Biomimetic synthesis of silver nanoparticles using the fish scales of Labeo rohita and their application as catalysts for the reduction of aromatic nitro compounds

Article abstract of DOI:10.1016/j.saa.2014.04.065

In this article, a cleaner, greener, cheaper and environment friendly method for the generation of self assembled silver nanoparticles (Ag NPs) applying a simple irradiation technique using the aqueous extract of the fish scales (which is considered as a waste material) of Labeo rohita is described. Gelatin is considered as the major ingredient responsible for the reduction as well as stabilisation of the self assembled Ag NPs. The size and morphology of the individual Ag NPs can be tuned by controlling the various reaction parameters, such as temperature, concentration, and pH. Studies showed that on increasing concentration and pH Ag NPs size decreases, while on increasing temperature, Ag NPs size increases. The present process does not need any external reducing agent, like sodium borohydride or hydrazine or others and gelatin itself can play a dual role: a 'reducing agent' and 'stabilisation agent' for the formation of gelatin-Ag NPs colloidal dispersion. The synthesized Ag NPs were characterised by Ultraviolet-Visible spectroscopy (UV-Vis), Transmission electron microscopy (TEM) and Selected area electron diffraction (SAED) analyses. The synthesized Ag NPs was used to study the catalytic reduction of various aromatic nitro compounds in aqueous and three different micellar media. The hydrophobic and electrostatic interaction between the micelle and the substrate is responsible for the catalytic activity of the nanoparticles in micelle.

Full text of DOI:10.1016/j.saa.2014.04.065

Accepted Manuscript
Bio mimetic synthesis of silver nanoparticles using the fish scales of Labeo
rohita and their application as catalysts for the reduction of aromatic nitro com-
pounds
Tanur Sinha, M. Ahmaruzzaman, A.K. Sil, Archita Bhattacharjee
PII:
S1386-1425(14)00638-6
http://dx.doi.org/10.1016/j.saa.2014.04.065
SAA 12029
DOI:
Reference:
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
Received Date:
Revised Date:
Accepted Date:
31 December 2013
7 April 2014
13 April 2014
Please cite this article as: T. Sinha, M. Ahmaruzzaman, A.K. Sil, A. Bhattacharjee, Bio mimetic synthesis of silver
nanoparticles using the fish scales of Labeo rohita and their application as catalysts for the reduction of aromatic
nitro compounds, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://
dx.doi.org/10.1016/j.saa.2014.04.065
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Bio mimetic synthesis of silver nanoparticles using the fish scales of Labeo
rohita and their application as catalysts for the reduction of aromatic nitro
compounds
Tanur Sinha, M. Ahmaruzzaman*, A. K. Sil, Archita Bhattacharjee
Department of Chemistry, National Institute of Technology, Silchar-788010, Assam,
India.
*
Corresponding Author. Tel.: +919435522348
E-mail- md_2002@rediffmail.com (M. Ahmaruzzaman)

ABSTRACT: In this article, a cleaner, greener, cheaper and environment friendly method for
the generation of self assembled Silver nanoparticles (Ag NPs) applying a simple irradiation
technique using the aqueous extract of the fish scales (which is considered as a waste
material) of Labeo rohita is described. Gelatin is considered as the major ingredient
responsible for the reduction as well as stabilisation of the self assembled Ag NPs. The size
and morphology of the individual Ag NPs can be tuned by controlling the various reaction
parameters, such as temperature, concentration, and pH. Studies showed that on increasing
concentration and pH Ag NPs size decreases, while on increasing temperature, Ag NPs size
increases. The present process does not need any external reducing agent, like sodium
borohydride or hydrazine or others and gelatin itself can play a dual role: a „reducing agent‟
and „stabilisation agent‟ for the formation of gelatin–Ag NPs colloidal dispersion. The
synthesised Ag NPs were characterised by Ultraviolet-Visible spectroscopy (UV-Vis),
Transmission electron microscopy (TEM) and Selected area electron diffraction (SAED)
analyses. The synthesised Ag NPs was used to study the catalytic reduction of various
aromatic nitro compounds in aqueous and three different micellar media. The hydrophobic
and electrostatic interaction between the micelle and the substrate is responsible for the
catalytic activity of the nanoparticles in micelle.
Keywords: Silver nanoparticles, Fish scales, gelatin, nitro-compounds, reduction, micelle

1
. INTRODUCTION
Noble metal nanoparticles (NPs) especially silver nanoparticles (Ag NPs) have shown
enormous interest to many research groups for their fascinating applications in the field of
electronics, catalysis, and pharmaceuticals etc. [1] These applications are based on different
.
chemical and physical properties attributed with nanomaterials due to their unique electronic
structure and extremely large surface area which were not observed from those of the bulk
and atomic species. However, various methods have been developed [2] for the synthesis of
Ag NPs in organic solvents, using toxic reducing agents like sodium borohydride and N, N-
dimethyl formamide.
Hence, for cleaner preparation of these noble metal nanoparticles, various biomimetic
approaches [3-4] are being explored. Green technology, in the niche of preparative protocols
of nanomaterial stands for switching to environmentally benign starting material and
reducing agent, water as the reaction medium and selection of non-toxic substances of Ag
NPs stability [5] with minimal wastage in terms of energy and raw materials [6-27].
Hence, in this paper, we develop an ecofriendly, clean, non-toxic, facile chemically
preparative method, for the generation of Ag NPs using the extract of fish scales of Labeo
rohita, acting as reducing as well as stabilising agent. The effects of various reaction
parameters, such as temperature, pH and concentration of the extract in the synthesis of Ag
NPs were also investigated. We have also studied the catalytic activity of these synthesised
Ag NPs in the reduction of several aromatic nitro compounds in aqueous and micellar
medium. The present synthesis process and the catalysis reaction are simple, environment
friendly, and robust.

2
. EXPERIMENTAL DETAILS
2
.1Materials:
Silver nitrate (AgNO ), p-nitro phenol (4-NP), o-nitro phenol (2-NP), p-nitro aniline (4-NP),
3
sodium borohydride (NaBH ), lithium dodecyl sulphate (LDS), cetyl pyridinium chloride
4
(CPC), and triton x-100 (TX-100) of A.R Grade was purchased from Sigma-Aldrich and used
as received. Double distilled water was used in all experiments.
2
.2 Preparation of fish scale extract:
0
2
g of Lobeo rohita fish scales were thoroughly washed and boiled for 20 min at 60 C in 100
ml double distilled water. The resulting solution was filtered and used for further
experiments.
2
.3 Synthesis of silver nanoparticles:
1
0 ml of 0.1M aqueous solution of silver nitrate was taken in a container and 10 ml of
different concentrations of fish scale extract (10, 20, 30 and 40%) were added separately and
0
heated at 70 C for 10 min followed by slow cooling of the solutions to room temperature.
The solution was then allowed to stabilize for 3 days. After 3 days the solutions with brown
sediments were formed at the bottom of the container which indicated the presence of Ag
NPs .These were then centrifuged, filtered and the residues were washed several times with
double distilled water to remove unbound polymers to yield the Ag NPs. Also samples of Ag
NPs were prepared by adding 10 ml of 0.1M aqueous solution of silver nitrate to10 ml of
0
1
0% of fish scale extract and heating at different temperatures (60, 70, 80, 90 and 100 C)
separately. Another set of samples of Ag NPs were prepared by heating 10 ml of 0.1M
0
aqueous solution of silver nitrate and 10 ml of 10% of fish scale extract at 70 C and keeping
the pH of the stock solutions at ( 0.5, 1, 1.5, 2, 3 and 4) separately.

2
.4 Characterization of silver nanoparticles:
The UV-Vis absorption spectra of the synthesised Ag NPs were recorded on Cary 100 Bio
spectrophotometer (max in nm) equipped with 1-cm quartz cell. The TEM, HR-TEM images,
and SAED pattern were recorded using JEOL-JEM 2100 transmission electron microscope
operated at an accelerating voltage of 200 kV. The TEM samples of Ag NPs were prepared
by placing the solution drops over the carbon coated copper grids and allowing the solvent to
evaporate at room temperature.
2
.5 Catalytic activity of the synthesised silver nanoparticles:
-
In a standard quartz cuvette, having 1 cm path length, 2 ml of water and 60 µl of ( 6.07x10
3
M ) nitro compounds (4-nitro phenol, 2-nitro phenol, and 4-nitro aniline) were taken
separately and the cuvette was then placed in a UV-Vis spectrometer and the absorbance was
recorded. To each of these nitro compound solutions, 350 µl of aqueous NaBH (0.1M) was
4
added and the absorbance was noted. Thereafter, 150 µl of 10% Ag NPs solution was added
to that mixture and the absorbance was recorded up to 20 m when the peak due to the nitro
compounds was no longer observed and the appearance of a new peak at ~ 430 nm was
noticed.
3
. RESULTS AND DISCUSSION
3
.1 UV-Vis analysis:
The Ag NPs exhibits a unique UV-Visible adsorption band derived from collective oscillation
of conduction electrons upon interaction with electromagnetic radiation, which is known as
localized surface plasmon resonance (LSPR). The shifting of these bands provides
information of the particle size, chemical surrounding, adsorbed species on the surface and

dielectric constant. Fig. 1 shows the UV-Visible spectra of the synthesised Ag NPs with
different concentration of fish scale extract (10, 20, 30, and 40%).
The spectra revealed the presence of Ag NPs in all the mixtures and the optical properties
exhibited by the Ag NPs (shown in the inset of Fig. 1) clearly indicates that the particles may
have different size or shape (Morphology). At low concentration, the absorption band (~420
nm) is originating mostly from surface Plasmon vibrations of spherical Ag NPs. However
with increase in concentration, the absorption bands become broader with increased intensity
and shifted to longer wavelength region (~450 nm). The broad absorption indicates the
aggregation of spherical NPs or a change in the dimension of spherical anisotropic
nanostructures, while an increased intensity reflects that the percentage of population of the
particles are increasing with increase in concentration of the extract in the solution.
Fig. 2 represents the UV-visible spectra of Ag NPs formed at different temperatures (60, 70,
0
8
0, 90, and 100 C). The spectra revealed the formation of Ag NPs possessing different size
0
and morphology. At low temperature (60 C), a band due to the surface Plasmon resonance of
Ag NPs appeared at about ~ 420 nm and shifted to longer wavelength (red shift) with an
0
increase in broadness of the band at high temperature (100 C). Usually, a red shift is
associated with an increase in particle size or with the withdrawal of electron density from
the surface [28-33]. Further, the optical properties exhibited by the solutions of Ag NPs
(
shown in inset of Fig. 2) indicated that the particles may have different size or shape
morphology). This increase in broadness reflects a change in dimension of spherical
(
anisotropic nanostructures or aggregation of Ag NPs.
Fig. 3 shows the UV-visible spectra of Ag NPs formed at different pH (3 and 4). At low pH (
pH= 3), a band due to the surface Plasmon resonance of Ag NPs appeared at about ~ 440 nm
which shifted to shorter wavelength (blue shift) at high pH (pH= 4). Usually, a blue shift is
associated with a decrease in particle size or donation of electron density from the surface

[
34]. These results show that concentration, temperature and pH are important factors for the
formation and size distribution of nanoparticles.
3
.2 TEM and SAED studies:
The morphology and size of the Ag NPs have been analyzed using TEM and SAED pattern.
Fig.4 (a) and Fig. 4 (c) corresponds to the TEM images of the synthesized Ag NPs using 10%
and 40% fish scale extract respectively. The SAED pattern of Fig. 4(a) and Fig. 4(c) is shown
in Fig. 4(e) and Fig. 4(f), and Fig. 4(b) and Fig. 4(d) are the magnified images of Fig. 4(a)
and Fig. 4(c), respectively. Fig. 4(a) and Fig. 4(c) display the formation of spherical Ag NPs.
It is apparent from the TEM images that spherical Ag NPs are polydispersed in nature with
the average size of spherical NPs ~17.47, and ~16.5 nm. Thus with increase in concentration
of the extract, particle size of Ag NPs decreases. The polycrystalline nature of nanoparticles
can be evidenced from the concentric diffraction rings in a SAED pattern as shown in Fig.
4
(e) and Fig. 4(f), respectively. The spots in Fig. 4(e) could be indexed corresponding to the
reflections from the (111) and (200) lattice planes, while the spots in Fig. 4(f) could be
indexed corresponding to the reflections from the (111), (200) (220), and (311) lattice planes.
The lattice spacing calculated from Fig. 4(b) and Fig. 4(d) is almost 0.26 and 0.23 nm and
corresponds to (111) lattice plane of face centered cubic (FCC) structure of silver [35].
0
0
TEM images of the synthesized Ag NPs at 60 C and 100 C are presented in Fig. 5(a), and
Fig. 5(b), respectively. The SAED pattern of Fig. 5(a) and Fig. 5(b) is shown in Fig. 5(c) and
Fig. 5 (d). The magnified images of Fig. 5(a) and Fig. 5(b) are presented in Supplementary
material as shown in Fig. A (1) and Fig. A (2), respectively. Fig. 5(a) and Fig. 5(b) displayed
the formation of spherical Ag NPs. It is apparent from the TEM image that spherical Ag NPs
are polydispersed with an average size of spherical NPs ~9.36, and ~22.96 nm. Hence,
average particle size of Ag NPs increases with increase in temperature. The concentric
diffraction rings in SAED pattern shown in Fig. 5(c) and Fig. 5(d) reveals the polycrystalline

nature of particles. The spots could be indexed corresponding to the reflections from the
111), (200), and (111), (200), (220), and (311) lattice planes as shown in in Fig. 5(c) and Fig.
(d), respectively. The fringe spacing calculated from Fig. A (1) and Fig. A (2) are 0.21 nm
(
5
and 0.23 nm, respectively and corresponds to (111) lattice plane of face centered cubic (FCC)
structure of silver [35].
TEM images of the synthesized Ag NPs at pH = 3 and pH= 4 are presented in Fig. 6(a) and
Fig. 6(b), respectively. Fig. 6 (c) and Fig. 6(d) are the SAED pattern of Fig.6 (a) and Fig.6
(b), respectively. Fig. B (1) and Fig. B (2) (shown in supplementary material) are the
magnified images of Fig. 6(a) and Fig. 6(b), respectively. Fig. 6 (a) and 6(b) indicated the
formation of spherical Ag NPs. It is apparent from the TEM image that spherical Ag NPs are
polydispersed in nature with the average size of spherical NPs ~46.14 nm, and ~10.33 nm.
Therefore, on increase in pH of the solution, average particle size decreases. Polycrystalline
nature of particles can be evidenced from the concentric diffraction rings in a SAED pattern
shown in Fig. 6(c) and Fig. 6(d). The spots could be indexed corresponding to the reflections
from the (111), (200), (220) and (311) lattice planes in Fig. 6(c) and (111), (200), (220)
lattice planes in Fig. 6(d) respectively. The lattice spacing calculated from Fig. B(1) and Fig.
B(2) are found to be 0.25nm and 0.23 nm, respectively, both of which corresponds to (111)
lattice plane of face centered cubic (FCC) structure of silver [35]. From all the TEM images it
is found that the Ag NPs are predominantly spherical in shape. These spherical NPs also
consist of dark regions surrounded by a faint thin shell with a capping agent possibly of
organic material (gelatin) present in the extract. Hence, from the above it is apparent that
particle size can be tuned by controlling the reaction parameters.
3
.3 Probable mechanism for the formation of Ag NPs:
It is well known that aqueous AgNO is not a stable chemical and can be decomposed easily
3
either by heating or by UV light illumination [36]. Under such conditions, Ag (0) is the sole

product after realising oxygen and nitrogen dioxide and in such case the Ag (0) formed non-
uniform micron-size Ag particles in the absence of any stabilising agent. So, the presence of
stabilising agent is very much essential for the synthesis of Ag NPs. Here, fish scale extract
serves as both reducing as well as stabilising agent.
Fish scales are biocomposites of high ordered, type–I collagen fibres (41-84%) and
hydroxyapatite Ca (OH) (PO ) [37-39]. Type-I collagen is a heterotrimeric copolymer
1
0
2
4 6
composed of two α (I) and one α (I) polypeptide chains, containing approximately 1050
1
2
amino acids each. Each polypeptide chain has a conformation of a left handed, polyproline-II
type helix, which are twisted together into a right handed coil, a triple helix which
–
represents a quaternary structure, being stabilised by numerous hydrogen bonds and
intramolecular vander waals interactions [40] as well as by some covalent bonds [41], and
further associated into right –handed micro fibrils and fibrils, being further assembled into
collagen fibres [42] with unusual strength and stability.
0
When the aqueous solution of these fish scales is heated at 60 C for 20 min, the collagen
present in these scales get denatured to a mixture of random–coil single, double and triple
strands [43] which is accompanied by change in physical and chemical properties due to
collapse of the triple helical structure . Such modified collagen is called denatured collagen
(gelatin) and can be evidenced from Fig. C (Supplementary material), where a peak at ~270
nm corresponds to the absorption of denatured collagen or gelatin [44]. These collagens are
made up of polypeptide chains, containing amino acids, having variable compositions,
particularly for the minor constituents, depending on raw materials and processes used,
proximate value by weight are [45] glycine 21%, proline 12%, hydroxyproline 12% ,
glutamic acid 10% , alanine 9%, arginine 8%, aspartic acid 6%, lysine 4%, serine 4% ,
leucine 3%, valine 2%, phenyl alanine 2%, threonine 2%, isoleucine 1%, hydroxylysine 1% ,
methionine and histidine <1 % with tyrosine < 0.5% , and contain functional groups such as

suspended double bonds,-NH , -SH and –COOH endowing it as a reducing as well as
2
stabilising agent.
It is believed that the mechanism for the formation of Ag NPs proceeds via 2 steps as shown
in the scheme 1. In the first step, when AgNO solution is added to the fish scale extract, they
3
+
formed an instantaneous gelatin-Ag complex where the positively charged Ag (I) ions were
initially bound with the negatively charged group of gelatin via electrostatic interaction which
can be evidenced by the shift of the absorption of the UV-visible spectra (Fig. D) (
Supplementary material) by 5-6 nm as well as dramatic increase in the absorption intensity as
compared to the absorption band of pure gelatin (Fig. C) (Supplementary material). In
addition, a weak hump appeared at around 350-400 nm due to the formation of Ag (I)-gelatin
complex. The next step involves the heating of the solution mixture for 10 min, so that the
formed complex starts reducing and ultimately self-assembled Ag NPs were formed after
cooling for 3 days at room temperature (Fig.7). we believe that the reduction mechanism of
all the Ag (I) ions proceed via the autocatalytic growth route, where once a metal atom is
evolved as a nucleation centre it can act as a catalyst for the reduction of the remaining metal
ions present in the solution via autocatalysis [46].
Thus, it was observed that gelatin itself acted as a reducing as well as stabilising agent for the
formation of self-assembled Ag NPs without the addition of reducing agent like NaBH4,
sugar, hydrazine etc.
3
.4 Catalytic reduction of 4-NP, 2-NP and 4-NA using self-assembled Ag NPs on gelatin as a
catalyst in aqueous medium:
To investigate the catalytic efficiency of self-assembled Ag NPs on gelatin in depth, the
reduction of 4-NP was monitored in the presence of NaBH as a model reaction [47-48]. The
4

+
Gelatin
+
Ag+
Gelatin-Ag complex
Heat,10min,
Cooled for
days
3
Self-assembled
Silver nanoparticle
Scheme 1
Mechanism for the formation of self-assembled Ag NPs
0
0
-
reduction of 4-NP [E (4-NP/4-AP) =-0.76V] by NaBH [E (H BO /BH ) = -1.33V] is
4
3
3
4
thermodynamically feasible but kinetically restricted in the absence of a catalyst.
It is observed that 4-NP in aqueous medium has a maximum absorption at 317 nm as shown
in Fig.8. However, when freshly prepared NaBH solution is added, there was a red shift
4
from 317 nm to 403 nm, and the light yellow colour of the solution changes to intense yellow
(Fig. E) (Supplementary material), due to the formation of 4-nitrophenolate ions in alkaline
condition. The peak at 403 nm remains unchanged even for a couple of days in the absence of
any catalyst.
Addition of 150 µl of 10% Ag NPs solution into the reaction mixture caused a gradual fading
of the characteristic yellow colour due to 4-NP and finally complete bleaching of the yellow
colour of the 4-NP solution was observed. This decolourisation was quantitatively monitored
spectrophotometrically with time and it has been noted as a successive decrease of peak
height. This is due to the reduction of 4-NP to 4-amino phenol (4-AP). A new peak centred at

4
20 nm due to the surface Plasmon resonance of Ag NPs was observed after completion of
the reduction (at 1200 s). In the mean time, a new peak at 298 nm appeared with progressive
increase in intensity (Fig.9), which is attributed to the typical absorption of 4-AP. This results
suggests that the catalytic reduction of 4-NP exclusively yielded 4-AP without any side
products [49].
In the reduction process, the overall concentration of NaBH was 0.1M and 4-NP was
4
-3
6
.07×10 M. Considering much higher concentration of NaBH compared to 4-NP, the
4
pseudo–first-order kinetics has been applied with respect to 4-NP to determine the catalytic
activity of Ag NPs. The absorbance of 4-NP is proportional to its concentration in solution;
the absorbance at time t (A ) and time t=0 (A ) are equivalent to the concentration at time t
t
0
(
(
(
C ) and time t=0 (C ). The rate constant (k) has been determined from the linear plot of ln
t 0
-4 -1
A /A ) versus reduction time in seconds, and the constant was estimated to be 6.0 ×10 s
t
0
Fig. 10).
Reduction of 2-nitro phenol (2-NP) and 4-nitro aniline (4-NA) (each having concentration
-3
6
.07×10 M), was also examined using 0.1 M NaBH and 10% Ag NPs as catalyst (spectra
4
not shown). The yellow colour due to 2-NP and 4-NA was discharged during the course of
the reduction and their absorbance values at their respective λmax positions decreased
gradually in presence of NaBH and Ag NPs as catalyst. It was found that two new peaks
4
gradually appeared for 4-nitro aniline (4-NA) at 300 and 209 nm, both of which are due to p-
phenylenediamine in alkaline aqueous solutions [50]. Similarly, in aqueous alkaline solution,
a new peak was observed at 406 nm due to 2-nitrophenolate ion and the peak gradually
disappeared during the course of NaBH reduction. A new peak at 298 nm appeared due to
4
the formation of 2-amino phenol (2-AP). The spectra of these products were compared with
the pure compounds and their rate constants were determined. It was found that their
reduction rates followed the order 4-NP˃ ꢀ-NP˃ ꢁ-NA.

3
.5 Mechanism of reduction of 4-NP to 4-AP by Ag NP in presence of NaBH4:
The reaction of NaBH with metal surfaces and its decomposition in presence of metal
4
nanoparticles attracts the limelight of research due to its applications in fuel cells [51-52].
The mechanism of reduction of 4-NP to 4-AP by NaBH in presence of Ag NPs is discussed
4
in terms of Langmuir- Hinshelwood model [51, 53] as illustrated in Fig. 11.
On the surface of Ag NPs, the borohydride ions get adsorbed and transfer a surface- hydrogen
species to the surface of the nanoparticles [51, 54]. It is a reversible step and can be explained
in terms of a Langmuir isotherm [51]. The 4-NP molecules are adsorbed on the surface of the
nanoparticles at the same time. It is also a reversible step and can also be explained in terms
of Langmuir isotherm [51]. The adsorption/desorption equilibriums and diffusion of reactants
to the nanoparticles are considered to be fast [51]. The reduction of 4-NP is the rate
determining step which occurs due to the reaction of adsorbed 4-NP with the nanoparticles
surface-bound hydrogen atoms (Fig.11). When the final product, 4-AP undergoes desorption,
the metal surface is set free and the catalytic cycle begins again [51].
3
.6 Catalytic reduction of 4-NP using self-assembled Ag NPs on gelatin as a catalyst in
micellar medium:
Micelles not only increase the suspendibility of metal nanoparticles (stabilisation) but also
refresh the surface of catalyst particles [55-57] and thereby facilitate in studying the reaction
kinetics [58] for heterogeneous catalysis. It is also used in various ratios to control the
particle shape and size [59]. However, the exact mechanism of interaction between metal
particles and the surfactant stabilizer has not yet been resolved [60-61]. To study the
interactions between the substrates and the catalyst, the reduction of 4-NP was meticulously
examined in three different micelles.

In micelles, a weak hydrophobic force operates between the substrate (4- nitrophenolate ion)
and the micelle. This hydrophobic interaction restricts the easy approach of the substrate
towards the metal nanoparticle surfaces. In addition, micelles build wrapper–like barriers
-
surrounding the metal particles, whereas BH4 tends to remain outside the dynamic barrier
due to lack of hydrophobic part. Accordingly, lowering the reduction rate in micelles in
comparison to the reduction rate in aqueous medium for a particular metal is perceivable.
Adsorption of the phenolate ion onto the nanoparticle surface does not get disturbed, when
the aqueous system is replaced by an anionic micellar solution, lithium dodecyl sulphate
(
LDS). This is due to the negatively charged micellar surface that prevented incorporation of
the substrate (4-NP) into the LDS micelle. As a result, the reduction rate in LDS rendered
itself comparable to that in aqueous medium. Due to the presence of both hydrophobic and
hydrophilic interactions between the phenolate ion and the positively charged micellar
surface in cationic micelle cetyl pyridinium chloride (CPC), adsorption of the substrate onto
the catalyst was retarded which decreased the rate to a considerable extent. Whereas
hydrophobic interaction becomes operative in non-ionic micelle, Triton-X-100 (TX-100),
between the substrate and the uncharged micellar surface, which give rise to an intermediate
rate of reaction. Therefore, the rate of reaction in three different micelle followed the
sequence of H O~LDS˃ ˃ritonꢂ-100˃ ꢃPꢃ. ˃he absorbance of ꢁ-NP as a function of time in
2
three different micelle (just above CMC) has been depicted in Fig.12. From the shift of acid-
base equilibrium [62], the location of the substrate, phenolate ions in micelle can be
-
quantified. Finally, Fig.13 schematically represents the location of the substrate (4-NP), BH ,
4
and Ag NPs in three different micellar surfaces and electron transfer reaction.

4
. CONCLUSION
In this paper, for the first time we have successively developed an environment friendly,
green, cheap and simple method for the synthesis of self-assembled silver nanoparticles using
a waste material extract (fish scale extracts) of Labeo rohita which itself played a dual role of
stabilising and reducing agent. Thus, the present method does not require the addition of any
external reducing agents, such as sodium borohydride or hydrazine or others. The sizes of the
Ag NPs were tuned just by controlling the various reaction parameters, such as temperature,
concentration and pH. It is observed from the TEM images that spherical Ag NPs are
polydispersed in nature with the average size of spherical NPs of ~16.5, and ~17.47 nm using
1
0% and 40% fish scale extract, respectively. It was also found that average size of spherical
Ag NPs increased with increase in temperature and found to be ~9.36, and ~22.96 nm, at
0
0
6
0 C and 100 C, respectively. The TEM studies indicated that the average size of Ag NPs are
~46.14 nm, and ~10.33 nm, at pH=3 and 4, respectively. The results showed that
concentration, temperature and pH are important factors for the formation and size
distribution of nanoparticles. These Ag NPs were utilized as a catalyst for the reduction of
several aromatic nitro-compounds into their corresponding amino derivatives. This procedure
for the synthesis of Ag NPs is useful for large-scale synthesis. This study gives a clear insight
of heterogeneous catalysis even in micellar media.
ACKNOWLEDGEMENTS
Tanur Sinha wants to acknowledge TEQIP-II of NIT Silchar for providing the financial
assistance and SAIF–NEHU Shillong for providing the TEM facilities.
REFERENCES
1
. N.L. Rosi, C.A. Mirikin, Chem. Rev. 105 (2005) 1547-1562.

2
3
4
5
6
. X. Chen, H.J. Schluesener, Toxicological Let. 176 (2008) 1-12.
. V.K. Sharma, R.A. Yngard, Y. Lin, Adv. Colloid Interface Sci. 145 (2009) 83-96.
. H. Korbekandi, S. Iravani, S. Abbasi, Crit. Rev. Biotechnol. 29 (2009) 279-306.
. P. Raveendran, J. Fu, S.L. Wallen, J. Am. Chem. Soc. 125 (2003) 13940-13941.
. K.F. Schmidt, “Green nanotechnology: It‟s easier than you think” Presentation given at
Green Nanotechnology Event hosted by the projects on Emerging Nanotechnologies at the
Woodrow Wilson International Center for Scholar, 2007.
7
8
9
1
1
1
1
1
1
1
. A. Mittal, J. Mittal, A. Malviya, V.K.Gupta, J. Colloid Interface Sci. 344 (2010) 497-507.
. V.K.Gupta, S. Agarwal, T. A. Saleh, J. Hazard. Mater. 185 (2011) 17-23.
. A. Mittal, L. Kurup ( Krishnam), V.K.Gupta, J. Hazard. Mater. 117 (2005) 171-178.
0. V.K.Gupta, A. Mittal, L. Kurup, J. Mittal, J. Colloid Interface Sci. 304 (2006) 52-57.
1. V.K.Gupta, A. Mittal, L. Krishnam, J. Mittal, J. Colloid Interface Sci. 293 (2006) 16-26.
2. V.K.Gupta, R. Jain, S. Varshney, J. Hazard. Mater.142 (2007) 443-448.
3. V.K.Gupta, R. Jain, S. Varshney, J. Colloid Interface Sci.312 (2007) 292-296.
4. V.K.Gupta, I. Ali, V.K. Saini, Water res. 41 (2007) 3307-3316.
5. V.K.Gupta, A. Rastogi, J. Hazard. Mater.163 (2009) 396-402.
6. V.K.Gupta, A. Mittal, A. Malviya, J. Mittal, J. Colloid Interface Sci. 335 (2009) 24-33.
1
1
7. S. Karthikeyan, V.K.Gupta, R. Boopathy, A. Titus, G. Sekaram, J.Mol. Liq. 173 (2012)
53-163.

1
1
2
2
8. V.K.Gupta, I. Ali, T. A. Saleh, A. Nayak, S. Agarwal, RSC Adv. 2 (2012) 6380-6388.
9. T. A. Saleh, V.K.Gupta, Environ Sci Pollut Res. 19 (2012) 1224-1228.
0. A.K. Jain, V.K.Gupta, S. Jain, Suhas, Environ. Sci. Technol. 38 (2004) 1195-1200.
1. V.K.Gupta, S. Sharma, Ind. Eng. Chem. Res. 42 (2003) 6619-6624.
2
8
2. V.K.Gupta, B. Gupta, A. Rastogi, S. Agarwal, A. Nayak, J. Hazard Mater. 186 (2011)
91-901.
2
2
2
3. A.K. Jain, V.K.Gupta, A. Bhatnagar, Suhas, Sep. Sci. Technol.38 (2003) 463-481.
4. A. Mittal, V.K.Gupta, A. Malviya, J. Mittal, J. Hazard. Mater. 151 (2008) 821-832.
5. V.K.Gupta, R. Jain, A. Mittal, M. Mathur, S. Sikarwar, J. Colloid Interface Sci.309
(2007) 464-469.
2
2
2
2
3
3
6. A. Mittal, J. Mittal, D. Kaur, V.K.Gupta, J. Colloid Interface Sci. 343 (2010) 463-473.
7. V.K.Gupta, A. Rastogi, A. Nayak, J. Colloid Interface Sci.342 (2010) 533-539.
8. T. Linnert, P. Mulvaney, A. Henglein, J. Am. Chem. Soc. 112 (1990) 4657-4664.
9. P. Mulvaney, A. Henglein, J. Phys. Chem. 94 (1990) 4182-4188.
0. T. Linnert, P. Mulvaney, A. Henglein, J. Phys. Chem. 97 (1993) 679-682.
1. A. Henglein, J. Phys. Chem. 97 (1993) 5457-5471.
3
2. F. Strelow, A. Henglein, J. Phys. Chem. 99 (1995) 11834-11838.
3. A. Henglein, Chem. Mater. 10 (1998) 444-450.
3

3
4. S. Raza, N. Stenger, S. Kadkhodazadeh, S. V. Fischer, N. Kostesha, A.P. Jauho, A.
Burrows, M . Wubs, and N. A. Mortensen, Nanopho- tonics. 2 (2013) 131-138.
3
3
5. M.G. Guzman, J. Dille, S. Godet, World Acad. of Sci. Eng & Technol. 67 (2008) 357-
64.
3
3
3
6. S. Kundu, Phys.Chem.Chem.Phys. 15 (2013) 14107-14119.
7. H. Onozato, N. Watabe, Cell Tissue Res. 201(1980) 303-316.
8. L. Zulberberg, J.B. Hahn, J.Y. Sire, Cell Tissue Res. 253 (1988) 597-607.
3
3
9. T. Ikoma, H. Kobayashi, J. Tanaka, D. Walsh, S. Mann, J. Struct. Bio. 142 (2003) 327-
33.
ꢁ
0. J. Brinckmann, H. Notbohm, P.K. Mueller, and Editors, “ꢃollagen Primer in Structure,
Processing And Assembly”, Springer ꢀꢁ7 (ꢀ005) 1- 252.
ꢁ
4
4
1. R.D. Harkness, “ꢃollagen”, Science Progress. 5ꢁ (1966) ꢀ57-274.
2. L. He, C. Mu, J. Ski, Q. Zhang, B. Shi, Q. Lin, Int. J. Biol. Macromol. 48 (2011) 354-359.
3. B.K. Simpson, S. Benjakul, P. Kittiphattanabawon, J.M. Regenstein, “Food Biochemistry
st
and Food processing”. Second Edition, 2012, 21 Chapter (Fish gelatin).
ꢁ
4
4
4
4
ꢁ. L. O‟Donovan, P. A. De Bank, J. Mater.ꢃhem. ꢀꢀ (ꢀ01ꢀ) ꢀ1878-21884.
5. A. Sharma, P. Dour, P. Gupta, World J. Microbial Biotechnol. 22 (2006) 1049-1054.
6. A. Henglein, A. Chem. Rev. 89 (1989) 1861-1873.
7. J. Liu, G. Qin, P. Raveendran, Y. Ikushima, Chem. Eur. J. 12 (2006) 2131-2138.
8. A. Murugadoss, A. Chattopadhyay, J. Phys. Chem. C. 112 (2008) 11265-11271.

4
2
9. Q. An, M. Yu, Y. Zhang, W. Ma, J. Guo, C. Wang, J. Phys. Chem. C. 116 (2012) 22432-
2440.
5
0. J.P. Phillips, H. Feuer, B.S. Thyagasrajan, Org. Electron. Spect. Data. 10 (1968) 50-58.
5
8
1. S. Wunder, F. Polzer, Y. Lu, Y. Mei, M. Ballauff, J. Phys. Chem. C. 114 (2010) 8814-
820.
5
5
5
5
5
5
5
2. B.H. Liu, P.Z. Li, J. Power Sources. 187 (2009) 527-534.
3. M.A. Vannice, Reactions; Springer Science+ Business Media: Philadelphia, PA, 2005.
4. G. Guella, B. Patton, A. Miotello, J. Phys. Chem. C. 111 (2007) 18744-18750.
5. T. Pal, T.K. Sau, N.R. Jana, Langmuir. 13 (1997) 1481-1485.
6. T. Pal, N.R. Jana, T.K. Sau, Corros. Sci. 39 (1997) 981-986.
7. R. Breslow, Acc. Chem. Res. 24 (1991) 159-164.
8. N.R. Jana, T.K. Sau, T. Pal, J. Phys. Chem. B. 103 (1998) 115-121.
5
7
9. M.A. Markowitz, D.N. Dunn, G.M. Chow, J. Zhang, J. Colloid Interface Sci. 210 (1999)
3-85.
6
6
6
0. H. Hirai, H. Alzawa, J. Colloid Interface Sci. 161 (1993) 471-474.
1. D. Yogev, S. Efrima, Langmuir. 7 (1991) 267-271.
2. T. Pal, N.R. Jana, Langmuir. 12 (1996) 3114-3121.

LIST OF FIGURES:-
1
. Fig: 1 UV-Vis absorption spectra recorded of synthesized Ag NPs with different
concentrations of fish scale extract (10, 20, 30, and 40 %) solution treated to 0.1 M
AgNO solution. The colours of the solution shown in the inset after 3 days in
3
increasing order of concentration from left to right.
2
. Fig: 2 UV-Vis absorption spectra recorded for silver NPs at different temperatures (60,
0
7
1
0, 80, 90 and 100 C ) when 10 ml of 0.1 M silver nitrate solution treated to 10 ml of
0% extract. The colours of the solution after 3 days in increasing order of temperature
from left to right.
3
4
. Fig: 3 UV-Vis absorption spectra recorded for silver NPs at different pH( 3 and 4 )
0
when 10 ml of 0.1 M silver nitrate solution treated to 10 ml of 10% extract at 70 C .
The colours of the solution after 3 days in increasing order of pH from left to right.
. Fig: 4 TEM images of Ag NPs synthesized with different concentrations of fish scale
extract (a) 10% (b) magnified image of (a), (c) 40% (d) magnified image of (c) , (e)
and (f) selected area electron diffraction (SAED) pattern of the TEM image shown in
(a) and (c) respectively.
5
6
. Fig: 5 TEM images of the Ag NPs synthesized when 10% extract solution treated to
0
0
0
.1 M silver nitrate solution at different temperatures (a) 60 C, (b) 100 C (c) and (d)
SAED pattern of TEM image shown in (a) and (b) respectively.
. Fig:6 TEM images of the Ag NPs synthesized when 10% extract solution treated to 0.1
0
M silver nitrate solution heated at 70 C at (a) pH=3, (b) at pH=4, (c) and (d) SAED
pattern of TEM image shown in (a) and (c) respectively.
7
8
. Fig: 7 UV-Vis absorption spectra recorded of Ag NPs.
-3
. Fig: 8 UV-Vis absorption spectra recorded of 60µl (6.07×10 M) 4-NP in aqueous
solution.

-
3
9
1
1
1
. Fig: 9 UV-Vis absorption spectra recorded of 60µl (6.07×10 M) 4-NP+ 350 µl
(
0.1M) NaBH + 150 µl 10% Ag NPs in aqueous solution.
4
0. Fig: 10.Plot of ln (A /A ) against time for pseudo –first –order reduction kinetics of 4-
t
0
NP in the presence of excess NaBH (0.1M) in aqueous solutions.
4
1. Fig: 11 Representation of Langmuir- Hinshelwood (LH) model for the mechanism of
the reduction of 4-NP to 4-AP by NaBH in presence of Ag NPs.
4
2. Fig: 12 Absorbance vs time (secs) plot for the reduction of 4-NP prompted by Ag NPs
-3
-3
as catalyst Concentration of LDS=CPC=TX-100=60×10 M, 4-NP=6.07×10 M,
NaBH =0.1M&AgNP=10%.
4
1
3. Fig: 13 Schematic representation of electron transfer process in three different micelle.

2.5
2.0
1.5
1.0
0.5
0.0
At40perc
At30perc
At20perc
At10perc
350
400
450
500
550
600
650
Wavelength (nm)
Fig: 1 UV-Vis absorption spectra recorded of synthesized Ag NPs with different
concentrations of fish scale extract (10, 20, 30, 40 %) solution treated to 0.1 M AgNO3
solution. The colours of the solution shown in the inset after 3 days in increasing order of
concentration from left to right.

At60C
At70C
At80C
At90C
At100C
2
1
0
400
500
600
700
Wavelength (nm)
Fig:2 UV-Vis absorption spectra recorded for silver NPs at different temperatures (60, 70,
0
8
0, 90 and 100 C) when 10 ml of 0.1 M silver nitrate solution treated to 10 ml of 10%
extract. The colours of the solution after 3 days in increasing order of temperature from left
to right.

1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
AtpH4
AtpH3
400
500
600
700
Wavelength (nm)
Fig: 3 UV-Vis absorption spectra recorded for silver NPs at different pH(3and 4 ) when 10 ml of
0
0
.1 M silver nitrate solution treated to 10 ml of 10% extract at 70 C . The colours of the solution
after 3 days in increasing order of pH from left to right.

Fig: 4 TEM images of Ag NPs synthesized with different concentrations of fish scale
extract (a) 10% (b) magnified image of (a), (c) 40% (d) magnified image of (c) , (e) and
(
f) selected area electron diffraction (SAED) pattern of the TEM image shown in (a) and
c) respectively.
(

Fig: 5 TEM images of the Ag NPs synthesized when 10% extract solution treated to 0.1 M
0
0
silver nitrate solution at different temperatures (a) 60 C and (b) 100 C and (c) and (d)
SAED pattern of TEM image shown in (a) and (b) respectively.

Fig:6 TEM images of the Ag NPs synthesized when 10% extract solution treated to 0.1 M
0
silver nitrate solution heated at 70 C at (a) pH=3, (b) at pH=4, (c) and (d) SAED pattern of
TEM image shown in (a) and (b) respectively.

2.5
2.0
1.5
1.0
0.5
0.0
250
300
350
400
450
500
550
600
Wavelength (nm)
Fig: 7 UV-Vis absorption spectra recorded of Ag NPs.