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Investigation of antibacterial properties silver nanoparticles prepared via green
Chemistry Central Journal 2012, 6:73doi:10.1186/1752-153X-6-73
Kamyar Shameli (firstname.lastname@example.org})
Mansor Bin Ahmad (email@example.com})
Seyed Davoud Jazayeri (firstname.lastname@example.org})
Parvaneh Shabanzadeh (email@example.com})
Parvanh Sangpour (firstname.lastname@example.org})
Hossein Jahangirian (email@example.com})
Yadollah Gharayebi (firstname.lastname@example.org})
20 May 2012
10 July 2012
27 July 2012
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Investigation of antibacterial properties silver
nanoparticles prepared via green method
Mansor Bin Ahmad1
Seyed Davoud Jazayeri3
Email: parvanh email@example.com
1 Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400
UPM Serdang, Selangor, Malaysia
2 Materials & Energy Research Center, Alborz, Karaj, P.O. Box: 31787/316, Iran
3 Institute of BioSciences, Universiti Putra Malaysia, 43400 Serdang, Selangor
Darul Ehsan, Malaysia
4 Department of Chemical Engineering, Faculty of Engineering, Islamic Azad
University, Malard Branch, Iran
5 Department of Chemistry, Islamic Azad University Behbahan Branch,
University Street, Behbahan 6361713198, Iran
* Corresponding author. Materials & Energy Research Center, Alborz, Karaj, P.O.
Box: 31787/316, Iran
This study aims to investigate the influence of different stirring times on antibacterial activity
of silver nanoparticles in polyethylene glycol (PEG) suspension. The silver nanoparticles
(Ag-NPs) were prepared by green synthesis method using green agents, polyethylene glycol
(PEG) under moderate temperature at different stirring times. Silver nitrate (AgNO3) was
taken as the metal precursor while PEG was used as the solid support and polymeric
stabilizer. The antibacterial activity of different sizes of nanosilver was investigated against
Gram–positive [Staphylococcus aureus] and Gram–negative bacteria [Salmonella
typhimurium SL1344] by the disk diffusion method using Müeller–Hinton Agar.
Formation of Ag-NPs was determined by UV–vis spectroscopy where surface plasmon
absorption maxima can be observed at 412–437 nm from the UV–vis spectrum. The
synthesized nanoparticles were also characterized by X-ray diffraction (XRD). The peaks in
the XRD pattern confirmed that the Ag-NPs possessed a face-centered cubic and peaks of
contaminated crystalline phases were unable to be located. Transmission electron microscopy
(TEM) revealed that Ag-NPs synthesized were in spherical shape. The optimum stirring time
to synthesize smallest particle size was 6 hours with mean diameter of 11.23 nm. Zeta
potential results indicate that the stability of the Ag-NPs is increases at the 6 h stirring time of
reaction. The Fourier transform infrared (FT-IR) spectrum suggested the complexation
present between PEG and Ag-NPs. The Ag-NPs in PEG were effective against all bacteria
tested. Higher antibacterial activity was observed for Ag-NPs with smaller size. These
suggest that Ag-NPs can be employed as an effective bacteria inhibitor and can be applied in
Ag-NPs were successfully synthesized in PEG suspension under moderate temperature at
different stirring times. The study clearly showed that the Ag-NPs with different stirring
times exhibit inhibition towards the tested gram-positive and gram-negative bacteria.
Silver nanoparticles, Green chemistry, Polyethylene glycol, Antibacterial activity, Reaction
Silver nanoparticles (Ag-NPs) have been known for its inhibitory and bactericidal effects in
the past decades . Antibacterial activity of silver containing materials can be applied in
medicine for reduction of infections on the burn treatment [2,3], prevention of bacteria
colonization on catheters [4,5] and elimination of microorganisms on textile fabrics [6,7] as
well as disinfection in water treatment . Besides that, Ag-NPs were also being reported in
the literature to exhibit a strong cytoprotective activity towards human immunodeficiency
virus (HIV) infections . Polyethylene glycol (PEG) is frequently used in the polymer
blends production to improve the biocompatibility of its film due to its wide range of
molecular weights, excellent solubility in aqueous medium, low toxicity, chain flexibility and
biocompatibility properties. Although PEG has non biodegradability properties, it is readily
excreted from the body and forms non-toxic metabolites . Besides that, PEG was able to
act both as reducing agent and stabilizer . In several research studies [12,13], researchers
proposed that longer polymer chain of PEG exhibits higher reducing activity and provides
higher stability in forming Ag-NPs. These can effectively prevent agglomeration of Ag-NPs.
There are numerous techniques to perform antibacterial and antimicrobial susceptibility tests.
The techniques include agar disk diffusion, broth dilution (macrodilution and microdilution),
agar dilution and E test method (modification of the disk diffusion and the agar dilution
method) . Agar disk diffusion is a traditional and routine method for antimicrobial
susceptibility tests . It has advancement to be used in this project because of its
reliability, low cost and simplicity [16,17]. Mueller Hinton agar is chosen among the culture
media because it gives satisfactory growth for most nonfastidious organisms like
Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli and it shows good
bacteria culture reproducibility .
There are many synthetic routes that have been developed to synthesize Ag-NPs due to the
applications found tremendously in wide range of fields. Among the synthetic routes includes
chemical reduction [19,20], thermal decomposition , electrochemical , sonochemical
, photochemical , microwave , radiation assisted process [26,27] and currently by
green chemistry synthesis [28-31].
Chemical reduction method is widely used to synthesize Ag-NPs because of its readiness to
generate Ag-NPs under gentle conditions and its ability to synthesize Ag-NPs on a large scale
. However, these chemical synthesis methods employ toxic chemicals in the synthesis
route which may have adverse effect in the medical applications and hazard to environment.
Therefore, preparation of Ag-NPs by green synthesis approach has advantages over physical
and chemical approaches as it is environmental friendly, cost effective and the most
significant advantage is that conditions of high temperature, pressure, energy and toxic
chemicals are not required in the synthesis protocol .
In this work, we reported “green” synthesis of Ag-NPs using sugar and PEG. This method
was performed by reducing the silver nitrate (AgNO3) in different stirring times of reaction at
moderate temperature with sugar and PEG used as green reducing agent and polymeric
stabilizer. The antibacterial activity of silver/polyethylene glycol [Ag(PEG)] were tested with
Mueller-Hinton agar disc diffusion method against Staphylococcus aureus (S. aureus), and
Salmonella typhimurium SL1344 (S. typhimurium SL1344).
Results and discussion
In this research, the PEG was appropriate as a stabilizer and polymeric media for reducing the
AgNO3 using sugar as a green reducing agent. As shown in Figure 1, after 1 and 3 h, the
colourless solution turned to yellow which indicates the initial formation of Ag-NPs.
Similarly, when the time of reaction was increased to about 6 h, the colour changed to the
light brown. However, when the solution was further stirred for a period of 48 h at a
temperature of 25, the colour of the solution change to dark brown and then gray. These
observations show that with the increase time of reaction, particle size and aggregation of
silver nanocrystal gradually increased.
Figure 1 Photograph of Ag-NPs prepared at different times of reaction in PEG solution
in the moderate temperatures for 1, 3, 6, 24 and 48 h (a–e), respectively
Sugar as an aldehyde can reduce silver ions to Ag-NPs and through this process oxidizes
itself gluconic acid . The possible chemical equations for preparing the Ag-NPs are:
(aq) (aq) aq
2[Ag PEG ]CH OH(CHOH) CHO2[Ag PEG ] CH OH(CHOH) COOH (2)
After dispersion of silver ions in the PEG aqueous solution matrix (Equation 1), PEG reacted
with the Ag to form a PEG complex [Ag(PEG]+, which reacted with sugar to form
[Ag(PEG)] due to the reduction of silver ions through the oxidation of sugar to gluconic acid
The formation of Ag-NPs in the polymeric media was further determined by using the UV–
visible spectroscopy, which was shown on the surface plasmon resonance (SPR) bands.
Figure 2 (A–C) shows that Ag-NPs started forming when [Ag(PEG)]+ reacted with suger at a
moderate temperature. However, the [Ag(PEG)]+ peak was not observed at beginning (0 h) of
the reaction until after about 1 h of the reaction time, the absorbance peaks could be seen at
different stirring times after the reaction started. Generally, the SPR bands are influenced by
the size, shape, morphology, composition and dielectric environment of the prepared
nanoparticles [35,36]. Previous studies have shown that the spherical Ag-NPs contribute to
the absorption bands at around 400 nm in the UV–visible spectra . From this research, the
SPR band characteristics of Ag-NPs were detected around 412–437 nm (Figure 2A, B),
which strongly suggests that the Ag-NPs were spherical in shape and have been confirmed by
the TEM results of this study. As shown, when the stirring time of reaction was increased, the
intensity of the SPR peak also gradual increase until 24 h but after 48 h the SPR peak change
to broad shape and intensity decreased, this phenomenon is related to the increased size and
also agglomeration of silver nano-crystals . Therefore this shows that the reduction of the
silver ions to silver atoms continued and resulted in an increase in the concentration of Ag-
Figure 2 The Ultraviolet–visible spectra curve of Ag-NPs prepared in PEG solution at
different times in the moderate temperatures (A–C)
Thus, there is a normal case in this situation for the SPR absorption band for the particles,
which agreed with the TEM results, whereby red–shifts were observed as size increased in
the during the reaction after 1, 3, 6, 24 and 48 h respectively. This can be explained by the
multilayer Mie theory model, which theorizes that the chemical interaction caused the
lowered electron conductivity in the outermost atomic layer and consequently caused the red–
shifts . As seen from the Figure 2C, it can be observed that 24 h had large absorbance
compared to 48 h because the particle size of Ag-NPs after 48 h were larger than those at 24
h. Also, absorption spectra of larger metal colloidal dispersions can exhibit broad peaks or
additional bands with the lower absorbance in the UV-visible range due to the excitation of
plasma resonances or higher multipole plasmon excitation . This phenomenon could be
due to the fact that, after reaching a certain particle size, the stabilizer was not able to
withhold the nanoparticle’s size effectively, which resulted in its very large size.
The TEM images and their corresponding particle size distributions of Ag-NPs at different
periods of time are shown in Figure 3. The TEM images and their size distributions revealed
that, the mean diameters and standard deviation of Ag-NPs were about 10.60 ± 3.75,
11.23 ± 7.91, 15.30 ± 7.64 and 25.31 ± 9.44 nm for 3, 6, 24 and 48 h (A–D), respectively. The
total numbers of Ag-NPs counted for each TEM images were about 32, 107, 226 and 64 for
3, 6, 24 and 48 h respectively. These results approved that with increase in time of reaction at
a moderate temperature, mean diameters and standard deviations of the Ag-NPs gradually
Figure 3 Transmission electron microscopy image and the particle size distribution for
Ag-NPs in PEG for the stirring times of 3, 6, 24 and 48 h, respectively (A–D)
Powder X–ray diffraction
Figure 4 shows the XRD patterns of Ag-NPs formed in the 6 h, 24 h, and 48 h from stirring
time of reaction, which indicates the formation of the silver crystalline structure. The XRD
peaks in the wide angle range of 2θ (30° < 2θ < 80°) ascertained that the peaks in 38.04°,
44.08°, 64.36° and 77.22° can be attributed to the 111, 200, 220, and 311 crystalline
structures of the face centered cubic (fcc) synthesized silver nano–crystal, respectively (Ag
XRD Ref. No. 00–004–0783) . The intensities of 111, 200, 220 and 311 reflections due to
the Ag-NPs phase were also found to increase along with the increased Ag-NPs in the
polymeric media (Figure 4a–c). The peaks showed that the main composition of
nanoparticles was silver and clearly no obvious other peaks present as impurities were found
in the XRD patterns. Therefore, this gives clear evidence for the presence of Ag-NPs in the
Figure 4 X-ray diffraction patterns of Ag-NPs synthesized in PEG after 6, 24 and 48 h
The average particle size of silver nanoparticles can be calculated using Debye–Scherrer
Where K is the Scherrer constant with value from 0.9 to 1 (shape factor), where λ is the X-ray
wavelength (1.5418 Å), β1/2 is the width of the XRD peak at half height and θ is the Bragg
angle. From the Scherrer equation the average crystallite size of Ag-NPs for 6, 24 and 48 h
times of reaction are found to be around 10–25 nm, which are also in line with the
observation of the TEM results discussed later.
Zeta potential measurement
As shown in the Figure 5, the Ag-NPs obtained possess a positive zeta potential value. Zeta
potential is an essential parameter for characterization of stability in aqueous Ag-NPs
suspensions. A minimum of ±30 mV zeta potential values is required for indication of stable
nano-suspension . At the 6, 24 and 48 h of stirring times zeta potential were equals to
54.5 ± 7.8, 42.4 ± 4.7 and 28.3 ± 3.2 mV respectively. So, these results clearly indicates that
the particles are fairly stable at the 6 h stirring time of reaction, but the stability decreased
when the reaction time was increased to 24 h and 48 h respectively.
Figure 5 Zeta potential for [Ag (PEG)] suspension after 6, 24 and 48 h from stirring
FT-IR chemical analysis
The interaction of Ag-NPs obtained with PEG and gluconic acid products by reduction of
sugar compound were confirmed by FT-IR spectra. Intense absorptions are observed at 1730,
1630 and 1007 cm−1. The IR band at 1730 cm−1 is characteristic of the C = O stretching mode
of the carboxylic acid group for gluconic acid. The bands due to C–O stretching mode got
merged in the very broad envelope centered on 1268 and 1007 cm−1 arising from C–O, C–O–
C stretches and C–O–H bends vibrations of Ag-NPs in PEG. Also, the aliphatic C–H
stretching, in 1413 and 1344 cm−1 were due to C–H bending vibrations (Figure 6a) . After
the bio-reaction of sugar with the AgNO3 in the PEG matrix, the created peak in 1730 cm−1
certified to the binding of –C = O for carboxylic acid in gluconic acid, and the shift in the
peak at 1007 cm−1 towards lower frequency compared to peak in 1094 cm−1 for PEG is
attributed to the binding of C–C–O and C–C–H groups with nanoparticles . The broad
peaks in 503, 407 and 291 cm−1 are related to Ag-NPs banding with oxygen from hydroxyl
groups of PEG chains (Figure 6b). On the other hand, as for the sugar spectrum (Figure 6c),
the absorption bands at 3246 cm−1 was due to the O–H stretching band, 2901 cm−1 was due to
the aliphatic C–H stretching, 1442, 1374 and 1339 cm−1 were due to C–H bending vibrations,
and also the combination band of O–C–H and C–O–H deformation is calculated from 1442 to
1339 cm−1. Then the plane C–H and O–H deformation from 1220 to 998 cm−1 can be
observed. The region from 1145 to 554 cm−1 contains C–O and C–C groups’ vibration modes
are present and the carbohydrates generally shows their characteristic bands .
Figure 6 Fourier transform infrared spectra for PEG (a), [Ag (PEG)] after 48 h (b)
from stirring times and sugar (c)
Thus, as shown hydroxyl group of PEG as capping agent can make a cover in the surface of
Ag-NPs. This is possible because the surface of Ag-NPs is positively charged. Certainly, we
suppose that colloidal stabilization for [Ag (PEG)] occur due to the presence of van der waals
forces between the oxygen negatively charged groups present in the molecular structure of
the PEG, and the positively charged that surround the surface of the inert Ag-NPs [45,47].
Therefore, the FT-IR spectra showed the existence molecular interactions between the Ag-
NPs with the chain of polymeric media . As shown in the Figure 6, schematic illustrated
the interaction between the charged of Ag-NPs that capped with PEG [28,48].
Inhibition zone values were obtained for PEG, [Ag(PEG)]+ (A0) and [Ag (PEG)] suspension
at the different stirring times 3 (A2), 6 (A3), 24 (A4) and 48 h (A5) and tested against S.
aureus and S. typhimurium. The results and images of inhibition zones are presented as the
average values in Table 1 and Figure 7, respectively. Table 1 shows that the AgNO3 and Ag-
NPs in PEG suspension gave high and similar antibacterial activity against Gram-negative
and Gram-positive bacteria. Because of their size, Ag-NPs can easily reach the nuclear
content of bacteria and they present the large and impressive surface area; thus, the contacts
with bacteria were the greatest [49,50]. This could be the reason behind their excellent
antibacterial effect. In the polymeric matrix systems, some researcher argue that silver ions
released from the surface of Ag-NPs are responsible for their antibacterial activity [51,52]. In
the aqueous phase systems, the results show that the antibacterial activity of Ag-NPs at 3 and
6 h stirring times in S.aureus is higher than that of the Ag+ ions. Similarly, the antibacterial
activity of Ag-NPs in S. typhimurium is generally higher than that of the Ag+ ions. With the
exception of the Ag-NPs at 3 h stirring time, the activity decreased with increase in stirring
time (6, 24 and 48 h). The high activity at the 6 h stirring time Ag-NPs is perhaps related to
large surface area of the nanoparticles . The diameters of inhibition zone in the agar plate
are given in mm. The tests were replicated three times for each treated samples and the
results are presented in Table 1. The solution of PEG (10 mg/ml) did not show any
antibacterial activity. The [Ag(PEG)]+ (A0) suspension for all tested bacteria shows high
antibacterial activity and interestingly these effects in the [Ag (PEG)] (A2–A5) were
increased with the decreasing size of Ag-NPs. However, a higher Ag-NPs loading doesn’t
improve the antibacterial activity .
Table 1 Average inhibition zone and standard deviation for PEG, [Ag (PEG)]+ (A0) and
[Ag (PEG)] suspension (A2–A5) at different stirring times (3, 6, 24 and 48 h),
A0 A2 A3
11.65 ± 0.56
9.67 ± 0.33
12.78 ± 0.12
9.44 ± 0.36
13.64 ± 0.29
11.51 ± 0.43
10.64 ± 0.39
9.71 ± 0.14
10.62 ± 0.36
Abbriviation: NA, Not Appear; CTX, Chloramphenicol; C, Cefotaxime
Figure 7 Comparison of the inhibition zone test between Gram-positive and Gram-
negative bacteria [S. aureus and S. typhimurium] for PEG, [Ag (PEG)]+ (A0) and [Ag
(PEG)] suspension at different stirring times [A2–A5 (3, 6, 24 and 48 h)]
In summary, we have described a simple and green method of colloidal Ag-NPs synthesis by
using green reducing agents which requires no special physical conditions. Ag-NPs were
successfully synthesized under moderate temperature (45 °C) at different stirring times of
reaction. The formation of Ag-NPs was confirmed in the UV-visible absorption spectra,
which showed the SPR band characteristics of Ag-NPs in the range of 412–437 nm. The
XRD results confirmed that the Ag-NPs possessed a face-centered cubic crystal structure
(fcc). In addition, this also revealed that Ag-NPs were the main composition present in the
nanocomposites without any contamination peaks. The TEM images showed that the Ag-NPs
were in spherical shape and the average diameters of the particles were 10.60, 11.23, 15.30
and 25.31 nm for the stirring times of 3, 6, 24 and 48 h, respectively. FTIR spectrum
suggested the complexation present between PEG and Ag-NPs to form metallopolymer [Ag
(PEG)] and the stability of the Ag-NPs was confirmed with the zeta potential measurements.
The antibacterial activities of [Ag (PEG)] at the different particle size of Ag-NPs were
showed antibacterial activity against the Gram-positive and Gram-negative bacteria. These
results show that the antibacterial activities of Ag-NPs in PEG can be modified with the size
of Ag-NPs and it decreases with the increase in the particle size. Needless to say, further
studies are required to investigate the biological effects of [Ag (PEG)] suspension on the
types of bacteria for potential widening of this subject area.
All reagents in this effort were analytical grade and were used as received without further
purification. AgNO3 (99.98 %) was used as a silver precursor, and was provided by Merck,
Germany. PEG (Mw 3,350) used as a stabilizer for the preparation of Ag-NPs which was
purchased from Sigma–Aldrich (USA). Meanwhile, the sugar was used as a green reducing
of silver ions to Ag atoms and was obtained from BDH Chemical Ltd., Poole, UK. All
solutions were freshly prepared using double distilled water and kept in the dark to avoid any
photochemical reactions. All glassware used in experimental procedures were cleaned in a
fresh solution of HNO3/HCl (3:1, v/v), washed thoroughly with double distilled water, and
dried before use.
Synthesis of Ag-NPs by using green method
The preparation of Ag-NPs in the PEG matrix is quite directly forward. In a typical synthesis,
a 10 mL of a 1.0 M solution of AgNO3 was added to 200 mL of a 0.1 wt.% aqueous solution
of soluble PEG to obtain the clear solution
components, 20 mL of a 1.0 M aqueous solution of sugar was then added and further stirred.
The solution obtained was distributed into 5 cuvettes, which were stirred and maintained at
different periods of time: 1 (a), 3 (b), 6 (c), 24 (d), and 48 h (e), respectively. Throughout the
reduction process, all solutions were kept at a constant temperature of 60 °C in the dark to
avoid any photochemical reactions. All solution components were purged with nitrogen gas
prior to use. Subsequently, reduction proceeded in the presence of nitrogen to eliminate
oxygen. The obtained colloidal suspensions of [Ag (PEG)] were then centrifuged at 20000
rpm for 15 min and washed four times to remove silver ion residue. The precipitate
nanoparticles were then dried overnight at 40 °C under vacuum overnight to obtain the Ag-
After complete dissolution of these
Evolution of antibacterial activity
The in vitro antibacterial activity of the samples was evaluated by utilizing the disc diffusion
method using Müeller–Hinton Agar (MHA) with determination of inhibition zones in
millimeter (mm), which conform with recommended standards of the National Committee for
Clinical Laboratory Standards (NCCLS; now renamed as Clinical and Laboratory Standards
Institute, CLSI, 2000). Salmonella typhimurium (S. typhimurium SL1344) and
Staphylococcus aureus (S. aureus) (ATCC 25923) were used for the antibacterial effect
assay. Briefly, the sterile paper discs (6 mm) impregnated with 20 μl of PEG,
and [Ag (PEG)] (3, 6, 24 and 48 h) with different treatment times were suspended in sterile
distilled water and were left to dry at 37 °C for 24 h in a sterile condition. The bacterial
suspension was prepared by making a saline suspension of isolated colonies selected from
tryptic soy agar plate, the agar plates were grown for 18 to 24 h. The suspension was adjusted
to match the tube of 0.5 McFarland turbidity standard using the spectrophotometer of 600
nm, which equals to 1.5 × 108 colony–forming units (CFU)/ml. The surface of MHA was
completely inoculated using a sterile swab, which steeped in the prepared suspension of
bacterium. Finally, the impregnated discs were placed on the inoculated agar and incubated at
37 °C for 24 h. After incubation, the diameter of the growth inhibition zones was measured.
Chloramphenicol (30 μg) and Cefotaxime (30 μg) were used as the positive standards in order
to control the sensitivity of the bacteria. All tests were done in triplicate.
Characterization methods and instruments
The prepared Ag-NPs were characterized by using the X–ray diffraction (XRD), transmission
electron microscopy (TEM), ultraviolet–visible spectroscopy, Fourier transform infrared
(FT–IR) spectroscopy and zeta potential measurements. The XRD patterns were recorded at a
scan speed of 2° min–1. Meanwhile, the structures of the produced Ag-NPs were examined
using Shimadzu PXRD–6000, powder X–ray diffraction. Moreover, TEM observations were
carried out using the Hitachi H–7100 electron microscopy, whereas the particle size
distributions were determined using the UTHSCSA Image Tool software (Version 3.00). To
make sure the formation of Ag-NPs, the colloids solutions were tested for their optical
absorption property using a Shimadzu H.UV, 1650 PC UV–visible spectrophotometer over
the range of 300 to 700 nm. The FT–IR spectra were however recorded over the range of
200–4000 cm−1 utilizing the Series 100 Perkin Elmer FT–IR 1650 spectrophotometer. The
zeta potential measurements were also performed using a Zetasizer Nano–ZS (Malvern
We declare that we have no competing interests.
KS carried out the synthesis, and characterization of the compounds, acquisition of data,
analysis and interpretation of data collected. SDJ carried out the antibacterial experiments.
MBA and PS involved in drafting of manuscript, revision of draft for important intellectual
content and give final approval of the version to be published. All authors read and approved
the final manuscript.
The authors thank from University Putra Malaysia (UPM) for its financial support (RUGS,
Project No. 9199840). The authors are also grateful to the staff of the Department of
Chemistry UPM for their help in this research, Institute of Bioscience (IBS/UPM) for
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