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Synthesis of amino acid esters of the ruthenium naphthalene complex [(C5Me4CH2OH)Ru(C10H8)]+
Acta Polytechnica 58(6):365–369, 2018
doi:10.14311/AP.2018.58.0365
© 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 UVvis 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, chemistry, 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 different 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 AgNPs 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 concern 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 typically prepared via reduction of a silver precursor
using chemical or physical means is dispersion instability against aggregation. One of the possibilities to enhance stabilization of nanoparticles is addition 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 exhibited 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. Functional groups of the biological materials are responsible
for the reduction of the Ag+ and subsequent stabilization 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 minerals [5]. Natural polymer on algae is considered to be
suitable for stabilization of inorganic silver nanoparticles.
We have recently reported that AgNPs biosynthesized 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 synthesized AgNPs were performed by two different ways:
using sodium citrate and both sodium citrate and
gelatin as a reducing and capping agent. This study
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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 temperature 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 ambient temperature to allow reducing the silver ions
into AgNPs.
Chemically synthesized AgNPs were prepared using 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 aqueous AgNO3 (0.29 mM). In the case of chemically synthesized 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 AgNPs 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 temperature and the end point of the reaction was the appearance 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 diskdiffusion 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 sterile swabs (6 mm) placed on agar plates seeded with
microorganisms. The minimum inhibitory concentration (MIC) was read after 7 days of incubation at the
ambient temperature and light mode (12 : 12).
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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 analyzed using an UV–vis spectra from 300 to 800 nm
with UNICAM UV/vis Spectrometer UV4. The absorbance was recorded on days 7 and 120.
Transmission Electron Microscope (TEM; JEM2000FX, 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 either 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 solution 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 indicated 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 spectrum (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 absorption 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 proceeded [12]. Shift of UV-visible spectrum (Fig. 1b)
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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 authors [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 absorption peaks typically located around 400 nm observed
on day 120 in the solutions with biologically synthesized AgNPs as well as gelatin/sodium citrate-AgNPs
(Fig. 1ab) indicated the presence of stable nanoparticles. The broad absorption band observed in citrateAgNPs, (Fig. 1c) indicated much less uniform and
stabilized nanoparticles. The SEM and TEM micrographs (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 peptides and proteins present in gelatin are able readily
interact with metals and hydrophilic ligands protect
gelatin coated AgNPs in aqueous solution. The coating serves to provide proper gap between the silver
core [12, 13].
The silver nanoparticles, which were reached using biological approaches, showed on SEM and TEM
micrographs (Fig. 2a) spherical particles with average particle size of 15 nm. The role of active compounds of biomass responsible for the process of AgNPs formation and stabilization was confirmed by
the work of several authors [6, 15, 23]. Their results 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
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Anna Mražíková, Oksana Velgosová, Jana Kavuličová et al.
gelatin/sodium citrate-AgNPs (from 4 to 55 nm) might
be caused by presence of a higher gelatin concentration 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 chemically synthesized AgNPs against the green algae
P. kessleri were observed in all three cases. The results
revealed that chemically synthesized sodium citrateAgNPs caused only particular inhibition (Fig. 2c)
against algae. Double zone of inhibition observed
around the swabs impreganated with sodium citrateAgNPs 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 eradication 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 easily 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 antimicrobial 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 chemical 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 displayed enhanced antialgal effects in comparison with
citrate-AgNPs. The UV-vis, SEM and TEM analyses 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 AgNPs. 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).
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