Biochemical changes in cyanobacteria during the synthesis of silver nanoparticles.

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 11/25/14 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

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Biochemical changes in cyanobacteria during the synthesis of silver

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nanoparticles

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L. Cepoi1, L. Rudi1, T. Chiriac1, A. Valuta1, I. Zinicovscaia2,3*, Gh. Duca4,

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E. Kirkesali3, M. Frontasyeva3, O. Culicov3,5, S. Pavlov3, I. Bobrikov3

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Institute of Microbiology and Biotechnology of the Academy of Science of Moldova, 1, Academiei Str., 2028 Chisinau, R. Moldova

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Institute of Chemistry of the Academy of Science of Moldova, 3, Academiei Str.,Chisinau, Moldova 3

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Joint Institute for Nuclear Research, 6 Joliot-Curie Str., 141980, Dubna, Russia, [email protected]

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Academy of Science of Moldova, 1, Stefan cel Mare Ave., 2001, Chisinau, Moldova

National R&D Institute for Electrical Engineering ICPE-CA, Splaiul Unirii, Nr. 313, District 3, 030138, Bucharest Romania

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_____________________

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Corresponding author:

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I. Zinicovscaia,

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Phone: +74962162436

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Fax: +74962165085

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E-mail: [email protected]

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Abstract:

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The methods of synthesis of silver nanoparticles by cyanobacteria Spirulina platensis and Nostoc

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linckia were studied. The complex of biochemical, spectral and analytical methods was used for

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biomass characterization and assessment of the changes of its main components (proteins, lipids,

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carbohydrates, and phycobilin) during nanoparticle formation. The size and shape of silver

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nanoparticles in the biomass of both types of cyanobacteria were determined. Neutron activation

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analysis was used to study the accumulation dynamics of the silver quantity. The analytical results

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suggest that the major reduction of silver concentration in solutions and increase in biomass occur

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within the first 24 hours of experiments. While in this time interval minor changes in the Nostoc

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linckia and Spirulina platensis biomass took place, a significant reduction of the level of proteins,

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carbohydrates, phycobiliproteins in both cultures and lipids in Spirulina platensis was observed after

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48 hours. At the same time, the antiradical activity of the biomass decreased. The obtained results

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show the necessity of determination of optimal conditions for the biomass-solution containing silver

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ions interaction, at which nanoparticle formation takes place and no biomass degradation occurs at

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silver nanoparticle formation by the studied cyanobacteria.

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Keywords: silver, nanoparticles, Spirulina platensis, Nostoc linckia, optical and analytical methods,

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biochemical analysis

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Introduction

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Microorganisms such as bacteria, microalgae, yeast and fungi have been used in recent years

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to develop non-toxic and environment friendly methods to synthesize nanoparticles (Seshadri et al.

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2012; Brayner et al. 2007; Maruthamuthu et al. 2012; Lengke et al. 2007). It has been reported that

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some microalgae can produce silver, gold, palladium and platinum nanoparticles intra- and

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extracellularly (Brayner et al. 2007; Lengke et al. 2007; Jenaa et al. 2013; Luangpipat et al. 2011;

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Mubarak et al. 2011). Among microorganisms, cyanobacteria are of great interest for nanoparticle

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formation research because they are a potential source of novel metabolites that have great

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importance from a biotechnological and industrial point of view (Galhan et al. 2011; Sudha S.S., et

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al. 2013; Roychoudhury R., Pal R., 2014).

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Cyanobacteria Spirulina platensis is widely used as a matrix for pharmaceuticals with some

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essential elements as well as a biologically active food additive for humans and animals. The ability

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to bio-transform and endogenously add the desired essential nutrients, such as Se, I, Cr, and other

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into a product that can be properly used by a human organism is a distinctive feature of Spirulina

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platensis. (Mosulishvili et al. 2002, 2007).

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Members of cyanobacteria belonging to the family Nostocaceae are considered the most

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impressive “biochemical factories” of the biological world. The genus Nostoc is a valuable source of

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a wide spectrum of secondary metabolites, such as antioxidant enzymes, phycobiliproteins,

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vitamins, and phenolic compounds that have many uses as anti-cancer, anti-HIV, antimalarial,

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antifungal and/or antimicrobial drugs (Galhan et al. 2011).

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Some metals are essential elements for living cells, being components of enzymes, hormones

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and vitamins. In this regard living organisms elaborated the mechanisms of active metal uptake from

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the environment. Microalgae have the ability to accumulate and assimilate metals at low 3


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concentrations from the solutions. The effectiveness of these processes is enabled by optimal

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surface/volume ratio, the large number of highly specific binding groups on the cell wall and by the

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efficient uptake and retention of metals. Besides, essential metals necessary for survival, microalgae

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can accumulate from the aquatic environment toxic, radioactive and precious metals (Arunakumara

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and Xuecheng 2008; Zhou et al. 2012; Rajamani et al. 2007; Cecal et al. 2012a, 2012b).

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In case of microalgae, metal toxicity primarily results from metal binding to sulphydryl

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groups in proteins, the destruction of the protein structure, or displacement of the essential

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functional elements in enzymes (Arunakumara and Xuecheng 2008). Reductive-oxidative reactions,

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which result in the change of a metal’s oxidation state, are one of the mechanisms associated with

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metal toxicity reduction by cyanobacteria. Depending on metabolism, particularity of specific

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species, this process can also lead to nanoparticle synthesis.

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Among noble metal nanomaterials, silver nanoparticles have received considerable attention due to their attractive physicochemical properties (Elechiguerra et al. 2005).

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Silver has been known to have a disinfecting effect and has been found in applications

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ranging from traditional medicines to culinary items. It has been reported that silver nanoparticles

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are non-toxic to humans and most effective against bacteria, virus and other eukaryotic

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microorganism at low concentrations and without any side effects (Savithramma et al. 2011). As

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antimicrobial agents, silver nanoparticles are considered as an alternative to silver ions, which are

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obtained from silver nitrate. However, silver ions have the disadvantage of forming complexes, and

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therefore the effect of the ions remain only for a short period of time. This disadvantage has been

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overcome through the use of silver nanoparticles, which are inert. Silver nanoparticles exhibit

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antimicrobial function by binding to reactive groups in bacterial cells, resulting in their precipitation

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and inactivation. [Sahayaraj and S. Rajesh 2011; Parashar et al. 2009).

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Silver nanoparticles have a broad range of applications, such as in nonlinear optics, textile

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engineering, biotechnology, bioengineering, water treatment cancer diagnostics and treatment,

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spectrally selective coating for solar energy absorption, bio-labeling, intercalation materials for

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electrical batteries, as optical receptors, and catalysts in chemical reactions (Seshadri et al. 2012;

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Jenaa et al. 2013; Singh and Balaji Raja 2011; Prabhu and Poulose 2012; Priyadarshini et al. 2013;

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Firdhouse and Lalitha 2012).

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At the same time, nanoparticle accumulation can affect the viability of the cells. It has been

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shown that silver nanoparticles have an inhibitory effect on the components of photosystem PS II

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(Oukarroum et al. 2012). The interaction of microalgae cells with silver nanoparticles leads to the

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accumulation of oxygen, which results in the destruction of the chlorophyll structure and metabolic

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disturbances (Saison et al. 2010).

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Thus, Spirulina platensis (Arthrospira) and Nostoc linckia can be used as nanofactories, as

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well as a source of different bioactive substances. Considering the assessment of quality of the

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obtained nanomaterials and effectiveness of biotechnological process, it is very important to strike a

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balance between biochemical composition and presence of nanoparticles in the biomass. This can be

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achieved by determining the optimal conditions in which nanoparticle formation takes place and no

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biomass degradation occurs.

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The aim of the present study was (i) the formation and characterization of silver

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nanoparticles produced by the cyanobacteria Spirulina platensis and Nostoc linckia and (ii) the

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determination of changes in the antioxidant activity of protein, lipid, phycobiliprotein, and

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carbohydrate content as well as biomass extract.

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A complex of biochemical, optical and analytical methods was used for the characterization

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of silver nanoparticles in the biomass of the studied cyanobacteria, as well as for assessing the

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changes in the contents cyanobacteria’s important components.

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Materials and Methods

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Materials

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Cyanobacterial biomass preparation

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To carry out the experiment, algological pure cultures of cyanobacteria Spirulina platensis

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CNM-CB-02 strain and Nostoc linckia CNM-CB-03 from the National Collection of Nonpathogenic

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Microorganisms (Institute of Microbiology and Biotechnology, Academy of Sciences of Moldova)

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were used. The cultivation of Spirulina platensis cells was carried out in an open-type tank with a

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volume of 60 L in the SP-1 nutritive medium (Rudic 2007) at a temperature of 32-35ºС, illumination

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∼ 3000-4000 lux, рН 8-9 and at constant mixing. The culture of Nostoc linckia CNM-CB-03 (from

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the same collection) is grown in laboratory conditions on mineral medium Gromov 6 (Gromov and

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Titova 1983) stirring daily, continuous illumination (a light intensity of 2000-3000 lx), at a

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temperature of 25-30 oC and the optimum pH of 6.0-7.0.

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On stationary growth phase (6th day for S. platensis and 10th day for N. linckia) the cyanobacteria

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biomass was separated from the culture medium. To avoid precipitation of Ag+ ions through

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reaction with Cl- and PO43- ions presented in culture medium cyanobacteria biomass was washed

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with 3.5% ammonium acetate. Obtained biomass (1g for S. platensis and 0.3 g for N. linckia) were

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resuspended in 250 ml Erlenmeyer flasks with 100 mL of 100 mg/L aqueous silver nitrate (AgNO3)

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solution for synthesis of silver nanoparticles. The resulting mixtures were put into the shaker

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repeatedly at 28-30 °C for different periods of time (24-72 hours). Biomass separated from culture

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medium, washed with ammonium acetate and re-suspended in distillate water served as the control. 6


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For UV-Vis spectral analysis, a 2-ml sample was taken after different time intervals and absorbance was measured. For biochemical studies the native biomass was used.

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For SEM, X-ray analysis, NAA, and XRD the Nostoc linkia biomass was harvested by

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centrifugation at 12000g for 8 min, and this wet biomass was dried at 105 oC; Spirulina platensis

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biomass was dried from solution at 105 oC.

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Methods

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UV-vis Spectrometry

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The UV-vis (Ultraviolet-Visual) spectra of the samples were recorded by UV-VIS

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Spectrophotometer T80+, TG Instruments Ltd with a wavelength range of 300-800 nm, with the

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interval of 1 nm.

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Scanning Electron Microscopy (SEM)

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Scanning Electron Microscopy (SEM) was carried out using the Quanta 3D FEG.

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Operational features of the microscope used in the experiment: magnification 5000–150000x;

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voltage 1–30 kV.

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Energy-Dispersive Analysis of X-Rays (EDAX)

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Microprobe analysis of silver nanoparticles was conducted with the energy-dispersive X-ray

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analysis spectrometer (EDAX, USA). The acquisition time ranged from 60 to 100 s, and the

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accelerating voltage was 20 kV.

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X-ray Diffraction (XRD)

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XRD measurements of the microbial biomass were performed at Empyrean Multi-Purpose

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Research X-Ray Diffractometer. The Cu anode (CuKα: λ= 1.541874 Å) was used as a source of

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radiation.

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Neutron Activation Analysis (NAA)

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The silver concentrations were determined by means of neutron activation analysis (NAA) at

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the reactor IBR-2 of the Frank Laboratory of Neutron Physics of the Joint Institute for Nuclear

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Research (Dubna, Russia). The experimental equipment and irradiation conditions of samples are

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described elsewhere (Frontasyeva and Pavlov 1999). The silver content was determined using the

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633.0 keV γ-line of

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approximately 1.6×1013n cm-2 s–1. The NAA data processing and determination of elemental

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concentrations were performed using Genie 2000 and the software developed at FLNP JINR

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(Dmitriev and Pavlov 2013).

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Ag by irradiation for 60 s under a thermal neutron fluency rate of

Biochemical analysis of biomass components

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The protein content was determined according to methodology proposed by Lowry et al. (1951).

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Phycobiliproteins content was assessed following Boussiba and Richmond (1980) methodology

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modified by Rudic and Bulimaga (1998). Carbohydrates were determined by applying Antron

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reagent (Filipovich et al. 1975) and lipids were determined spectrophotometric using fosfovanilinic

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reagent (Johnson et al. 2007). Determination of antioxidative activity

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For antioxidant tests ethanolic and water extracts were prepared. For each sample 10 mg of the

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biomass was mixed with 10 ml ethanol or water for 60 min. After filtration the extracts were kept at

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0 °C.

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The antioxidative activity was determined by application of non-biological tests with radicals

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ABTS (2.2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2.2-diphenyl-1-

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picrylhydrazyl) (Cepoi et al. 2009).

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Result and Discussion

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NAA was used to determine the change in the total silver concentration in biomass of

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cyanobacteria after 24, 48, and 72 hours of interaction with silver nitrate. The NAA results for

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Nostoc lincia (a), as well as Spirulina platensis (b) biomass are shown in Fig. 1. For both types of

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cyanobacteria, the total concentration of silver increase identically, and the main changes occur

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during the first 24 hours of reaction.

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The data obtained by NAA illustrate the fact that during the first day the metal concentration

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increases rapidly and then does not change significantly for the next few days. It is known that in the

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first ‘rapid’ phase the metal ions are mainly adsorbed onto the surface of microorganisms by the

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functional amino, carboxylic, sulfhydryl, phosphate, and thiol groups that can bind metal ions. In the

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second ‘slow’ phase, the metal ions are reduced to metallic form by biomolecules on the microbial

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surface or partially transported through the cell membrane into the cytoplasm and accumulated

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intracellularly (Chojnacka et al. 2005). The same dynamics were observed in the present study.

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It can be suggested that a part of adsorbed silver can be transformed to nanoparticles. The

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formation of silver nanoparticles by the studied cyanobacteria was assessed by registration of the

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specific absorption peaks on the biomass absorption spectra.

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The addition of cyanobacterial biomass to silver nitrate solution resulted in the color change

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of the solution from green to yellow-green for Spirulina platensis and from brown to read-brown for

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Nostoc linckia. These color changes arose because of the excitation of surface plasmon vibrations

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with the silver nanoparticles. The UV-Vis absorption spectra of the suspensions of the studied

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cyanobacteria for different incubation time are shown in Fig. 2. The absorption spectra of Ag

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nanoparticles formed in the reaction media have absorbance maxima at 438 nm. The intensity of the

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peak increases as a function of the reaction time.

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The reduction of Ag ions and the resulting growth of Ag (0) must have been driven by some

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active species in the algal biomass. According to Mie theory, only a single SPR band is expected in

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the adsorption spectra of spherical nanoparticles, whereas anisotropic particles could give rise to two

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or more SPR bands depending on the shape of the particles (Sosa et al. 2003). In the present case, as

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confirmed by SEM images, a single band was observed that gives evidence for the presence of

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spherical shaped silver nanoparticles.

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Fig. 3 presents the SEM image of S. platensis cells after interacting with silver nitrate

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solution for different incubation time. The SEM images show that most of the particles are

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spherical-like and create big agglomerates. The obtained images revealed the great number of silver

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nanoparticles formed by the biomass of S. platensis extracellularly.

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The EDAX X-ray spectra proved the presence of silver nanoparticles in Spirulina platensis

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cells that were treated with silver nitrate solution. The energy of X-rays is characteristic of the

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elements from which these X-rays are emitted. A spectrum of the energy versus relative counts of

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the detected X-rays is presented in Fig. 4.

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One peak of Ag was observed for biomass of Spirulina platensis. The signals from C, K, Ca,

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S, P, and Mg atoms were also recorded. These signals are likely to be due to X-ray emission from

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the proteins and enzymes present in the cell walls of the biomass.

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In case of Nostoc linckia (Fig.5) the nanoparticles are well-distributed on the bacterial

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surface. Cyanobacteria Nostoc cells are surrounded with an exopolysaccharidic layer that can remain

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covalently linked or loosely attached to the cell surface, or be released into the surrounding

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environment (Pereira et al. 2009). It is suggested that exopolysaccharides play the role of reducing

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and stabilizing agent, as these complex polymers can also contain acetylated amino sugar moieties,

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as well as non-carbohydrate constituents, such as phosphate, lactate, acetate and glycerol (De Vuyst

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and Degeest 1999).

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Fig.6 shows the EDAX spectrum of biomass Nostoc linckia with silver nanoparticles. In this

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spectrum one peak of Ag was observed. The signals from C, K, Na, Cl, Ca, S, P, and Mg atoms to

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be due to X-ray emission from the cell walls were also recorded.

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The X-ray diffraction method was used for examining the samples of Spirulina platensis and

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Nostoc linkia biomass with the silver nanoparticles. In Fig.7 XRD patterns of the silver

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nanoparticles synthesized by Spirulina platensis and Nostoc linckia refined by Rietveld method

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(Young, 1993) are shown. The stars on the figure indicate impurity phases. The grey color shows

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regions of the pattern excluded from the refinement.

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In all cases the diffractograms showed the presence of silver crystals of cubic form (Space group is Fm3m) with the lattice parameter 4.098±0.004Å characteristic of Ag.

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A number of Bragg’s reflections corresponding to the structure of silver are seen here: four

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characteristic peaks (111), (200), (220) and (311). The obtained results clearly show that silver

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nanoparticles formed by reduction of Ag (I) ions are crystalline in nature. The Scherrer equation was

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used for an approximate estimation of the size of nanoparticles using the line broadening on the

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diffractogram:

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d = Kλ/βcosθ,

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where K is the shape factor; for cubic crystals it is 0.9–1;

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λ is the X-ray wavelength for CuKα; β is the line broadening at half the maximum intensity in

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radians;

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θ is the Bragg angle, and d is the size of a nanoparticle in nm.

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It is important to realize that the Scherrer formula is applicable to grains less than 0.15 µm in

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size. For the approximate estimation of the size of nanoparticles all silver peaks were used. The

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calculations were carried out in Full Prof program (Roisnel and Rodriguez-Carvajal, 2001).

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The results obtained show that at the concentration of AgNO3 100 mg/L the size of silver nanoparticles in Spirulina platensi are ≈6 nm and in Nostoc linkia ≈4-5 nm.

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In order to determine the changes of biochemical characteristics of Spirulina and Nostoc

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biomass after the process of silver nanoparticle formation, the contents of the essential parameters

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(proteins, lipids, carbohydrates, and phycobilins) were monitored. The results obtained are shown in

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Figs. 8-13.

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The content of proteins in the Nostoc biomass after interaction with AgNO3 decreases

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slightly in the first 24 hours (Fig. 8а). In the next 24 hours the protein content was reduced by 45 %

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however during this time period up to 72 hours there was not a significant change in protein levels.

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During 72 hours the protein content of Nostoc was reduced by 51 %. Consequently, the Nostoc

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biomass was exposed to an extensive process of protein degradation.

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As a result of exposure to silver nitrate, the protein contents of the Spirulina biomass was

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reduced by 22.7 % in the first 24 hours of reaction (Fig. 8b) followed by a 71.7 % reduction after 48

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hours. After 72 hours the proteins content in the biomass constituted 11.2 %. This drastic decrease

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of proteins content indicated biomass degradation.

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For cyanobacteria the decrease in phycobilin content was most pronounced (Fig. 9a). For the

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Nostoc biomass it was decreased by 83% during 24 hours. After 24 hours of interaction with silver

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nitrate phycoerythrin was reduced by 86%, phycocyanin by 62% and allophycocyanin by 68%.

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After 48 hours of exposure the phycoerythrin content was reduced by 89 %, phycocyanin by 99 %

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and allophycocyanin by 75 %. After 72 hours the phycoerythrin content was reduced by 92 %. The

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similar results were obtained for Spirulina biomass (Fig. 9b). After 72 hours of reaction the

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phycoerythrin content was very small: 0.12 % of phycocyanin and 0.09 % of allophycocyanin.

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The carbohydrate content in Nostoc biomass was reduced from 41.6 to 38.6 % in the first 24

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hours and reached 32 % after 72 hours (Fig. 10a). It is suggested that carbohydrates perform a

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protective function which is weakened during the experiment. In contrast to proteins, carbohydrates

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show a high rate of resistance. A different picture appears in case of the Spirulina biomass (Fig.

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10b). In Spirulina, carbohydrates have a structural function in the form of endopolysaccharides

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Their degradation is very rapid and coincides with lipid degreadation. After 72 hours of exposure to

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silver nitrate the carbohydrate content was reduced by 84 %. Polysacharides constitute 2.4 % in the

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biomass compared to the initial 14.8 %.

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Lipids from Nostoc execute mainly the structural functions of membranes of functional

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components. The low rate of lipid degradation confirms this fact. For Nostoc biomass in the first 48

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hours of reaction with silver nitrate the lipid content does not change (Fig. 11a). After 72 hours the

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content of lipids changed only by 9.7 %. In case of Spirulina biomass the lipid content degradation

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was evident. As a result of interaction with silver nitrate during 24 hours the lipid content decreased

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by 26.5 %, after 48 and 72 hours by 59 %, and 62 %, respectively (Fig. 11b).

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Nostoc and Spirulina biomass are also a sourse of components with antioxidant activity

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(Cepoi et al. 2009). It is well known, that cyanobacteria cells respond to the presence of toxic metals

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by changing their antioxidative status. This thereby affects both the survability of the cells and the

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quality of the biomass obtained in these conditions. To determine antiradical activity, the ethanolic

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and water extracts were obtained from the Nostoc and Spirulina biomass.

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The antiradical capacity of ethanolic and water extracts was evaluated by applying tests with

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DPPH radical and ABTS cation-radical. The results for the extracts from the Nostoc biomass after

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exposure to silver nitrate are presented in Fig. 12.

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The ABTS test indicates a more pronounced reduction of the antiradical capacity of the water extract from the Nostoc biomass in comparison with the ethanolic extract.

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Both tests showed essential changes in antiradical activity of ethanolic and water extracts

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after the first 24 hours of contact of the Nostoc biomass with silver ions. This indicates the ability of

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the Nostoc biomass to resist oxidative stress caused by high metal concentration After 48 hours

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antiradical activity related to cation-radical ABTS extracts from the Nostoc biomass sharply

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decreased by 12.5 % from the initial value for ethanol extracts and by 66 % for water ones.

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In case of Spirulina biomass (Fig.13) the antiradical activity of water and ethanol extracts

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was reduced substantially in 48 hours of reaction. The ABTS test indicates the reduction of water

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extract activity by 62.5 % in comparison with 51.5 % shown by the DPPH test. The activity of the

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ethanolic extract after interaction of the biomass with silver nitrate decreased by 84.4 % in the

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ABTS test and by 35.8 % in the DPPH tests. The reduction of Spirulina biomass antiradical activity

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was more evident in comparison with the Nostoc biomass.

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Conclusions

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The results of the presented studies show that Spirulina platensis and Nostoc linkia are active

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accumulators of silver ions from the solution. A part of the accumulated silver is converted into

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nanoparticles. The silver nanoparticles in both cases are mainly formed extracellularly. The shape of

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the majority of the nanoparticles is spherical and on average their size is: ≈ 6 nm for Spirulina

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platensi and ≈ 4-5 nm for Nostoc linkia.

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Biochemical tests performed on the essential parameters of the biomass for the studied

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cyanobacteria showed the toxic effect of silver ions on the cells. Silver nanoparticles are more toxic

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for Spirulina platensis than for Nostoc linckia.

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This drastic decrease of protein, carbohydrate, lipid and phycobilin content indicated

321

Spirulina biomass degradation. The reduction of Spirulina biomass antioxidative activity was more

322

evident in comparison with Nostoc linckia biomass. For Nostoc linckia less modification of

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biochemical components than for Spirulina platensis were observed.

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It should be mentioned that during the first 24 hours of biomass interaction with silver nitrate

325

solution the composition of the biomass changed insignificantly (except the phycobiliprotein

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content).

327 328

In addition, high antiradical activity of ethanolic and water extracts from the biomass remains unchanged.

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The results of the performed investigation show that for biotechnological purposes the time

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of silver nanoparticle synthesis using cyanobacteria should be minimized to avoid biomass

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destruction.

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Acknowledgements

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Presented work was partially supported by the JINR GRANT No.13-402-03

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No conflict of interest.

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Fig. 1. Total silver concentrations in biomass of: (i) Spirulina platensis and (ii) Nostoc linckia

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Fig. 6. EDAX spectrum of Nostoc linckia with silver nanoparticles

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linkia

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Fig. 8. Change of protein content in (a) Nostoc linckia and (b) Spirulina platensis biomass after

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exposure to silver nitrate Fig. 9. Change of phycobilin content in (a) Nostoc linckia and (b) Spirulina platensis biomass after exposure to silver nitrate Fig. 10. Change of carbohydrate content in (a) Nostoc linckia and (b) Spirulina platensis biomass after exposure to silver nitrate Fig. 11. Change of lipid content in (a) Nostoc linckia and (b) Spirulina platensis biomass after exposure to silver nitrate Fig. 12. The antiradical capacity of extracts from Nostoc linckia biomass after exposure to silver nitrate 21


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Fig. 13. Antiradical capacity of extracts from Spirulina platensis biomass after exposure to silver nitrate

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Biochemical Evaluation of Silver Nanoparticles in Wistar Rats.

Synthesis of silver nanoparticles and antibacterial property of silk fabrics treated by silver nanoparticles.

Bacteriagenic silver nanoparticles: synthesis, mechanism, and applications.

Synthesis of monodisperse, quasi-spherical silver nanoparticles with sizes defined by the nature of silver precursors.

Screening of cyanobacterial extracts for synthesis of silver nanoparticles.

Optimization of Biological Synthesis of Silver Nanoparticles using Fusarium oxysporum.

Green synthesis of hyaluronan fibers with silver nanoparticles.

Facile and green synthesis of silver nanoparticles using oxidized pectin.

Sonochemical synthesis of silver nanoparticles using starch: a comparison.

Green synthesis of silver nanoparticles by Phoma glomerata.

Green synthesis of silver nanoparticles using Andean blackberry fruit extract.

Green Synthesis of Robust, Biocompatible Silver Nanoparticles Using Garlic Extract.

Investigation of silver nanoparticles synthesis using aminated β-cyclodextrin.

Antifungal activity of silver nanoparticles obtained by green synthesis.

Synthesis, characterization and biological applications of mycosynthesized silver nanoparticles.

Synthesis and characterization of silver nanoparticles from (bis)alkylamine silver carboxylate precursors.

Photosensitized synthesis of silver nanoparticles using Withania somnifera leaf powder and silver nitrate.

Silver nanoparticles: Synthesis methods, bio-applications and properties.

Silver Nanoparticles: Synthesis, Characterization, Properties, Applications, and Therapeutic Approaches.

silver nanocomposites.

The role of tannic acid and sodium citrate in the synthesis of silver nanoparticles.

Advances in Metabolic Engineering of Cyanobacteria for Photosynthetic Biochemical Production.

Biochemical changes during the initial stages of industrial lead exposure.

Green synthesis, optical properties and catalytic activity of silver nanoparticles in the synthesis of N-monosubstituted ureas in water.