstituents surrounding the surface of AgNPs11. Bibliographic reports indicate that at the ultrastructure level the AgNP have three mechanisms of action: (1) cell wall and membrane damage, (2) intracellular penetration and damage and (3) oxidative stress. [8],[12].

It has been reported that most AgNP exhibit a greater antibacterial activity against Gram-negative bacteria such as E.coli than Gram-positive bacteria such as S. aureus. This is mainly due to the thickness of the peptidoglycan layer, since in Gram positive this layer is thicker and more compact. In Gram-negative bacteria, the petidoglucan layer is thinner and is bound to lipopolysaccharides (LPS), which have a significant contribution to the action of AgNP in which electrostatic interactions take place facilitating adhesion and internationalization to the periplasm of these nanomaterials [12].

CONCLUSIONS

It was demostrated that the green extracts obtained from totumo, papa and kiwi have the protein, polysaccharide and phytochemical components necessary to carry out the reduction of substrates such as silver nitrate salt and thus producing nanoparticles. It was also possible to check the microbicidal activity of these nanoparticles against strains of pathogenic microorganisms very common in the food and clinical sector. For this reason, synthesized AgNPs could be profiled as alternative disinfectants to mitigate the presence of these pathogens on surfaces and therefore in food.

References

[1] Stephen King, Helen jarvie y Peter Dobson, [Internet] consultado 25 de mayo del 2019 (Actualizado14 de mayo de 2019)  disponible en: https://www.britannica.com/science/nanoparticle

[2] Ahmed S, Ikram S, S SY. Journal of Photochemistry & Photobiology , B : Biology Biosynthesis of gold nanoparticles : A green approach. JPB [Internet]. Elsevier B.V.; 2016;161:141–53.

[3] Pirtarighat S, Ghannadnia M, Baghshahi S. Materials Science & Engineering C Biosynthesis of silver nanoparticles using Ocimum basilicum cultured under controlled conditions for bactericidal application. Mater Sci Eng C [Internet]. Elsevier; 2019;98(July 2018):250–5.

[4] Amini SM. PT US CR. Mater Sci Eng C [Internet]. Elsevier B.V; 2019;109809.

[5] Zikalala N, Matshetshe K, Parani S, Oluwafemi OS. Nano-Structures & Nano-Objects Biosynthesis protocols for colloidal metal oxide nanoparticles. Nano-Structures & Nano-Objects [Internet]. Elsevier B.V.; 2018;16:288–99.

[6]Raj S, Trivedi R. Green synthesis and characterization of silver nanoparticles using Enicostemma axillare ( Lam .) leaf extract. Biochem Biophys Res Commun [Internet]. 2018;503(4):2814–9. Available from: https://doi.org/10.1016/j.bbrc.2018.08.045

[7] Villamizar, R.; Monroy, L.. Using silver nanoparticles for control pathogenic microorganisms in foods. Alimentech. 2015. 13, 54-59.

[8] Roy A, Bulut O, Some S, Mandal AK, Yilmaz MD. Green synthesis of silver nanoparticles: biomolecule-nanoparticle organizations targeting antimicrobial activity. View. 2019;2673–702.

[9] Minerva, S.  [Tesis] Nanopartículas De Plata: Preparación, Caracterización Y Propiedades Con Aplicación En Inocuidad De Los Alimentos. [Internet] Consultado 25 Julio del 2019 Disponible en: http://e-spacio.uned.es/fez/eserv/bibliuned:master-Ciencias-CyTQ-Msanchez/Sanchez_Moreno_Minerva_TFM

[10] Hussain M, Iqbal N, Muhammad R, Sumaira I. Applications of Plant Flavonoids in the Green Synthesis of Colloidal Silver Nanoparticles and Impacts on Human Health. Iran J Sci Technol Trans A Sci [Internet]. 2017; Available from: https://doi.org/10.1007/s40995-017-0431-6

[11] José A. García J, Cantón, Gomez L, Martinez L. R,. Métodos básicos para el estudio de la sensibilidad a los antimicrobianos. [Internet]. España. Juan J. Picazo.  [consultado el 16 de agosto del 2019].Disponible en: https://www.seimc.org/contenidos/documentoscientificos/procedimientosmicrobiologia/seimc-procedimientomicrobiologia11.pdf

[12]Seetharaman P, Chandrasekaran R, Gnanasekar S. Biogenic gold nanoparticles synthesized using Crescentia cujete L . and evaluation of their di ff erent biological activities. Biocatal Agric Biotechnol [Internet]. 2017;11(March):75–82. Available from: http://dx.doi.org/10.1016/j.bcab.2017.06.004

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