IVR Number: - 201900044876

Scholar Name: - Saurabh Kumar Maurya

Title of Project

Rapid Biosynthesis of Silver

Nanoparticles

Inspire Mentorship Project

 ACKNOWLEDGEMENT
I would like to express my special thanks of gratitude to my teacher Dr.
Pawan Ghosh, who gave me this golden opportunity to do this wonderful
project titled Rapid Biosynthesis of Silver Nanoparticles, and also for
their support in completing the project. This project also helped me in
doing a lot of research and I came to know so many new things.

I would like to express my gratitude towards my parents and friends who

gave valuable suggestions and guidance for completions of my project.
This cooperations and healthy criticism came handy and useful with them.

Date: 14/10/2020 Name of student

Place: Jabalpur Saurabh Kumar Maurya

1
 Title Of Project

Rapid Biosynthesis of
Silver Nanoparticles

2
Table of Contents
1. AIM....................................................................................................4
2. INTRODUCTION..............................................................................4
3. EXPERIMENTAL..............................................................................7
Extracellular biosynthesis of Ag nanoparticles using culture
supernatant of bacillus subtilis.......................................................7
Combinatorial synthesis of nanoparticles using bacterial
supernatant and microwave irradiation........................................8
Nitrate reductase assay...................................................................9
Determination of the tryptophan/tyrosine residues.................10
4. RESULT AND DISCUSSION...........................................................10
Extracellular biosynthesis of silver nanoparticles using
culture supernatant of bacillus subtilis........................................10
Rapid extracellular biosynthesis of silver nanoparticles using
culture supernatant of bacillus subtilis and microwave
irradiation.......................................................................................13
5. CONCLUSION.................................................................................17
6. REFERENCES.................................................................................18

3
1.AIM
In the field of nanotechnology, the creation of processes for the rapid
and dependable synthesis of nanosized materials is crucial. There
have been reports of using microorganisms to make silver
nanoparticles, but the process is slow. Using a combination of the
culture supernatant of Bacillus subtilis and microwave (MW)
irradiation in water in the absence of a surfactant or soft template, we
present a novel, simple, and "green" combinatorial synthesis strategy
for the synthesis of metallic nanostructures of noble metals like silver
(Ag). It was found that exposure of culture supernatanant of Bacillus
subtilis and microwave irradiation to silver ion lead to the formation
of silver nanoparticles. The silver nanoparticles were in the range of
5-60 nm in dimension. The nanoparticles were examined using UV-
Visible Spectroscopy, and Transmission Electron Microscopy (TEM)
analyses. This method produces nanoparticles in a very short amount
of time, does not require the use of harmful chemicals, and the
nanoparticles remain stable for several months. The primary
conclusion is that the bio-reduction approach to the production of
nanoparticles is an effective alternative to the electrochemical ones.

2. INTRODUCTION
Nanotechnology gives the capacity to design the properties of
materials by controlling their size, and this has driven research toward
a huge number of possible purposes for nanomaterials. Unusual
optical, thermal, chemical, and physical properties are present in
metallic nanoparticles.The reduction of materials’ dimension has
pronounced effects on the physical properties that may be
significantly different from the corresponding bulk material. Some of
the physical properties exhibited by nanomaterials are due to (i) large
surface atom, (ii) large surface energy, (iii) spatial confinement,
4
and (iv) reduced

5
imperfections. One key aspect of nanotechnology concerns the
development of rapid and reliable experimental protocols for the
synthesis of nanomaterials over a range of chemical compositions,
sizes, high monodispersity and large scale production.
A variety of techniques have been developed to synthesize metal
nanoparticles, including chemical reduction using a number of
chemical reductants including NaBH4, N2H4, NH2OH, ethanol,
ethylene glycol and N,N-dimethyformamide (DMF)1-5, aerosol
technique6 , electrochemical or sonochemical deposition7, 8,
photochemical reduction9 , and laser irradiation technique10. A lot of
interest has been created by the term "green nanotechnology". In a
broad sense, this term includes a wide range of possible applications,
from nanotechnology-enabled, environmentally friendly
manufacturing processes that reduce waste products (ultimately
leading to atomically precise molecular manufacturing with zero
waste); the use of nanomaterials as catalysts for greater efficiency in
current manufacturing processes by minimizing or eliminating the use
of toxic materials (green chemistry principles); the use of
nanomaterials and nanodevices to reduce pollution (e.g. water and air
filters); and the use of nanomaterials for more efficient alternative
energy production (e.g. solar and fuel cells).
With the flourishing demand of “green” nanoparticle synthesis
processes11, the field of nanoparticle synthesis has recently
developed new routes. These include employing microorganisms,
such as: Pseudomonas stutzeri12 Verticilium sp13 , Fusarium
oxysporum14,15
, Thermomonospora sp16 and, also, alfalfa and geranium plants17,18 .
Biosynthetic methods employing either biological microorganisms or
plant extracts have emerged as a simple and viable alternative to
chemical synthetic procedures and physical methods14-21. Although
it is known that microorganisms such as bacteria, yeast and now fungi
play an important role in remediation of toxic metals through
reduction of the metal ions, this was considered interesting as
nanofactories very recently.
6
The presence of hydrogenase in the F. oxysporium broth was
demonstrated. Some bacteria reduce Fe3+ oxides by producing and
secreting small, diffusible redox compounds that can serve as electron
shuttle between the microbe and the insoluble iron substrate22.This
extracellular enzyme shows an excellent redox properties and it can
act as an electron shuttle in the metal reduction. It was evident that
electron shuttles or other reducing agents (e.g. hydroquinones)
released by microorganisms are capable of reducing ions to
nanoparticles23. However, biological nanoparticles synthesis
generates nanoparticles at a much slower rate. In the studies synthesis
of silver nanoparticles using the cell mass of bacteria24-25 and
fungi13 or their leached cell components14, the time required
completing the reaction ranged from 24 to 120 h; this lengthy reaction
is the one major drawback of the biological synthesis. Biological
synthesis would have greater commercial viability if the nanoparticles
could be synthesis more rapidly and in the large production scale.
Despite stability and a green method of production, particles are not
monodispersible and the rate at which they form is not comparable to
chemical synthesis methods. Thermal factors have been demonstrated
to affect the size and uniformity of nanoparticles.
Controlling particle growth rate is possible through knowledge of a
material’s properties and controlling of the reaction by changes to the
time and temperature. The ability of any material to absorb
microwave energy is expressed by its dielectric loss factor combined
with the dielectric constant. The microwave radiation heats up a
material through its dielectric loss, which converts the radiation
energy into thermal energy. The main challenges frequently
encountered by the researchers of nanoparticle synthesis are (i)
controlling the particle size/shape and (ii) achieving the
monodispersity. The synthesis of nanoparticles with controlled
monodispersity is a recent demand by the material developers for the
advancement of nanotechnology. Herein, we describe a novel
combinatorial synthesis approach which is rapid, simple and “green”
for the synthesis of metallic nanostructures of noble metals such as
silver (Ag), by using a combination of culture
7
supernatanant of Bacillus subtilis and microwave (MW) irradiation in
water in absence of a surfactant or soft template.
We chose Bacillus subtilis because it is a ubiquitous bacterium that
can be found in a variety of ecological niches, including soil, water,
and air. These bacterium do not have a history of being pathogenic
when they come into contact with other organisms in the
environment.

3. EXPERIMENTAL
Bacillus subtilis stock cultures were maintained by subculturing at
monthly intervals. Growth medium used was Luria Broth (LB)
medium (1% Bacto-tryptone [Difco], 0.5% yeast extract [Difco], 1%
NaCl, pH 7.5). 250 mL of Luria broth (LB) was prepared, sterilized,
and inoculated with fresh batch of the bacteria, Bacillus subtilis. The
culture flasks were incubated for 36 h at 37ºC with shaking at 150
rpm. After incubation period, the cultures were centrifuged at 5,000
rpm for 10 minutes and the supernatant were collected. These
supernatant were used as the starting material for synthesis of
nanoparticles.

Extracellular biosynthesis of Ag
nanoparticles using culture supernatant of
bacillus subtilis
In a typical synthesis of silver nanoparticle extracellularly, 50 mL
aqueous solution of 1 mM silver nitrate (AgNO3) was treated with 50
mL of Bacillus subtilis supernatant solution in a 250 mL Erlenmeyer
flask (pH adjusted to 8.5). The whole mixture was put into a shaker at
40°C (200 rpm) for 5 days and maintained in the dark. Control
experiments were conducted with uninoculated media, to check for
the role of bacteria in the synthesis of nanoparticles. The reduction of
Ag+ ions was monitored by sampling an aliquot (2 mL) of the
solution at intervals of 24 h. and measuring the UV-Vis spectra of the
8
solution. Absorption measurements were carried out on a Genesys 10
UV-VIS

9
spectrophotometer. The spectra were recorded at room temperature
using a one-centimetre quartz cuvette. The transmission electron
microscopy (TEM) analysis of extracellular synthesized silver
nanoparticles were prepared by drop-coating biosynthesized silver
nanoparticles solution on carbon-coated copper TEM grids (40 µm ×
40 µm mesh size). Samples were dried and kept under vacuum in
desiccators before loading them onto a specimen holder. TEM
measurements were performed on a JEOL model 1200EX electron
microscope operated at an accelerating voltage at 120 kV.

Combinatorial synthesis of nanoparticles

using bacterial supernatant and microwave
irradiation
As mentioned above, 50 mL aqueous solution of 1 mM silver nitrate
(AgNO3) was treated with 50 mL of Bacillus subtilis supernatant
solution in a 250 mL Erlenmeyer flask (pH adjusted to 9.0). The
whole mixture was put in a domestic microwave oven (National
Model N N- GD 576M). The sample was subjected to several short
burst of microwave irradiation at frequency of 2.45 GHz, at power
output of about 100W in a cyclic mode (on 15 s, off 15 s) to prevent
overheating as well as aggregation of metals. The irradiation process
was conducted for a minimum of 5 up to maximum of 15 cycles. The
reduction of Ag+ ions was monitored by sampling an aliquot (2 mL)
of the solution after 5, 7, 9, 12 and 15 cycles and measuring the UV-
Vis spectra of the solution. Absorption measurements were carried out
on a Genesys 10 UV-VIS spectrophotometer. The spectra were
recorded at room temperature using a one-centimetre quartz cuvette.
Samples for transmission electron microscopy (TEM) were prepared
as described previously.

1
0
 Nitrate reductase assay
Nitrate reductase converts nitrate to nitrite. The enzyme activity was
measured using the method described by Harley where the activity
was measured by putting in the substrate for the enzyme (nitrate) and
then measuring the amount of nitrite after 1 h. The net increase in
nitrite at 1 h is the amount of nitate reductase activity. The reagents
were: assay medium: 30 mM KNO3 and 5% propanol in 0.1 M
phosphate buffer pH 7.5; nitrite solution: 25 µM NaNO2 (Nitrite)
solution; nitrite assay reagents: sulfanilamide solution: 1% (w/v) in
25% (v/v) HCl and N-(1- napthy) ethelenediamine dihydrochloride
solution (NEED): 0.02% (w/v) in distilled water. Five tubes each
containing 10 mL of the Bacillus subtilis culture supernatant, taken
from the 120 h reaction were prepared. Tubes ‘a’ and ‘b’ (duplicates)
were place in a boiling water bath sample to kill the enzyme as soon
as 10 mL assay medium was added (i.e. at time-zero).
After boiling 5 minutes, the tubes were kept at room temperature until
the nitrite was measured. 10 mL of assay medium were added to tubes
‘c, d, e’ (triplicates) and incubate in the dark for 60 minutes before
placing the tubes in the boiling water bath for 5 minutes. After the
samples had cooled, 5 mL of sulfanilamide solution was added to
each. Then 5 mL of NEED solution was added and quickly mixed by
inverting the tubes. After 20 minutes the absorbance at 540 nm was
measured using a Genesys 10 UV-VIS spectrophotometer. The
amount of nitrite found in tubes ‘a’ and ‘b’ were subtracted from that
in tubes ‘c, d', and ‘e’ to calculate the amount produced by enzyme
activity during 60 min. Nitrite standard curve was prepared by placing
known amounts of nitrite (0 to 250 nmol NO2) followed by 5 mL of
sulphanilamide and 5 mL of NEED solution. After mixing and
incubation for 20 min at room temperature absorbance was measured
at 540 nm. The enzyme activity was calculated based on the increase
in nitrite over 60 min for the amount of sample (10 mL) and
expressed as nmol nitrite/hour/mL.

1
1
 Determination of the
tryptophan/tyrosine residues
Presence of tryptophan/tyrosine residues in proteins released in the
bacterial culture supernatant was analyzed spectrophotometrically by
measurement of absorbance in the 250 – 300 nm wavelength regions.
The measurements were carried out on a Genesys 10 UV-VIS
spectrophotometer. The spectra were recorded at room temperature
using a one-centimetre quartz cuvette.

4. RESULT AND DISCUSSION

Extracellular biosynthesis of silver
nanoparticles using culture supernatant of
bacillus subtilis
Extracellular production of silver nanoparticles by the culture
supernatants of Bacillus subtilis with aqueous silver nitrate solution,
1mM was investigated. The picture of test tubes of silver nitrate
solution after exposure to culture supernatant of Bacillus subtilis is
shown in the inset of Figure 1. The appearance brown colour clearly
indicates the formation of silver nanoparticles in the reaction
mixture19. The characteristics brown colour of colloidal silver
solution is due to the excitation of surface plasmon vibrations in the
nanoparticle and provides a convenient spectroscopic signature of
their formation. Several hydroquinones with excellent redox
properties were reported that could be act as electron shuttle in metal
reductions22, 23. Thus, it was evident that electron shuttle or others
reducing agents released by Bacillus subtilis are capable of reducing
silver ions to silver nanoparticles. On the other hands, the reduction of
silver ions did not occur in the absence of bacterial cells. This
10
clearly indicates that the

10
reducing agents that are released into the cultures of the
aforementioned Bacillus subtilis are involved in the reduction process.

Figure 1. UV-Vis spectra recorded as a function of time of reaction of 1 mM

aqueous solution of AgNO3 with Bacillus subtilis culture supernatant. The inset
of the figure shows a test tube of the silver nanoparticle solution formed at the
end of the reaction.
From the UV-Vis spectra recorded for the aqueous silver nitrate - B.
subtilis reaction medium as a function of time (Figure 1) it can be
observed that the silver surface plasmon band occurs at around 410
nm and steadily increase in intensity as a function of time of reaction.
The surface plasmon band in the silver nanoparticles solution remains
close to 410 nm throughout the reaction period, suggesting that the
particles are dispersed in the aqueous solution with no evidence for
aggregation. After completion of the reaction, the silver nanoparticles
solution was tested for stability. It was observed that the nanoparticle
solution of silver was extremely stable for more than six months with
no signs of aggregation even at the end of this period.
The particles are thus stabilized in solution by the capping agent that
is likely to be proteins secreted by the biomass. A representative TEM
picture recorded from the silver nanoparticle film deposited on a
carbon coated copper TEM grid is shown in Figure 2. This picture
shows individual silver particles as well as a number of aggregates.
The morphology of the nanoparticles is highly variable, with spherical
and occasionally triangular nanoparticles observed in the
11
micrograph.

12
Under observation of such images in an optical microscope, these
assemblies were found to be aggregates of silver nanoparticles in the
size range 5–50 nm. The nanoparticles were not in direct contact even
within the aggregates, indicating stabilization of the nanoparticles by
a capping agent. This corroborates with the previous observation by
Ahmad et al14 in their study on F. oxysporum.
As discussed earlier, the silver nanoparticle solution, synthesized by
the reaction of Ag + ions with Bacillus subtilis culture supernatant, is
exceptionally stable and the stability is likely to be due to capping
with proteins secreted by the bacteria. The separation between the
silver nanoparticlesseen in the TEM image could be due to capping
by proteins and would explain the UV-Vis spectroscopy
measurements, which is characteristic of well-dispersed silver
nanoparticles.

Figure 2. TEM micrograph at 100,000 times magnification recorded from a

drop- coated film of an aqueous solution incubated with Bacillus subtilis culture
supernatant and reacted with 1 mM Ag+ ions for 120 h at 40°C. The scale bar
corresponds to 100 nm.

13
 Rapid extracellular biosynthesis of silver
nanoparticles using culture supernatant of
bacillus subtilis and microwave irradiation
The previous method was extended Rapid extracellular production of
silver nanoparticles by the culture supernatants of Bacillus subtilis
with aqueous silver nitrate solution was extended by incorporating
microwave irradiation. The advantage of using microwave radiation is
that it provides uniform heating around the nanoparticles and can
assist the digestive ripening of such particles without aggregation.
The microwave radiation heats up a material through its dielectric
loss, which converts the radiation energy into thermal energy. From
the UV- Vis spectra for microwave irradiated 1 mM AgNO3 solution
in the presence of culture supernatants of Bacillus subtilis (Figure 3) it
can be observed that the silver surface plasmon band occurs at around
410 nm and steadily increase in intensity as a function of time of
reaction. It can be seen that surface plasmon absorption band of Ag
particles was observed just after 5 cycles of irradiation and increased
with further irradiation. The absorbance observed at 12th cycle (180 s
of irradiation) was roughly similar to that obtained by the
nonmicrowave reaction incubated for 72h at 40°C. The surface
plasmon band in the silver nanoparticles solution remains close to 410
nm throughout the reaction period, suggesting that the particles are
dispersed in the aqueous solution with no evidence for aggregation.
Not only the heating is faster through microwave radiation, but also
the temperature distribution of the solution is more uniform. As such,
this has led to the fast reaction rate and narrow size distribution of the
Ag nanoparticles. This rapid microwave heating also provides
uniform nucleation and growth conditions, leading to homogeneous
nanomaterials with smaller sizes. Power dissipation is fairly uniform
throughout with “deep” inside-out heating of the polar solvents, which
leads to a better crystallinity.

14
Figure 3. UV-Vis spectra recorded as a function of time of reaction of 1 mM
aqueous solution of AgNO3 with Bacillus subtilis culture supernatant irradiated
with microwave. The irradiation period was 5 cycles up to 15 (microwave
exposure was 15 s per cycle)
It can be seen from Figure 3 that the bands are sharper and more
symmetrical, which reflect more uniform size distribution. The major
advantage noticed is the time of reaction; the reaction proceeds at a
much faster rate when irradiated with microwave. A representative
TEM picture recorded from the silver nanoparticle film deposited on a
carbon coated copper TEM grid is shown in Figure 4. This picture
shows individual silver particles as well as a number of aggregates.
The morphology of the nanoparticles is variable, with majority of
them spherical.
By gentle heating, using microwave irradiation of an aqueous silver
nitrate solution containing Bacillus subtilis culture supernatant, we
had obtained relatively mono-disperse silver nanoparticles. Under
observation of such images in an optical microscope, these assemblies
were found to be aggregates of silver nanoparticles in the size range
5– 50 nm. The nanoparticles were not in direct contact even within
the aggregates, indicating stabilization of the nanoparticles by a
capping agent. This corroborates with the previous observation by
Ahmad et al14 in their study on F. oxysporum. As discussed earlier,
the silver nanoparticle solution, synthesized by the reaction of Ag +
ions with Bacillus subtilis culture supernatant, is exceptionally
stable and the
15
stability is likely to be due to capping with proteins secreted by the
bacteria.
The separation between the silver nanoparticles seen in the TEM
image could be due to capping by proteins and would explain the UV-
Vis spectroscopy measurements, which is characteristic of
welldispersed silver nanoparticles. The nanoparticles present in the
aqueous medium were quite stable, even up to 5 months of incubation
at 25°C with no signs of aggregation even at the end of this period.

Figure 4. TEM micrograph at 100,000 times magnification recorded from a

drop- coated film of 1 mM aqueous solution of AgNO3 with Bacillus subtilis
culture supernatant irradiated with microwave. The scale bar corresponds to 100
nm.
The particles are thus stabilized in solution by the capping agent that
is likely to be proteins secreted by the biomass. This is an important
aspect of synthesis of nanoparticles, since the lack of sufficient
stability of many nanoparticles preparation has to some extent
impeded the development of the real world applications of
nanomaterials. Application of the biological systems for synthesis of
silver nanoparticles has already been reported earlier16, However, the
exact reaction mechanism leading to the formation of silver
nanoparticles by all these organisms is yet to be elucidated. Ahmad et
al14 have reported that certain NADH dependent reductase was
16
involved in reduction of silver ions in case of F. oxysporum.

17
While the above experiments clearly establish that the reduction of the
Ag+ ions occurs extracellularly, it would be important to identify the
reducing agents responsible for this. The average protein contains
3.5% phenylalanine, 3.5% tyrosine and 1.1% tryptophan: these three
amino acids contribute the majority of the proteins absorbance and
fluorescence properties in the 250-300 nm wavelength range,
although if the protein contains significant numbers of disulphide
bonds these too will contribute to the absorption properties in the
wavelength range 250-280 nm. On the other hand the peptide bond
and other amino acids contribute to absorbance properties in the 210 –
220 nm regions. The Figure 5 shows the UV-Vis spectrum in low
wavelength region recorded from the reaction of 10-3 M AgNO3 with
culture supernatant of Bacillus subtilis after 12th cycle of microwave
irradiation.
The presence of the absorption band around 275 nm is attributed to
aromatic amino acids of proteins. It is well known that the absorption
band at around 275 nm arises due to electronic excitations in
tryptophan and tyrosine residues in the proteins. This observation
indicates the release of proteins into solution by B. subtilis and
suggests a possible mechanism for the reduction of the metal ions
present in the solution. In this study, the UV–Vis spectrum study
provides some clues regarding the mechanism of synthesis of silver
nanoparticles. There were two absorbance peaks found in the UV
range corresponding to 220 and 275 nm. While the peak at 220 nm
may be due to absorption by amide bond, the other peak at 275 nm
may be attributed to the tryptophan and tyrosine residues present in
the protein. This indicates secretion of some proteinic components
into the medium by the bacterial biomass which might play important
role in the reduction of the metal ions in the form of nanoparticles.
Consequently the proteins also may bind to the nanoparticles and
enhance the stability.

18
Figure 5. UV-Vis spectra in the low wavelength region recorded from the
reaction of 10-3 M AgNO3 with culture supernatant of Bacillus subtilis after
12th cycle of microwave irradiation.
Previous studies14-16, 19 have indicated that NADH- and NADH-
dependent enzymes are important factors in the biosynthesis of metal
nanoparticles. The reduction seems to be initiated by electron transfer
from the NADH by NADH-dependent reductase as electron carrier.
Many fungi that exhibit these characteristic properties, in general, are
capable of reducing Au(III) or Ag(I)29. Besides these extracellular
enzymes, several naphthoquinones31 and anthraquinones23 with
excellent redox properties, were reported in F. oxysporum that could
be act as electron shuttle in metal reductions 22. The exact mechanism
of the reduction of metal ions is yet to be elucidated for bacteria. In
this study nitrate reductase activity was detected in the culture
supernatant of Bacillus subtilis and was about 152 nmol/ h/ mL
culture supernatant of Bacillus subtilis. It appears that the reductase
together with electron shuttling compounds and other
peptides/proteins may be responsible for the reduction of Ag+ ions
and the subsequent formation of silver nanoparticles.

5. CONCLUSION
In conclusion, the Bacillus subtilis has shown potential for
extracellular synthesis of fairly monodispersed, silver nanoparticles in
the range of 5–50 nm. The kinetics of silver nanoparticles synthesis
19
using the cell

11
0
filtrates in combination with microwave irradiation indicates that the
rapid synthesis of nanoparticles would be suitable for developing a
“green nanotechnology” biosynthesis process for mass scale
production. The extracellular synthesis offers a great advantage over
an intracellular process of synthesis from the application point of
view. Since the nanoparticles formed inside the biomass would have
required additional step of processing for release of the nanoparticles
from the biomass by ultrasound treatment or by reaction with suitable
detergents. The extracellular synthesis of nanoparticle makes it
possible to harness and immobilize/deposit onto desired solid support
for the use of different practical purposes. In future, it would be
important to understand the biochemical and molecular mechanism of
the synthesis of the nanoparticles by the cell filtrate in order to
achieve better control over size and polydispersity of the
nanoparticles.

6. REFERENCES
[1]. Petit C, Lixon P, and Pileni M. P, J Phys Chem., 1993, 97, 12974-
12983.
https://www.frontiersin.org/articles/10.3389/fmicb.2017.00626/full
[2]. Heath J R, Knobler C M and Leff D V, J Phys Chem., B, 1997,
101, 189-197.
https://www.hindawi.com/journals/jchem/2009/734264/
[3]. Liz-Marzán L M and Lado-Touriňo I, Langmuir, 1996, 12, 3585-
1589.
https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s1295
1-018-0334-5
[4]. Komarneni S, Newalkar D Li, B, Katsuki H and Bhalla A S,
Langmuir, 2002, 18, 5959-5962.
11
1
https://www.sciencedirect.com/science/article/pii/S135951130700061
X

11
2
[5]. Santos I P and Liz-Marzán L M, Langmuir, 2002, 18, 2888-2894.
https://link.springer.com/article/10.1007/BF03353671
[6]. Harfenist S A, Wang Z L, Alvarez M M, Vezmar I and Whetten
R L, J Phys Chem., 1996, 100, 13904-13910.
[7]. Stiger R M, Gorer S, Craft B and Penner P M, Langmuir, 1999,
15, 790-798.
[8]. Pol V G, Srivastava D N, Palchik O, Palchik V, Slifkin M A, Weiss
A. M and Gedanken A, Langmuir, 2002, 18, 3352-3357.
[9]. Itakura T, Torigoe K and Esumi K, Langmuir, 1995, 11, 4129-
4134.
[10]. Abid J P, Wark A W, Brevet P F and Girault H H, Chem.
Commun., 2002, 792-793.
[11]. Huang H and Yang X, Carbohydr. Res., 2004, 339, 2627-2631.
[12]. Klaus-Joerger T, Joerger R, Olsson E and Granqvist C, Trends
Biotechnol., 2001, 19(1), 15-20.
[13]. Mukherjee P, Ahmad A, Manda l D, Senapati S, Sainkar S R,
Khan M I, Parishcha R, Ajaykumar P V, Alam M, Kumar R and
Sastry M, Nano Lett., 2001, 1(10), 515-519.
[14]. Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan M I,
Kumar R and Sastry M, Coll. Surf., B, 2003, 28(4), 313-318.
[15]. Mukherjee P, Senapati S, Mandal D, Ahmad A, Islam Khan M,
Kumar R and Sastry M, Chem Bio Chem., 2002, 3, 461-463.
[16]. Ahmad, A, Senapati S, Islam Khan M, Kumar R and Sastry M,
Langmuir, 2003, 19, 3550-3553.
[17]. Gardea-Torresdey J L, Gomez E, Peralta-Videa J R, Parsons J G,
Troiani H and Yacaman M J, Langmuir, 2003, 19, 1357-1361.
[18]. Shankar S S, Ahmad A and Sastry M, Biotechnol Prog., 2003,
19, 1627-1631.
[19]. Durán N, Marcato P D, Alves O L, Gabriel I H, Souza D E and
11
3
Esposito E, J Nanobiotechnology, 2005, 3, 1-7.

11
4
[20]. Mukherjee P, Ahmad A, Mandal D, Senapati S, Sainkar S R and
Khan M I, Nano Lett., 2001, 1, 515-9.
[21]. Joerger R, Klaus T and Granqvist C G, Adv Mater, 2000, 12,
407- 409.
[22]. Newman D K and Kolter R, Nature, 2000, 405, 94-97.
[23]. Baker R A and Tatum J H, J Ferment Bioeng, 1998, 85, 359-361.
[24]. Klaus T, Joerger R, Olsson E and Granqvist C G, Proc Natl Acad
Sci USA 1999; 96, 13611-4.
[25]. Nair B and Pradeep T, Cryst Growth Des, 2002, 2, 293-8

20