iron oxide nanoparticle analysis

8/12/2019 iron oxide nanoparticle analysis

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SYNTHESIS OF IRON OXIDE NANOPARTICLES,CHARACTERIZATION AND THEIR

ANTIMICROBIAL ACTIVITY

Thesis submitted to Biju Patnaik University of Technology for partialfulfillment of degree of Bachelor of Technology in Biotechnology

for Year 2011

SUBMITTED BY ABHIJIT DEHURI

REGD NO - 0701106142

SUPERVISED BYMr. SUDHANSU SEKHAR BEHERA

Lecturer

DEPARTMENT OF BIOTECHNOLOGY,

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

BHUBANESWER -7510032011


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DEP RTMENT 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

ertific te

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)


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DEP RTMENT 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

ertific te

This is to certify that the thesis entitled Synthesis of iron oxide

nanoparticles, characterization and their antimicrobial activit y 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


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DECL R TION

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 ofBiotechnology, 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)


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cknowledgements

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)


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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 2O and FeCl 3.6H 2O

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



4.3. Antimicrobial activity

5. DISCUSSION



5.3. Antimicrobial activity

6. CONCLUSION

7. REFERENCE


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LIST OF T BLES

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.


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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.


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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.


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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 biolo gical

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 Coulomb s 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 sci ence. 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. Nanotechnologyand nanoscience got a boost in the early 1980s with two major developments: the birth
http://en.wikipedia.org/wiki/Nanosciencehttp://en.wikipedia.org/wiki/Nanoscience


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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.
http://en.wikipedia.org/wiki/Cluster_(physics)http://en.wikipedia.org/wiki/Scanning_tunneling_microscopehttp://en.wikipedia.org/wiki/Scanning_tunneling_microscopehttp://en.wikipedia.org/wiki/Scanning_tunneling_microscopehttp://en.wikipedia.org/wiki/Scanning_tunneling_microscopehttp://en.wikipedia.org/wiki/Scanning_tunneling_microscopehttp://en.wikipedia.org/wiki/Fullereneshttp://en.wikipedia.org/wiki/Carbon_nanotubeshttp://en.wikipedia.org/wiki/Carbon_nanotubeshttp://en.wikipedia.org/wiki/Carbon_nanotubeshttp://en.wikipedia.org/wiki/Nanotechnologyhttp://en.wikipedia.org/wiki/Diameterhttp://en.wikipedia.org/wiki/Diameterhttp://en.wikipedia.org/wiki/Nanotechnologyhttp://en.wikipedia.org/wiki/Carbon_nanotubeshttp://en.wikipedia.org/wiki/Fullereneshttp://en.wikipedia.org/wiki/Scanning_tunneling_microscopehttp://en.wikipedia.org/wiki/Cluster_(physics)


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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


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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


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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 co precipitation. 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 .
http://en.wikipedia.org/wiki/Monodispersehttp://en.wikipedia.org/wiki/Monodispersehttp://en.wikipedia.org/wiki/Colloidhttp://en.wikipedia.org/wiki/Colloidhttp://en.wikipedia.org/wiki/Coprecipitationhttp://en.wikipedia.org/wiki/Iron%28II%29_hydroxidehttp://en.wikipedia.org/wiki/Iron%28II%29_hydroxidehttp://en.wikipedia.org/wiki/Iron%28II%29_hydroxidehttp://en.wikipedia.org/wiki/Suspension_%28chemistry%29http://en.wikipedia.org/wiki/Ionic_strengthhttp://en.wikipedia.org/wiki/Ionic_strengthhttp://en.wikipedia.org/wiki/Ionic_strengthhttp://en.wikipedia.org/wiki/Salt_%28chemistry%29http://en.wikipedia.org/wiki/Perchloratehttp://en.wikipedia.org/wiki/Chloridehttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Nitratehttp://en.wikipedia.org/wiki/Nitratehttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Chloridehttp://en.wikipedia.org/wiki/Perchloratehttp://en.wikipedia.org/wiki/Salt_%28chemistry%29http://en.wikipedia.org/wiki/Ionic_strengthhttp://en.wikipedia.org/wiki/Ionic_strengthhttp://en.wikipedia.org/wiki/Suspension_%28chemistry%29http://en.wikipedia.org/wiki/Iron%28II%29_hydroxidehttp://en.wikipedia.org/wiki/Coprecipitationhttp://en.wikipedia.org/wiki/Colloidhttp://en.wikipedia.org/wiki/Monodisperse


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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 oil in water

(o/w) or water in oil (w/o), depending on the dispersed and continuous phases. Water

in oil 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-samplemagnetometer (VSM), Energy Dispersive X-Ray Spectroscopy (EDX)
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magnetite nanoparticles coated by PEG. 2006 Wiley Periodicals, Inc. J Biomed Mater Res

80A: 333 341, 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 pre pared in microemulsions. They weresubsequently coated with silver using glucose as reducing agent. The presence of a relatively


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homogeneous coating of 2 nm was conrmed by transmission electron microscopy and X-ray

diff raction. 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.


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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 2O3 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 2O3 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).


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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 afunction 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/Io

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 O4 nanoparticles were analyzed for phase composition using X-ray powder

diffraction (XRD, Phi lips XPert PRO) over the 2 Theta range from 10 90 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.


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=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 phenomenonof 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 3O4 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


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(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

104-10 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 wereincubated 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 Bacill us subtil is 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.


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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.


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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)


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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:

DilutionRate

200nm

210 Nm

220 Nm

230 Nm

240 Nm

250 Nm

260nm

270nm

280nm

290nm

300nm

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 2.55010 - 1.928 1.733 1.624 1.545 1.517 1.504 1.497 1.419 1.409 1.408 1.401 1.400 1.37610 -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 0.10010 - 0.097 0.089 0.049 0.037 0.029 0.027 0.025 0.024 0.023 0.022 0.022 0.022 0.02210 -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:

DilutionRate

330nm

340 Nm

350 Nm

360 Nm

370 Nm

380 Nm

390nm

400nm

410nm

420nm

430nm

440 Nm

450 Nm

10 - 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.00010 -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-

0.185 0.254 0.179 0.179 0.179 0.169 0.153 0.133 0.121 0.109 0.096 0.083 0.07410 - 0.031 0.031 0.032 0.032 0.032 0.032 0.032 0.029 0.027 0.025 0.023 0.021 0.02010 - 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:

DilutionRate

200nm

210 Nm

220 Nm

230 Nm

240 Nm

250 Nm

260nm

270nm

280nm

290nm

300nm

310 Nm

320 Nm

10 - 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.00010 -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 4.00010 - 0.800 0.783 0.740 0.889 0.674 0.595 0.580 0.567 0.561 0.557 0.553 0.511 0.50110 - 0.229 0.227 0.207 0.194 0.191 0.117 0.114 0.112 0.112 0.111 0.111 0.087 0.08310 - 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:

DilutionRate

330nm

340 Nm

350 Nm

360 Nm

370 Nm

380 Nm

390nm

400nm

410nm

420nm

430nm

440 Nm

450 Nm

10 - 4.000 4.000 4.000 4.000 4.000 4.000 4.000 4.000 3.456 2.934 2.748 2.604 2.53410 -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 0.34010 - 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 0.00810 - 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


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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

10 3

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

10 3


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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

10 3

0

0.1

0.2

0.3

0.40.5

0.6

0.7

0.8

0.9

1

A b s o r

b a n c e

Wavelength

10 3


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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 ZoneGram negative 14mm No Inhibition Zone

Strain Sample 1 Sample 2

Gram positive 9mm No Inhibition ZoneGram negative 10mm 11mm


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Fig: Solutions of sample 1 and 2 forthe antimicrobial activity


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DISCUSSION


The mechanism of the formation of Fe 3O4 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 3O4. 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 3O4 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.


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Possible mechanism for antibacterial action of Fe 3O 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 7 8 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 3O4

nanoparticles. On the other hand, the cell wall in gram-positive bacteria is principally

composed of a thick layer (about 20 80 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.


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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.


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iron oxide nanoparticle analysis