SYNTHESIS OF IRON OXIDE NANOPARTICLES,

CHARACTERIZATION AND THEIR

ANTIMICROBIAL ACTIVITY


Thesis submitted to Biju Patnaik University of Technology for partial
fulfillment of degree of Bachelor of Technology in Biotechnology
for Year 2011


SUBMITTED BY
ABHIJIT DEHURI
REGD NO - 0701106142


SUPERVISED BY
Mr. SUDHANSU SEKHAR BEHERA
Lecturer






DEPARTMENT OF BIOTECHNOLOGY,
COLLEGE OF ENGINEERING AND TECHNOLOGY
(A constituent college of Biju Patnaik University of Technology)

BHUBANESWER -751003
2011




DEPARTMENT OF BIOTECHNOLOGY
COLLEGE OF ENGINEERING AND TECHNOLOGY
(A Constituent College of Biju Patnaik University of Technology, Odisha)
TECHNO CAMPUS, P.O.:- GHATIKIA, KALINGA NAGAR,
BHUBANESWAR-751 003, INDIA


Dr.H.N. Thatoi
Reader & Head

Certificate
This is to certify that the thesis entitled Synthesis of iron oxide
nanoparticles, characterization and their antimicrobial activity Submitted by
Mr. Abhijit Dehuri bearing registration no. 0701106142 for the degree of Bachelor
of Technology in Biotechnology to the College of Engineering & Technology ,
Bhubaneswar, Odisha , India in partial fulfillment of the requirements for the award
of the Bachelor of Technology in Biotechnology carried out under the supervision of
Mr. Sudhansu Sekhar Behera , Lecturer, Department of Biotechnology, CET is a
faithful record of bonafide research work

.


Place: Bhubaneswar
Date: (H.N.Thatoi)


DEPARTMENT OF BIOTECHNOLOGY
COLLEGE OF ENGINEERING AND TECHNOLOGY
(A Constituent College of Biju Patnaik University of Technology Orissa)
TECHNO CAMPUS, P.O.:- GHATIKIA, KALINGA NAGAR
BHUBANESWAR-751 003, INDIA


Mr.SUDHANSU SEKHAR BEHERA
Lecturer

Certificate
This is to certify that the thesis entitled Synthesis of iron oxide
nanoparticles, characterization and their antimicrobial activity Submitted by
Mr. Abhijit Dehuri bearing registration no. 0701106142 for the degree of Bachelor
of Technology in Biotechnology to the College of Engineering & Technology ,
Bhubaneswar, Odisha ,India is a faithful record of bonafide research work carried out
under my guidance and supervision and the results of the investigation reported in
the thesis have not been submitted for the award of any degree or diploma.



Place: Bhubaneswar
Date: Mr.SUDHANSU SEKHAR BEHERA




DECLARATION
I Mr. Abhijit dehuri, B.tech in Biotechnology, College of Engineering and Technology
(Biju Pattnaik University of Technology), Bhubaneswar, Orissa do hereby declared that,
the present study entitled Synthesis of iron oxide nanoparticles, characterization and their
antimicrobial activity submitted by me for the partial fulfilment of the B.tech in
Biotechnology is the original work carried out by me under the guidance and supervision of
Mr. Sudhansu Shekhar Behera and Mr. Swagat Ku Das, Lecturer, Department of
Biotechnology, College of Engineering and Technology (Biju Pattnaik University of
Technology), Bhubaneswar, Orissa.

This work is based on my original research work and no part of the thesis has so
far been submitted for the award of any other degree or diploma to the Biju Pattnaik
University of Technology, ODISHA, INDIA or elsewhere.




Place: Bhubaneswar, Orissa
Date: (Abhijit Dehuri)


Acknowledgements
I would like to avail this opportunity to express my sincere gratitude and profound
obligations to Dr. H. N. Thatoi, Reader and Head, Department of Biotechnology, College of
Engineering and Technology (BPUT), Bhubaneswar, Orissa for his valuable suggestions,
ungrudging help and unfailing encouragement, which sustained me throughout this strenuous
work.
I would also like to express my sincere gratitude to my supervisor Mr.Sudhansu
Sekhar Behera, and Mr.Swagat Ku Das, lecturer, Department of Biotechnology, College of
Engineering and Technology (Biju Pattnaik University of Technology), Bhubaneswar, Orissa
for his valuable suggestion, help and kind guidance.
I am highly grateful to my co-supervisor Mr.Jayant Ku. Patra for their kind support
and valuable suggestion during my research work.
I am grateful to the authorities of the College of Engineering and Technology
(Biju Pattnaik University of Technology), for extending laboratory facilities to carry out the
research work.
I have the pleasure to express my thanks to all the faculty members of
Department of Biotechnology, College of Engineering and Technology (Biju Pattnaik
University of Technology), Bhubaneswar, Orissa whose cordial and silent help put me in a
comfortable stage in my working laboratory. My friends contributed immensely by their
valuable suggestions and co-operation during this work.
My heartfelt gratefulness &special thanks to Madhusudan Barik for his immense
help valuable suggestions criticism and pain taking efforts in doing the experimental work
successfully.
I express my heartily devotion and foremost indebtedness to my beloved parents
whose blessings, inspiration, sacrifice, constant guidance and support are behind my success.
Place: Bhubaneswar
Date: (Abhijit Dehuri)

CONTENTS

1. INTRODUCTION
2. REVIEW OF LITERATURES
3. MATERIALS AND METHODS
3.1. Synthesis of iron oxide nanoparticles
3.1.1. Synthesis of nanoparticles by using FeCl
2
.4H
2
O and FeCl
3
.6H
2
O
3.1.2. Synthesis of nanoparticles by using hydrolysis method
3.1.3. Estimation of the antimicrobial activity of iron oxide nanoparticles
3.2. Characterization of nanoparticles
3.2.1. Characterization by using spectroscopy method
3.2.2. Characterization by using x-ray diffraction method
4. RESULTS
4.1. Synthesis of iron oxide nanoparticles
4.2. Characterization of nanoparticles
4.3. Antimicrobial activity
5. DISCUSSION
5.1. Synthesis of iron oxide nanoparticles
5.2. Characterization of nanoparticles
5.3. Antimicrobial activity
6. CONCLUSION
7. REFERENCE


LIST OF TABLES

Table 1: Absorbance of nanoparticles in UV range of sample 1.

Table 2: Absorbance of nanoparticles in visible range of sample 1.

Table 3: Absorbance of nanoparticles in UV range of sample 2.

Table 4: Absorbance of nanoparticles in visible range of sample 2.









LIST OF FIGURES

Figure 1: Antimicrobial activity of nanoparticles against gram negative bacteria.

Figure 2: Antimicrobial activity of nanoparticles against gram positive bacteria.

Figure 3: Synthesis of nanoparticles by using hydrolysis method.

Figure 4: Synthesis of nanoparticles by using ferric and ferrous chloride solution.

Figure 5: Solution of both sample 1 and sample 2 nanoparticles for the antimicrobial activity.







Abstract:
The iron oxide nanoparticles have been synthesized using aqueous solution of ferric and
ferrous ions with sodium salt. The size of the iron-oxide nanoparticles is controlled by the
concentration of sodium salt in the medium. The synthesis of iron-oxide nanoparticles found
by UV-Visible spectroscopy, which are valid with standard data. The antibacterial effect of
iron oxide nanoparticles used in the study was found more potent in Gram negative bacteria
as compared to Gram positive one.






INTRODUCTION
Nanotechnology involves the tailoring of materials at atomic level to attain unique properties,
which can be suitably manipulated for the desired applications. Most of the natural processes
also take place in the nanometer scale regime. Therefore, a conuence of nanotechnology and
biology can address several biomedical problems, and can revolutionize the eld of health
and medicine. Nanotechnology is currently employed as a tool to explore the darkest avenues
of medical sciences in several ways like imaging, sensing, targeted drug delivery and gene
delivery systems and articial implants. Hence, nanosized organic and inorganic particles are
nding increasing attention in medical applications due to their amenability to biological
functionalization. Based on enhanced effectiveness, the new age drugs are nanoparticles of
polymers, metals or ceramics, which can combat conditions like cancer and ght human
pathogens like bacteria .Nano-magnetic materials have been advocated for use in biomedicine
with grain sizes down to the nanoscale for longer than any other type of material due to the
intrinsic magnetic behavior, such as superparamagnetism, exhibited when grain sizes are
reduced. Technological advances, mainly in the field of information storage technology, from
the 1950s onwards have resulted in enormous research efforts towards techniques and
preparation of magnetic nanoparticles with well-defined properties. Therefore, since this time
there has been a wide availability and knowledge base concerning magnetic nanoparticles,
including techniques for the preparation of particles, which led to speculation that they may
have other applications, for example in biology and medicine. In addition, as magnetic
nanoparticles obey Coulombs law under an external magnetic field gradient, the fact that we
can, in theory, remotely control biological material opens up a wealth of possibilities.
History
The dawn of the journey into the nano world can be traced back to 1959, when Caltech
physicist Richard Feynman painted a vision of the future of science. In a talk titled Theres
Plenty of Room at the Bottom, Feynman hypothesized that atoms and molecules could be
manipulated like building blocks. The history of nanotechnology traces the development of
the concepts and experimental work falling under the broad category of nanotechnology.
Although nanotechnology is a relatively recent development in scientific research, the
development of its central concepts happened over a longer period of time. Nanotechnology
and nanoscience got a boost in the early 1980s with two major developments: the birth
of cluster science and the invention of the scanning tunnelling microscope (STM). These
developments led to the discovery of fullerenes in 1985 and the structural assignment
of carbon nanotubes a few years later.
Nanoparticle
In nanotechnology, a particle is defined as a small object that behaves as a whole unit in
terms of its transport and properties. Particles are further classified according to size: in terms
of diameter, fine particles cover a range between 100 and 2500 nanometers. Nanoparticles are
sized between 1 and 100 nanometers. Nanoparticles may or may not exhibit size-related
properties that differ significantly from those observed in fine particles or bulk materials
Properties of Nanoparticle
Nanoparticles often possess unexpected optical properties as they are small enough to confine
their electrons and produce quantum effects. For example gold nanoparticles appear deep red
to black in solution. Nanoparticles of usually yellow gold and grey silicon are red in colour.
And absorption of solar radiation in photovoltaic cells is much higher in materials composed
of nanoparticles than it is in thin films of continuous sheets of material. I.E. the smaller the
particles, the greater the solar absorption.
Other size-dependent property changes include quantum confinement in semiconductor
particles, surface Plasmon resonance in some metal particles and superparamagnetism in
magnetic materials.
Suspensions of nanoparticles are possible since the interaction of the particle surface with the
solvent is strong enough to overcome density differences, which otherwise usually result in a
material either sinking or floating in a liquid.
The high surface area to volume ratio of nanoparticles provides a tremendous driving force
for diffusion, especially at elevated temperatures. Sintering can take place at lower
temperatures, over shorter time scales than for larger particles.



Types of nanoparticle
Extensive libraries of nanoparticles, composed of an assortment of different sizes, shapes,
and materials, and with various chemical and surface properties, have already been
constructed. The classes of nanoparticles listed below are all very general and multi-
functional, however, some of their basic properties and current known uses in biotechnology,
and particularly nanomedicine, are described here.
Fullerenes: Buckyballs and Carbon tubes
Both members of the fullerene structural class, buckyballs and carbon tubes are carbon based,
lattice-like, potentially porous molecules.
Liquid Crystals
Liquid crystal pharmaceuticals are composed of organic liquid crystal materials that mimic
naturally-occuring biomolecules like proteins or lipids. They are considered a very safe
method for drug delivery and can target specific areas of the body where tissues are
inflammed, or where tumors are found.
Liposomes
Liposomes are lipid-based liquid crystals, used extensively in the pharmaceutical and
cosmetic industries because of their capacity for breaking down inside cells once their
delivery function has been met. Liposomes were the first engineered nanoparticles used for
drug delivery but problems such as their propensity to fuse together in aqueous environments
and release their payload, have led to replacement, or stabilization using newer alternative
nanoparticles.
Nanoshells
Also referred to as core-shells, nanoshells are spherical cores of a particular compound
surrounded by a shell or outer coating of another, which is a few nanometers thick.
Quantum dots
Also known as nanocrystals, quantum dots are nanosized semiconductors that, depending on
their size, can emit light in all colours of the rainbow. These nanostructures confine
conduction band electrons, valence band holes, or excitons in all three spacial directions.
Examples of quantum dots are semiconductor nanocrystals and core-shell nanocrystals,
where there is an interface between different semiconductor materials. They have been
applied in biotechnology for cell labelling and imaging, particularly in cancer imaging
studies.
Superparamagnetic nanoparticles
Superparamagnetic molecules are those that are attracted to a magnetic field but do not retain
residual magnetism after the field is removed. Nanoparticles of iron oxide with diameters in
the 5-100 nm range, have been used for selective magnetic bioseparations. Typical techniques
involve coating the particles with antibodies to cell-specific antigens, for separation from the
surrounding matrix.
Used in membrane transport studies, superparamagnetic iron oxide nanoparticles (SPION)
are applied for drug delivery and gene transfection. Targeted delivery of drugs, bioactive
molecules or DNA vectors is dependent on the application of an external magnetic force that
accelerates and directs their progress towards the target tissue. They are also useful as MRI
contrast agents.
Dendrimers
Dendrimers are highly branched structures gaining wide use in nanomedicine because of the
multiple molecular "hooks" on their surfaces that can be used to attach cell-identification
tags, fluorescent dyes, enzymes and other molecules. The first dendritic molecules were
produced around 1980, but interest in them has blossomed more recently as biotechnological
uses are discovered.
Nanorods
Typically 1-100 nm in length, nanorods are most often made from semiconducting materials
and used in nanomedicine as imaging and contrast agents. Nanorods can be made by
generating small cylinders of silicon, gold or inorganic phosphate, among other materials.
There are various types of nanoparticles used for different purposes namely gold,
silver, silicon, iron oxide, platinum, copper and manganese dioxide nanoparticles. The liquid
is usually either an intense red colour (for particles less than 100 nm), or a dirty yellowish
colour (for larger particles). (Buzea et al., 2007). [2] Environ Sci. Technol. 42 (11).
IRON OXIDE NANOPARTICLE
Iron oxide nanoparticles are iron oxide particles with diameters between about 1 and 100
nanometers. The two main forms are magnetite (Fe3O4) and its oxidized form maghemite (-
Fe2O3). They have attracted extensive interest due to their superparamagnetic properties and
their potential applications in many fields (although Cu, Co and Ni are also highly magnetic
materials, they are toxic and easily oxidized).
Applications of iron oxide nanoparticles include terabit magnetic storage devices, catalysis,
sensors, and high-sensitivity biomolecular magnetic resonance imaging (MRI) for medical
diagnosis and therapeutics. These applications require coating of the nanoparticles by agents
such as long-chain fatty acids, alkyl-substituted amines and diols. . In recent years, nano-
sized Fe3O4 particles have been used in drug delivery system (DDS), protein separation and
purification, enzyme and protein immobilization.
Synthesis of Nanoparticle
The preparation method has a large effect on shape, size distribution, and surface chemistry
of the particles. It also determines to a great extent the distribution and type of structural
defects or impurities in the particles. All these factors affect magnetic behavior. Recently,
many attempts have been made to develop processes and techniques that would yield
monodisperse colloids consisting of nanoparticles uniform in size and shape.
Coprecipitation
By far the most employed method is coprecipitation. This method can be further divided into
two types. In the first, ferrous hydroxide suspensions are partially oxidized with different
oxidizing agents The other method consists in ageing stoichiometric mixtures of ferrous and
ferric hydroxides in aqueous media, yielding spherical magnetite particles homogeneous in
size. The size and shape of the nanoparticles can be controlled by adjusting pH, ionic
strength, temperature, nature of the salts (perchlorates, chlorides, sulfates, and nitrates), or the
Fe(II)/Fe(III) concentration ratio
.

Microemulsions
A microemulsion is a stable isotropic dispersion of 2 immiscible liquids consisting of
nanosized domains of one or both liquids in the other stabilized by an interfacial film of
surface-active molecules. Microemulsions may be categorized further as oilinwater
(o/w) or waterinoil (w/o), depending on the dispersed and continuous phases. Water
inoil is more popular for synthesizing many kinds of nanoparticles. The water and oil are
mixed with an amphiphillic surfactant. The surfactant lowers the surface tension between
water and oil, making the solution transparent. The water nanodroplets act as nanoreactors for
synthesizing nanoparticles. The shape of the water pool is spherical. The size of the
nanoparticles will depend on size of the water pool to a great extent. Thus, the size of the
spherical nanoparticles can be tailored and tuned by changing the size of the water pool.
High-temperature decomposition of organic precursors
The decomposition of iron precursors in the presence of hot organic surfactants results in
samples with good size control, narrow size distribution (5-12 nm) and good crystallinity; and
the nanoparticles are easily dispersed. For biomedical applications like magnetic resonance
imaging, magnetic cell separation or magnetorelaxometry, where particle size plays a crucial
role, magnetic nanoparticles produced by this method are very useful. Viable iron precursors
include Fe (Cup)
3
,Fe(CO)
5
, or Fe(acac)
3
in organic solvents with surfactant molecules.
NANOPARTICLE CHARACTERIZATION TECHNIQUES
XRD analysis, UV-VISIBLE spectrophotometry, Transmission Electron Microscopy (TEM),
Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), electron diffraction
(ED) photography, Fourier transforms infrared spectrometer (FT-IR), and vibrating-sample
magnetometer (VSM), Energy Dispersive X-Ray Spectroscopy (EDX)




REVIEW OF LITERATURES
In 2007, Hironori Iida, Kosuke Takayanagi, Takuya Nakanishi, Tetsuya Osaka et.al
synthesized Fe3O4 nanoparticles with various sizes and magnetic properties by controlled
hydrolysis. Nanoparticles of Fe3O4 were synthesized by hydrolysis in an aqueous solution
containing ferrous and ferric salts at various ratios with 1,6-hexanediamine as a base. It was
found that the ferrous to ferric ratio inuences the reaction mechanism for the formation of
Fe3O4. When the ratio of ferrous to ferric ions was increased, the formation of large
hydroxide particles as a precursor of Fe3O4 was promoted, which resulted in an increase in
the size of Fe3O4 nanoparticles. As a result, the mean diameter of Fe3O4 nanoparticles
increased from 9to 37 nm as the molar percentage of ferrous ions with respect to the total
iron ions was increased from 33 to 100%. Furthermore, it was demonstrated that magnetic
properties of Fe3O4 nanoparticles can be controlled by adjusting the molar ratio of ferrous to
ferric ions as well as the particle diameter.
Jing Sun, Shaobing Zhou, Peng Hou,Yuan Yang, Jie Weng, Xiaohong Li, Mingyuan Li
et.al synthesized and characterized biocompatible Fe3O4 nanoparticles. In this study,
magnetite (Fe3O4) nanoparticles with a size range of 820 nm were prepared by the modied
controlled chemical coprecipitation method from the solution of ferrous/ferric mixed salt-
solution in alkaline medium. In the process, two kinds of surfactant (sodium oleate and
polyethylene glycol) were studied; then, sodium oleate was chosen as the apt surfactant to
attain ultrane, nearly spherical and well-dispersed (water-base) Fe3O4 nanoparticles, which
had well magnetic properties. The size and size distribution of nanoparticles were determined
by particle size analyser. And the magnetite nanoparticles was characterized by X-ray powder
diffraction (XRD) analysis, transmission electron microscopy (TEM), electron diffraction
(ED) photography, Fourier transform infrared spectrometer (FT-IR), and vibrating-sample
magnetometer (VSM). Also the effect of many parameters on the Fe3O4 nanoparticles was
studied, such as reaction temperature, pH of the solution, stirring rate and concentration of
sodium oleate. And the 5-dimethylthiazol-2-yl-2,5- diphenyltetrazolium bromide (MTT)
assay was performed to evaluate the biocompatibility of magnetite nanoparticles. The results
showed that the Fe3O4 nanoparticles coated by sodium oleate had a better biocompatibility,
better magnetic properties, easier washing, lower cost, and better dispersion than the
magnetite nanoparticles coated by PEG. 2006 Wiley Periodicals, Inc. J Biomed Mater Res
80A: 333341, 2007
D. Predoi et.al synthesized The iron oxide nanoparticles and iron oxide nanoparticles coated
with dextrin have been using aqueous solution of ferric and ferrous ions and mixtures of
dextrin with sodium salt. The size of the iron-oxide nanoparticles is controlled by the
concentration of sodium salt in the medium. An average size of iron oxide and iron oxide
coated with dextrin was found by transmission electron microscopy (TEM). The iron oxide
nanoparticles are nearly spherical with an average diameter of about 8.0 1 nm. The iron
oxide nanoparticles coated with dextrin appear as cluster-like aggregates. The average
diameter of these nanoparticles is about 5.1 nm. The attachment of the dextrin on the particle
surface was confirmed by FTIR spectroscopy and thermogravimetric analyses (TGA).
Ki Do Kim, Sung Soo Kim, Yong-Ho Choa, and Hee Taik Kim et.al synthesized Fe3O4
nanoparticles by co-precipitation of Fe3+ and Fe2+ with NH4OH, and then silica was coated
onto the surface of Fe3O4 by hydrolysis of TEOS. Coupling agent was also coupled with the
surface of the nanoparticles and protein was immobilized. Morphology, particle size, and
magnetic properties of the nanoparticles were characterized by TEM, DLS, and VSM,
respectively. As a result, silica coated Fe3O4 nanoparticles with an average size of 15 nm
were obtained and super-paramagnetic properties were achieved.
Nheim Tran, Aparna Mir, Dhriti Mallik, Avind Sinha, Suprava nayar, Thomas J
Webster et.al studied the bactericidal effect of iron oxide nanoparticles on Staphylococcus
aureus. In order to study the effects of iron oxide (IO) nanoparticles on Staphylococcus
aureus, IO nanoparticles were synthesized via a novel matrix-mediated method using
polyvinyl alcohol (PVA). The IO nanoparticles were characterized by transmission electron
microscopy and dynamic light scattering. Further, S.aureus were grown in the presence of
three different IO nanoparticle concentrations for four , 12, 24 hours. Live/dead assays were
performed and the results provide evidence that IO/PVA nanoparticles inhibited S.aureus
growth at the highest concentrations (3mg/ml) at all points.
E. Iglesias-Silva, J. Rivas, L.M. Leon Isidro, M.A. Lopez-Quintela et.al synthesized the
silver coated magnetite nanoparticle. They described the preparation of relatively
monodisperse silver-coated Fe3O4 nanoparticles by a two-step procedure. Fe3O4
nanoparticles of 9 2 nm in size were rst prepared in microemulsions. They were
subsequently coated with silver using glucose as reducing agent. The presence of a relatively
homogeneous coating of 2 nm was conrmed by transmission electron microscopy and X-ray
diraction. A preliminary study of the magnetic properties shows a large decrease of the
magnetization for the coated magnetite nanoparticles in comparison with the uncoated ones.










MATERIALS AND METHODS
Iron oxide nanoparticles were prepared by two methods.
FIRST METHOD:
Initially 8Ml of 1M FeCl
3
and 2mL of 2MFeCl
2
solution was added to a 100Ml beaker.
100Ml of 1.0M aqueous NaOH solution was slowly added to the beaker with continuous
stirring over a period of 5mins. After an initial brown precipitate and later a black precipitate
will be formed. The solution was observed under UV and Visible spectroscopy for
determining the nanoparticle. Then the solution was preserved in water bath at 65c for 24h.
Finally the solution was filtered through filter paper and allowed to dry in hot air oven to get
powder form.
SECOND METHOD:
The -Fe
2
O
3
hydrosol was synthesized by using hydrolysis method. The amount of stock
solution of FeCl
3
of 3M and HCl of 0.2M were mixed in a flask at the ratio of 1:3(v/v). The
deionized water was added till the final concentration of Fe
3+
is 0.01M.This mixture was
preserved in water bath at 96C for 24h and then quenched to room temperature. An orange-
red solution was formed which was the -Fe
2
O
3
nanoparticle hydrosol. Then the solution was
filtered through the filter paper and allowed to dry in hot air oven to get powder form
.
CHARACTERIZATION
PRINCIPLES:
There is various characterization techniques used for characterizing different
nanoparticles. Here we have discussed the basic principles of few techniques that have been
used in the experimental part of this project work. They are Absorption spectrophotometer
(UV-VIS), X-Ray diffraction (XRD).


A. Absorption spectroscopy
A spectrophotometer is an instrument, which is used to determine the percentage of
transmittance light radiation when light of certain frequency is passed through the samples.
The spectrophotometer records the intensity of absorption (A) or optical density (O.D) as a
function of wavelength. If suppose I
t
and I
o
are the intensities of incident and transmitted rays
of light respectively.
Then the absorbance A is defined as A=log I
t
/I
o

Or, A = cx
Where, x=sample path length (cm)
c=concentration (mol lit-1)
=molar extinction coefficient (mol
-1
cm
-1
lit)
This technique usually gives the preliminary concept of particle size and size
distribution such as mono or polydispersity.Usually a blue shift (decrease in wavelength) is
associated with a decrease in particles size and vice-versa.

B. X-Ray Diffraction:
The Fe
3
O
4
nanoparticles were analyzed for phase composition using X-ray powder
diffraction (XRD, Philips XPert PRO) over the 2 Theta range from 1090 degree at rate of
2.5degree/min, using Cu-K -a radiation (l 1.54060 A ). The identification of phases has
now turned into multifaceted probe for materials analysis and characterization. This method
can yield a greater deal of structural information about materials under investigation. The
random diffraction orientations of the individual crystal in a powder specimen are equivalent
to the solution of a single crystal about all possible axes during the X-ray exposure. The
reciprocal lattice therefore takes an all possible orientations relative to the incident beam but
its origin remains fixed at the end of the incident beam vector.
Condition for diffraction:
It should satisfy Braggs diffraction. To satisfy Braggs equation, it is necessary to
adjust d, , in such a way that
2dSin= n
Where,
d= perpendicular distance between lattice planes of miller indices.
= wavelength of the incident X-ray.
=glancing angle
Braggs law
When a chromatic intense beam of light falls on a parallel lattice plane of crystal, the
incident beam reflected secularly from various planes of crystal. In this case, the phenomenon
of reflection is known as crystal diffraction of scattering. Diffraction is essentially a
scattering phenomenon in which large numbers of atoms co-operate.
PROCEDURE:
Both the solution were prepared for serial dilution and observed under UV and visible
spectroscopy from 200nm to 450nm. Then the graph was plotted according to the absorbance.
The solution containing nanoparticles will give highest absorbence at 340nm.

DETERMINATION OF ANTIMICROBIAL ACTIVITY OF IRON
OXIDE NANOPARTICLES:
PRINCIPLES:
Stock solution:
The gram negative & gram positive bacteria such as E.coli and B.subtilis .respectively
were grown overnight in Luria Bertani (LB) broth at 37C. Bacterial cells were centrifuged at
6000 rpm for 15 min; washed cell pellets were resuspended in LB and optical density (OD)
was adjusted to 0.1 at 595 nm. (OD of 0.1 corresponds to a concentration of 10
8
CFU ml
-1
of
medium).The bactericidal activity of Fe
3
O
4
nanoparticles was estimated by determining the
minimal inhibitory concentration (MIC).
Dilution assays:
Dilution assays are standard methods used to compare the inhibition efficiency of
antimicrobial agents. The test extracts or compounds are mixed with suitable media that has
been inoculated with the test microorganism. It can be carried out in liquid media (broth
dilution assay) or in solid media (agar dilution assay). Growth inhibition is expressed by
Minimal Inhibitory Concentration (MIC) which is defined as the lowest concentration able to
inhibit any visible microbial growth. The Minimal Bactericidal or Fungicidal Concentration
(MBC or MFC) is determined by plating-out samples of completely inhibited dilution
cultures and assessing growth after incubation [Cos et al., 2006; Yin and Tsao et al., 1999;
Salie et al., 1996]. The inoculate concentrations of bacterial or fungal cultures are between
10
4
-10
8
CFU/mL [Camporese et al., 2003; Karaman et al., 2003].
In the agar plate dilution assay, the microbial cell suspension is spread over the
surface of the agar plate [Verstegui et al., 1996], inoculated on the center of the agar surface
[Sato et al., 2000; Quiroga, et al., 2001], by the streak method [Kumar et al., 2006] or mixed
with the media as in the broth dilution assay [Navarro and Delgado, 1999; Cos et al., 2002;
Pyun and Shin, 2005]. Bacterial cells (both gram-positive & gram-negative) were grown in
NB medium and 500 l of 24-h-old bacterial culture (0.1 OD) was spreaded over the NB agar
plates, supplemented with 0.3mg/ml of synthesized parent I.O nanoparticles. All plates were
incubated in BOD incubator for 24 h. Lower concentration to MIC cannot inhibit microbial
growth.
The I.O nanoparticles were suspended in triple distilled water to conduct the time-
dependent antibacterial study. E. coli and Bacillus subtilis cells were treated with 2.0 ml of
each concentration (0.3mg/ml) of I.O nanoparticle for 24h. Each treated bacterial culture was
serially diluted till 10
6
dilution factor and 100 l from each culture was homogeneously
spread in NB agar plates. All plates were incubated at 37C for 24 h and the number of
colonies grown on agar plate was counted.

Well diffusion assay:
The well diffusion assay is suitable for aqueous extracts because they are difficult to
dry on paper discs [Vlietinck, et al., 1995; Fazeli et al., 2007; Magaldi, et al., 2004; Tadeg,
etal., 2005]. However, the leaking of sample under the agar layer must be considered. Wells
with 8 mm diameter are cut in the agar plate using a cork borer and 100 L of sample is
loaded into the well [Fazeli et al., 2007; Patton et al., 2006]. Microbial cell suspension is used
in a similar way to the disc diffusion assay and the inhibition diameter is measured after
incubation.


Zone of inhibition test:
For the antimicrobial activity 50ml of nutrient agar was prepared and sterilized. 0.3mg/ml of
Iron oxide nanoparticle on E.coli and Bacillus subtilis was added. For this, 20 ml NB agar
was poured in well-rinsed, autoclaved petri plates and allowed to solidify. 1.0 ml of active
bacterial culture was homogeneously spread in the agar plates and 100 l of Iron oxide
nanoparticle solution filled in deep blocks, prepared by cutting the agar by gel puncture. The
plates were then placed in incubator at 37c for overnight growth. Then the inhibition zone
was measured against each bacteria.












RESULT
4.1. Synthesis of iron oxide nanoparticles:
Iron oxide nanoparticles were synthesized by the two methods.
First nanoparticle was synthesized by coprecipitation method using ferric and ferrous
chloride salts. And the second method was by using hydrolysis method. The iron oxide
nanoparticles synthesized was obtained in the form of dry brownish powder. These iron oxide
particles were used for the characterization for determining the nanoparticles.

Fig: Powder form of iron oxide nanoparticles (sample 1)

Fig: Powder form of iron oxide nanoparticles (sample 2)


4.2. UV-Visible spectroscopy:
These iron oxide nanoparticles are subjected for characterization by two methods
mainly UV and visible spectroscopy and x-ray diffraction technique. The nanoparticles
present in the solution should give highest absorbence at 230nm and 340nm.

TABLE-1: SOLUTION 1
UV WAVELENGTH:
Dilution
Rate
200
nm
210
Nm
220
Nm
230
Nm
240
Nm
250
Nm
260
nm
270
nm
280
nm
290
nm
300
nm
310
Nm
320
Nm
10
-1
4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 2.705 2.597 2.588 2.562 2.550
10
-2
1.928 1.733 1.624 1.545 1.517 1.504 1.497 1.419 1.409 1.408 1.401 1.400 1.376
10
-3
0.319 0.312 0.251 0.330 0.300 0.280 0.255 0.120 0.114 0.110 0.109 0.100 0.100
10
-4
0.097 0.089 0.049 0.037 0.029 0.027 0.025 0.024 0.023 0.022 0.022 0.022 0.022
10
-5
0.015 0.015 0.008 0.008 0.009 0.008 0.008 0.009 0.008 0.009 0.009 0.008 0.008

VISIBLE WAVELENGTH:
Dilution
Rate
330
nm
340
Nm
350
Nm
360
Nm
370
Nm
380
Nm
390
nm
400
nm
410
nm
420
nm
430
nm
440
Nm
450
Nm
10
-1
4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000
10
-2
1.262 1.237 1.236 1.235 1.232 1.190 1.093 1.022 0.942 0.877 0.794 0.711 0.650
10
-3
0.185 0.254 0.179 0.179 0.179 0.169 0.153 0.133 0.121 0.109 0.096 0.083 0.074
10
-4
0.031 0.031 0.032 0.032 0.032 0.032 0.032 0.029 0.027 0.025 0.023 0.021 0.020
10
-5
0.012 0.012 0.012 0.012 0.012 0.011 0.013 0.014 0.013 0.012 0.011 0.011 0.010

TABLE-2: SOLUTION 2
UV WAVELENGTH:
Dilution
Rate
200
nm
210
Nm
220
Nm
230
Nm
240
Nm
250
Nm
260
nm
270
nm
280
nm
290
nm
300
nm
310
Nm
320
Nm
10
-1
4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000
10
-2
4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000
10
-3
0.800 0.783 0.740 0.889 0.674 0.595 0.580 0.567 0.561 0.557 0.553 0.511 0.501
10
-4
0.229 0.227 0.207 0.194 0.191 0.117 0.114 0.112 0.112 0.111 0.111 0.087 0.083
10
-5
0.273 0.225 0.223 0.222 0.210 0.218 0.121 0.111 0.106 0.103 0.100 0.080 0.077

VISIBLE WAVELENGTH:
Dilution
Rate
330
nm
340
Nm
350
Nm
360
Nm
370
Nm
380
Nm
390
nm
400
nm
410
nm
420
nm
430
nm
440
Nm
450
Nm
10
-1
4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 3.456 2.934 2.748 2.604 2.534
10
-2
4.000 4.000 2.109 1.539 1.087 0.802 0.621 0.518 0.454 0.413 0.388 0.363 0.340
10
-3
0.484 0.540 0.472 0.423 0.365 0.309 0.258 0.221 0.190 0.162 0.136 0.115 0.099
10
-4
0.084 0.079 0.030 0.024 0.020 0.016 0.015 0.013 0.012 0.010 0.010 0.009 0.008
10
-5
0.080 0.076 0.038 0.028 0.023 0.019 0.018 0.017 0.016 0.014 0.014 0.014 0.011
GRAPH:
Sample-1: UV and Visible Wavelength



Solution 1: UV Wavelength



0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
A
b
s
o
r
b
a
n
c
e

Wavelength
103
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
A
b
s
o
r
b
a
n
c
e

Wavelength
103

Solution 1: Visible Wavelength



Solution 2: UV and Visible Wavelength



0
0.05
0.1
0.15
0.2
0.25
0.3
A
b
s
o
r
b
a
n
c
e

Wavelength
103
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
A
b
s
o
r
b
a
n
c
e

Wavelength
103


Solution 2: UV Wavelength



Solution 2: Visible Wavelength


0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
A
b
s
o
r
b
a
n
c
e

wavelength
103
0
0.1
0.2
0.3
0.4
0.5
0.6
A
b
s
o
r
b
a
n
c
e

Wavelength
103
1st READING:



2nd READING:



4.3. Antimicrobial activity of the nanoparticles:
The nanoparticles have their own antimicrobial property to inhibit the growth of
harmful and pathogenic bacteria. These iron oxide nanoparticles have their antimicrobial
activity against human pathogens. These particles have less antimicrobial activity as
compared to other nanoparticles. Iron oxide nanoparticles of sample 1 have their maximum
inhibition zone for gram positive bacteria than gram negative bacteria. And sample 2 have
maximum inhibition zone against gram negative bacteria. The sample 2 has 11mm inhibition
zone and sample 1 has 14mm inhibition zone.

FIGURE: 1st Reading (Sample-1) FIGURE: 2
nd
Reading (Sample-2)
Fig: Gram negative bacteria Fig: Gram positive bacteria

Strain Sample 1 Sample 2
Gram positive No Inhibition Zone No Inhibition Zone
Gram negative 14mm No Inhibition Zone
Strain Sample 1 Sample 2
Gram positive 9mm No Inhibition Zone
Gram negative 10mm 11mm










Fig: Solutions of sample 1 and 2 for
the antimicrobial activity

DISCUSSION
5.1. Synthesis of iron oxide nanoparticles
The mechanism of the formation of Fe
3
O
4
nanoparticles with ferrous and ferric salts
at the ratio of 1 to 2, by the coprecipitation reaction in which stoichiometric amounts of
ferrous and ferric ions react to produce Fe
3
O
4
. The size of the iron-oxide nanoparticles is
controlled by the concentration of sodium salt in the medium. The synthesized nanoparticles
are present in the form of brownish powder. The synthesis powder form Fe
3
O
4
is formed as a
result of the dehydration reaction of ferrous and ferric ions.

5.2. Characterization of nanoparticles:
The characterization was done by UV and visible spectroscopy. The absorbance was
measured in the spectrophotometer under different wavelength. The absorbance should be
higher at 230nm and 340nm.The absorbance of iron oxide nanoparticles has highest at the
above range due to charge transfer spectra.

5.3. Antimicrobial activity:
The different pathogenic strains of gram positive and gram negative bacteria are used
for the estimation of antimicrobial activity of nanoparticles. The antimicrobial activity against
gram positive and gram negative bacteria was observed. The inhibition zone of iron oxide
nanoparticles of sample 1 against gram positive bacteria was about 14mm in diameter. And
the inhibition zone of nanoparticles of sample 1 against gram negative bacteria was about
10mm and sample 2 was 11mm in diameter.



Possible mechanism for antibacterial action of Fe
3
O
4

nanoparticles:
The differences between gram-positive and gram-negative bacteria essentially rest in
the structure of their respective cell walls. The gram-negative bacteria have a layer of
lipopolysaccharide at the exterior, followed underneath by a thin (about 78 nm) layer of
peptidoglycan. Although the lipopolysaccharides are composed of covalently linked lipids
and polysaccharides, they lack strength and rigidity. Negative charges on the
lipopolysaccharides are attracted towards weak positive charges available on Fe
3+
of Fe
3
O
4

nanoparticles. On the other hand, the cell wall in gram-positive bacteria is principally
composed of a thick layer (about 2080 nm) of peptidoglycan, consisting of linear
polysaccharide chains cross-linked by short peptides to form a three dimensional rigid
structure. The rigidity and extended cross-linking not only endow the cell walls with fewer
anchoring sites for the I.O nanoparticles but also make them difficult to penetrate. Thus I.O
nanoparticles are more toxic to E. coli than B.subtilis.












CONCLUSION

The stable iron oxide nanoparticles were successfully synthesized by using two
different methods. The particles were characterized by using UV and visible spectroscopy
which gives a highest absorbence at 230nm and 340nm and is valid with standard
protocol.The antimicrobial activity was determined against gram positive and gram negative
bacteria which gives a highest inhibition zone of 14mm diameter against gram negative
bacteria.












REFERENCE
1. Baker C, Pradhan A, Pakstis L, Pochan D J and Shah S I 2005 Synthesis and
antibacterial properties of silver nanoparticles J. Nanosci. Technol. 5 2449
2. Brunner, Tobias J.; Wick, Peter; Manser, Pius; Spohn, Philipp; Grass, Robert N.;
Limbach, Ludwig K.; Bruinink, Arie; Stark, Wendelin J. (2006). "In Vitro
Cytotoxicity of Oxide Nanoparticles: Comparison to Asbestos, Silica, and the Effect
of Particle Solubility". Environmental Science & Technology.
3. Curtis A and Wilkinson C 2001 Nanotechniques and approaches in biotechnology
Trends Biotechnol. 19 97101.
4. Cristina Buzea, Ivan Pacheco, and Kevin Robbie (2007). "Nanomaterials and
Nanoparticles: Sources and Toxicity". Biointerphases 2: MR17.
5. Conductive Polymer / Solvent Systems: Solutions or Dispersions?, Bernhard
Wessling, 1996
6. D. Predoi National Institute of Materials Physics, P.O. Box. MG 07, 077125,
Magurele, Romania. Digest Journal of Nanomaterials and Biostructures Vol. 2, No. 1,
March 2007, p. 169 - 173
7. E. Iglesias-Silva, J. Rivas, L.M. Leon Isidro, M.A. Lopez-Quintela Laboratory of
Magnetism and Nanotechnology, IIT, University of Santiago de Compostela, E-15782
Santiago de Compostela, Galicia, Spain Physical-Chemistry Department, University
of the Basque Country, Faculty of Science and Technology, Barrio Sarriena, s/n E-
48940, Leioa, Bizkaia, Spain Available online 7 February 2007
8. Elaissari and V. Bourrel, J. Magn. Magn. Mater.,225, 151 (2001).
9. Farokhzad O C, Cheng J, Teply B A, Sherifi I, Jon S, Kantoff P W, Richie J P and
Langer R 2006 Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy
in vivo Proc. Natl Acad. Sci. USA 103 631520.
10. Graduate school of Frontier Sciences, University of Tokyo, Japan, http://www.ib.k.u-
tokyo.ac.jp/ib-E/index.html.
11. Gleiter H 2000 Nanostructured materials, basic concepts and microstructure Acta
Mater. 48 1-12.
12. Hironori Iida, Kosuke Takayanagi, Takuya Nakanishi, Tetsuya Osaka .Department of
Applied Chemistry, School of Science and Engineering, Waseda University, 3-4-1
Okubo, Shinjuku-ku, Tokyo 169-8555, Japan Institute for Biomedical Engineering,
Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, and
Japan .Received 18 January 2007; accepted 17 May 2007 .Available online 24 May
2007
13. H. Gries, W. Mutzel, C. Zurth, and H. J. Weinmanm,US pat. 5746999 (1998).
14. Jing Sun, Shaobing Zhou, Peng Hou, Yuan Yang, Jie Weng, Xiaohong Li, Mingyuan
Li School of Materials Science and Engineering, Key Laboratory of Advanced
Technologies of Materials, Ministry of Education, Southwest Jiaotong University,
Chengdu 610031, Peoples Republic of China Department of Microbiology, Huaxi
Basic Medicine and Forensic College, Sichuan University, Chengdu 610041, Peoples
Republic of China.
15. Ki Do Kim, Sung Soo Kim, Yong-Ho Choa, and Hee Taik Kim Department of
Chemical Engineering, Hanyang University, Gyeonggi 426-791, Korea. Received
August 3, 2007; Accepted November 20, 2007
16. K. D. Kim, H. J. Bae, and H. T. Kim, Colloids and Surf. A: Physicochem. Eng.
Aspects, 224, 119 (2003).
a. K. D. Kim and H. T. Kim, J. Sol-Gel Sci. And Tech., 25, 183 (2002). (b) I.
Bica, J. Ind. Eng. Chem., 12, 501 (2006). (c) I. Bica, J. Ind. Eng.Chem., 12,
620 (2006).
17. K. OGrady, J. Phys. D: Appl. Phys., 2003, 36, R165.
18. L. Xianqiao, M. Zhiya, X. Jianmin, and L. Huizhon, J. Magn. Magn. Mater, 270, 1
(2004).
19. Langer R 2001 Drug delivery: drugs on target Science 293 589.
20. M. Koneracka, P. Kopcansky, M. Antalik, M. Timko, C. N. Ramchand, D. Lobo, R.
V. Mehta, and R. V. Upadhyay, J. Magn. Magn. Mater., 201, 427 (1999).
21. M. S. Cho, S. T. Lim, I. B. Jang, H. J. Choi, and M. S. Jhon, IEEE Trans. Magn., 40,
3036 (2004).
22. M. H. Sousa, J. C. Rubim, P. G. Sorbrinho, and F.A. Tourinho, J. Magn. Magn.
Mater., 225, 67(2001).
23. M. Koneracka, P. Kopcansky, M. Antalik, M.Timko, C. N. Ramchand, D. Lobo, R. V.
Mehta, and R. V. Upadhyay, J. Magn. Magn. Mater., 201, 427(1999).
24. Morones J R, Elechiguerra J L, Camacho A, Holt K, Kouri J B Ramirez J T and
Yacaman M J 2005 The bactericidal effect of silver nanoparticles Nanotechnology 16
234653
25. Nheim Tran, Aparna Mir, Dhriti Mallik, Avind Sinha, Suprava nayar, Thomas J
Webster Department of Physics, Brown University, Providence, Rhode Island, USA;
Department of Material Science and Technology, National Metallurgical Laboratory,
Burmamnines 831007, Jamshedpur, India; Division of Engineering and Department
of Orthopedics, Brown University, Providence, Rhode Island, USA.
26. P. Tartaj, M. Puerto Morales, S. Veintemillas-Verdaguer, T. Gonzalez-Carreno and C.
J. Serna, J. Phys. D: Appl. Phys., 2003, 36, R182.
27. Panacek A, Kvitek L, Prucek R, Kolar M, Vecerova R, Pizurova N, Sharma V K,
Nevecna T and Zboril R 2006 Silver colloid nanoparticles: synthesis, characterization,
and their antibacterial activity J. Phys. Chem. B 110 1624853
28. Roy K, Mao H Q, Huang S K and Leong K W 1999 Oral gene delivery with
Chitosan-DNA nanoparticles generatesimmunologic protection in a murine model of
peanut allergy Nat. Med. 5 38791.
29. Sachlos E, Gotora D and Czernuszka J T 2006 Collagen scaffolds reinforced with
biomimetic composite nano-sized carbonate-substituted hydroxyapatite crystals and
shaped by rapid prototyping to contain internal microchannels Tissue Eng. 12 2479
87.
30. Stoimenov P K, Klinger R L, Marchin G L and Klabunde K J 2002 Metal oxide
nanoparticles as bactericidal agents Langmuir 18 667986.
31. Sondi I and Salopek-Sondi B 2004 Silver nanoparticles as antimicrobial agent: a case
study of E. coli as a model for gram-negative bacteria J. Colloids Interface Sci. 275
17782
32. Vaseashta A and Dimova-Malinovska D 2005 Nanostructured and nanoscale devices,
sensors and detectors Sci. Technol.Adv. Mater. 6 3128.
33. Waren C W and Nie S 1998 Quantum dot bioconjugates for ultra-sensitive
nonisotopic detection Science 281 20168.
34. Xu Z P, Zeng Q H, Lu G Q and Yu A B 2006 Inorganic nanoparticles as carriers for
efficient cellular delivery Chem.Eng. Sci. 61 102740.