دوپامین اصلی

Faculty of Engineering
Department of Chemical Engineering
M.Sc.Thesis
Title of the Thesis:

Preparation and Application of Graphene Oxide/Functionalized
Graphene Oxide Nanostructures as Antibacterial Agents
By:
Sara Azimi
Evaluated and approved by thesis committee: as
Dr. Jamshid Behin
Supervisor
Dr. Laleh Rajabi
Supervisor
Dr. Mojtaba Ahmadi
Internal Examiner
Dr. Ramin Abiri
External Examiner
March 2013
Faculty of Engineering
Department of Chemical Engineering
M.Sc.Thesis
Title of the Thesis:

Preparation and Application of Graphene Oxide/Functionalized
Graphene Oxide Nanostructures as Antibacterial Agents
Supervisors:
Dr. Jamshid Behin
Dr. Laleh Rajabi
By:
Sara Azimi
March 2013
In The Name of God

The Beneficent
The Merciful
Acknowledgment
Although this thesis bears only one authors name on its cover, it could be similar to
other work that has not been achieved without the support of a number of people.
First of all, I would like to thank the two pillars of my life, God and my parents.
I might not know where the lifes road will take me but walking with you, God, through
this journey has given me strength.
My dear parents, you have given me so much, thanks for teaching me that, I should
never surrender. You always told me to reach for the stars; I think I get my first one.
On the academic side, I would like to express my appreciation to my supervisors: Dr.
Jamshid Behin and Dr. Laleh Rajabi. I should thank you for your support and guiding
me throughout the entire process of the thesis. It was an honor to work with you.
My special gratitude goes to Dr. Ramin Abiri, the Head of Microbiology Department in
Kermanshah University of Medical Science. You certainly made this study happen.
There are not enough words to describe your kindness. Thanks for all your help,
inspiring discussions and attention.
I also owe thanks to Mr. Ali Derakhshan. We are so privileged at Polymer Research
center to have someone like you helping all the students.
Finally, I would like to thank my dear friend, Hanieh Karimnezhad for being such a
great classmate.
ABSTRACT
This thesis starts with the aim full synthesis and characterization of Graphene Oxide
nanomaterial (GO) and water soluble material in order to prepare two types of functionalized
GO nanomaterials. The GO nanomaterial was synthesized through oxidation of natural
graphite powder using Hummers method. Also, the soluble material was prepared using
extraction of leaves. The objective of synthesizing these nanomaterials was to evaluate their
antibacterial activity against Escherichia coli (E. coli), and thus, to find out the effect of these
functionalization on toxicity of GO towards E. coli. FTIR, SEM, EDX, UV/vis
spectrophotometer, Ultrasonic bath, Shaker incubator, Colony counter and Digital camera
were used to conduct the experiments and also to characterize. These analyses confirmed that,
novel synthesized GO nanomaterials were well functionalized. Under similar concentration
and incubation conditions, functionalized GO nanomaterials dispersion had the highest
antibacterial activity, sequentially followed GO. In addition, it was revealed that, the exposure
of E. coli to functionalized GO nanomaterials has led to losing cellular integrity and
consequently cell death.
Fabrication of these types of functionalized GO nanomaterials and evaluating their
antibacterial activity has not been reported elsewhere and thus, there is a large potential for
them to offer new opportunities for the development of antibacterial agents in medical fields.
Keywords: Functionalization, Graphene Oxide, Nanomaterials, Escherichia coli.
LIST OF CONTENTS
Content .......................... Page
1
Chapter 1: General Introduction

1.1.
Nanotechnology
1.2.
Graphite Oxide
1.2.1.Structural features and properties of Graphite Oxide

1.3.
Graphene Oxide (GO)
3
4
1.3.1. Solvent-based exfoliation
1.3.2. Thermal exfoliation
1.3.3. Microwave exfoliation
1.4. Chemical functionalization of Graphene Oxide
1.4.1. At the carboxylic acid group of Graphene Oxide
1.4.2. At the epoxy group of Graphene Oxide
1.4.3. Non-covalent functionalization of Graphene Oxide
1.5. Graphene Oxide applications
1.5.1. Transparent conductive films
1.5.2. Composites and paper-like materials
1.5.3. Energy-related materials
1.5.4. Graphene Oxide sheets at interfaces
1.5.5. Applications in biology and medicine
1.5.6. Graphene Oxide as antibacterial agent
1.6. Antibacterial agents
10
1.6.1. Definition of antibacterial agents
10
1.6.2. Mechanisms of antibacterial action
11
1.6.3. Chlorophyll as a natural antibacterial agent
11
1.6.3.1. Medical and antimicrobial effect of Chlorophyll and its

derivatives
11
1.6.3.2. Chlorophyll structure
12
1.6.3.3. Chlorophyll reactions
12
1.7. Escherichia coli (E. coli) as a model bacterium

A
14
1.8. Overview of spread plate as a colony counting methodology
15
1.9. Objective and scope of this research work
16
17
Chapter 2: Literature Review

2. 1. Literature review
18
2.1.1.Antimicrobial property of GO and its mechanism against

bacteria
18
2.1.2. Fabrication of modified antibacterial GO composites
20
24
Chapter 3: Experimental
3.1. Chemicals
25
3.2. Apparatus
25
3.3. Synthesis of GO nanomaterial
26
3.3.1. Synthesis of Graphite Oxide
26
3.3.2. Preparation of GO nanosheet suspension
26
3.4. Preparation of water soluble material
26
3.4.1. Material
26
3.4.2. Solvent method
27
3.5. Functionalized GO nanomaterials syntheses
28
3.5.1. Synthesis of Graphene Oxide nanomaterial

28
3.5.2. Synthesis of Graphene Oxide nanomaterial
28
3.6. Cell Preparation
29
3.7. Cell viability test
30
3.8. Characterization techniques
30
31
Chapter 4: Results And Discussion

4.1. Synthesis of Graphene oxide (GO) nanomaterial
32
4.2. Preparation of water soluble
32
4.3. FE-SEM and EDX Analysis of soluble material
33
4.4. Synthesis of Graphene Oxid nanomaterial
34
4.5. Synthesis of Graphene Oxid
35
4.6. Characterization of GO and functionalized GO nanomaterials
37
4.6.1. FT-IR analysis
37
4.6.2. UV-Visible analysis
39
4.6.3. FE-SEM and EDX analysis
40
4.7. Cell viability tests
42
4.7.1. Concentration-dependent antibacterial test
42
4.7.1.1. Concentration-dependent antibacterial tests
43
47
50
54
4.8. images of E. coli in control sample and treated E. coli with GO

material
58
4.9. Antibacterial mechanism
59
4.10. Antibacterial mechanism
62
Chapter 5: Conclusions
64
Suggestions
66
References
67
LIST OF FIGURES
Figure ... Page
Figure 1.1.
The structure of graphite oxide.
Figure 1.2.
Schematic illustrating the structural difference between layered
graphite oxide and exfoliated GO platelets.

Figure 1.3.
Functionalization of the carboxylic acid groups of GO,
showing the covalent attachment of porphyrins and fullerenes.

Figure 1.4.
Covalent functionalization of the epoxy groups of GO.
Figure 1.5.
SEM images of Escherichia coli after incubation with saline
10
solution for 2 h without GO materials (b) and Escherichia coli

cells after incubation with GO dispersion (40 g/mL) for 2 h
(d).
Figure 1.6.
Chlorophyll structure.
12
Figure 1.7.
Reaction of Chlorophyll a with acid.
13
Figure 1.8.
Reaction of Chlorophyll a with base.
13
Figure 1.9.
Synthesis of chlorophyllin.
14
Figure 1.10.
E. coli structure.
15
Figure 3.1.
Spinach leaves.
27
Figure 3.2.
Two-phase product.
28
Figure 3.3.
Grown E. coli cells in MH Agar medium.
29
Figure 4.1.
Synthesis of Graphene Oxide, the two dimensional multilayer
32
nanostructure.
Figure 4.2.
Extraction procedure of water soluble material.
33
Figure 4.3.
FE-SEM micrographs (a,b) and EDX spectrum (c) sediment.
34
Figure 4.4.
Synthesis of Graphene Oxide
35
Figure 4.5.
Synthesis of Graphene Oxide
36
Figure 4.6.
Three Synthesized materials used in comparative antibacterial
37
test.
Figure 4.7.
FT-IR spectra
39
Figure 4.8.
UV-Vis absorption spectra
40
Figure 4.9.
FE-SEM micrographs and EDX spectrum of GO
42
Figure 4.10.
Cell viability measurement
43
Figure 4.11.
Photograph of E. coli colonies cultivated on MH Agar plate
44
obtained from incubated suspensions with MH broth (control

sample).
Figure 4.12.
Photographs of E. coli colonies cultivated on MH agar plates
44
obtained from incubated suspensions with concentration of

material.
Figure 4.13.
Photographs of E. coli colonies cultivated on MH Agar plate
45

material.
Figure 4.14.
45

material.
Figure 4.15.
46
obtained from incubated suspensions concentration.

Figure 4.16.
46
obtained from incubated suspensions concentration.

Figure 4.17.
Cell viability measurement after incubation with concentration
47
of GO.
Figure 4.18.
48
obtained from incubated suspensions concentration of GO.

Figure 4.19.
48
obtained from incubated suspensions with concentration of GO.

Figure 4.20.
49

Figure 4.21.
49

Figure 4.22.

E
50

Figure 4.23.
51
of GO.
Figure 4.24.
51

Figure 4.25.
52

Figure 4.26.
52

Figure 4.27.
53

Figure 4.28.

obtained from incubated suspensions with concentration of GO
material.
Figure 4.29.
53

of GO.
Figure 4.30.
54
Photograph of E. coli colonies cultivated on MH Agar plate

obtained from incubated suspensions with MH broth (control
sample).
Figure 4.31.
55

material.
Figure 4.32.
55
Photographs of E. coli colonies cultivated on MH Agar plates

material.
Figure 4.33.
56

material.
Figure 4.34.
56

material.
57
F
Figure 4.35.

material.
Figure 4.36.
57
images of E. coli after incubation with MH broth for 2 h

without other materials (a, c), E. coli cells after incubation with
GO dispersion for 2 h (b) , E. coli cells after incubation with
GO dispersion (1000 g/mL) for 2 h (d).
58
Figure 4.37.
Antibacterial mechanism.
60
Figure 4.38.
Schematic of an example, demonstrating the important role of
Figure 4.39.
GO on structure.
61
Antibacterial mechanism.
63
Chapter 1
General Introduction
1.1.
Nanotechnology
Nanotechnology is the manipulation of matter on an atomic and molecular scale. It is able
to create many new materials and devices with at least one dimension sized from 1 to 100
nanometers. This field of science involves study of how to control the formation of two and
three dimensional assemblies of molecular scale building blocks into well-defined
nanostructures or nanomaterials [1]. Materials reduced to the nanoscale can show different
properties compared to what they exhibit on a macro scale. For instance, opaque substances
become transparent (copper), stable materials turn combustible (aluminum), insoluble
materials become soluble (gold). Additionally, a number of physical properties change when
compared to macroscopic systems. One example is the increase in surface area to volume ratio
altering mechanical, thermal and catalytic properties of materials. These novel materials play
an important role in industry and unusual properties of them have resulted in nanotechnology
advances in different areas such as electronics, biomedicine, pharmaceuticals, cosmetics,
environmental analysis, catalysis, medical and biological applications [2].
Apart from these applications, it has been investigated that nanostructures can be
generally categorized into inorganic nanoparticles including the ones based on metal oxides
(zinc oxide, iron Oxide, titanium dioxide, and cerium oxide), metals (gold, silver, zinc, and
iron), quantum dots (cadmium sulfide and cadmium selenide), and carbon-based materials
such as fullerenes, carbon nanotubes (CNTs), graphene, and graphene oxide (GO) generating
from graphite oxide [3].
1.2.
Graphite Oxide
The history of graphite oxide formation is extending back many decades to some of the
earliest studies. The first well-known example came in 1859 when British chemist B. C.
Brodie was exploring the structure of graphite by investigating the reactivity of flake graphite.
One of the reactions he performed involved adding potassium chlorate (KClO3) to a slurry of
graphite in fuming nitric acid (HNO3) [4]. Brodie determined that, the resulting material was
composed of carbon, hydrogen and oxygen, resulting in an increase in the overall mass of the
flake graphite.
Recently, various methods have been employed to modify the former version.
Importantly, they have all demonstrated that, the products of the oxidation reaction show
2
strong variance, depending not only on the particular oxidants used, but also on the graphite
source and reaction conditions. Hummer and Offeman method is the most common one, which
is applied in various approaches [5]. They developed a safer, quicker, and more efficient
method by reacting graphite with a mixture of potassium permanganate (KMnO4) and
concentrated sulfuric acid (H2SO4). The most common source of graphite used for chemical
reactions, including its oxidation, is flake graphite, which is a naturally occurring mineral that
is purified to remove heteroatomic contamination [6]. Trmel and Russ demonstrated the
ability of the reactants to oxidize unsaturated aliphatic double bonds over aromatic double
bonds [7]. Also, flake graphite contains numerous localized defects in its structure, that may
serve seed points for the oxidation process. Consequently, the complexity of flake graphite
and the defects make the elucidation of precise oxidation mechanisms very challenging.
1.2.1. Structural features and properties of Graphite Oxide

Aside from the operative oxidative mechanisms, considerable effort has been directed
towards understanding the structure of graphite oxide. They illustrated that, the oxidation
process preserves the layer structure of the parent graphite, but the layers are buckled, and the
interlayer spacing is about two times larger (0.6-0.8 nm) than that of graphite [8]. Moreover,
experimental observations have proved that, graphite oxide consists of single-atom-thick
carbon sheets with carboxylate groups on the periphery (figure 1.1), where they provide pH
dependent negative surface charge and colloidal stability. The basal surfaces contain hydroxyl
and epoxide functional groups, which are uncharged but polar. The basal planes also include
unmodified graphenic domains, which are hydrophobic and capable of - interactions
relevant to adsorption of dye molecules or some drugs. The result is an amphiphilic giant
sheet-like molecule, which can act like a surfactant and stabilize hydrophobic molecules in
solution [9].
Figure 1.1. The structure of graphite oxide [9].
1.3.
Graphene Oxide (GO)

Graphite oxide can be exfoliated using a variety of methods and yielding a material,
named as graphene oxide (GO). These techniques will be discussed in more details below.
1.3.1. Solvent-based exfoliation

In the this route, the hydrophilic nature and increased interlayer spacing of graphite
oxide facilitates direct exfoliation into water assisted by mechanical exfoliation such as ultrasonication or stirring, at concentrations up to 3 mg/ml, forming colloidal suspensions of GO.
Figure 1.2 illustrates the structural difference between layered graphite oxide and exfoliated
GO platelets. Mechanical stirring is an alternative route to produce single-layer GO platelets
of much larger lateral dimensions and aspect ratios when compared with GO platelets
produced by sonication. However, it has been reported that magnetic stirring exfoliates
graphite oxide very slowly and in low yield [10].
Figure 1.2. Schematic illustrating the structural difference between layered graphite oxide and exfoliated
GO platelets [10].
1.3.2. Thermal exfoliation

Graphite Oxide can also be exfoliated by rapid heating, yielding thermally expanded
graphite oxide (TEGO). In this exfoliation method, the dry powder is typically charged into a
quartz tube and subjected to thermal shock by heating to 400 C or higher. The rapid heating is
believed to cause various small molecule species (CO, CO2, water) to evolve and internal
pressure to increase, forcing the sheets apart and yielding a dry and high-surface area material
with a low bulk density [10].
1.3.3. Microwave exfoliation

Additionally, exfoliation process can be done by microwave radiation, yielding a
related material referred to as microwave expanded graphite oxide (MEGO). Importantly, it
was reported that, while GO platelets are likely to maintain the chemical structure of graphite
oxide, TEGO and MEGO are reduced [10, 11].
1.4. Chemical functionalization of Graphene Oxide

In this section, we will discuss the addition of other groups to GO platelets, using
various chemical reactions that provide for either covalent or non-covalent attachment to the
resulting material. Such approaches render GO a more versatile precursor for a wide range of
applications. Reactive oxygen containing groups of GO such as carboxylic acid, epoxy and
hydroxyl groups are ideal regions for chemical modification of this nanomaterial.
1.4.1. At the carboxylic acid group of Graphene Oxide

A wide range of reactions, utilizing carboxylic acids has been developed over the
course of the development of small molecule organic chemistry, and many of these reactions
have been applied to GO. The coupling reactions often require activation of the acid group
using thionyl chloride (SOCl2) [12-15]. Subsequent addition of nucleophilic species such as
amines or hydroxyls produce covalently attached functional groups to GO platelets via the
formation of amides or esters. Likewise, porphyrin-functionalized primary amines and
separately, fullerene-functionalized secondary amines have been attached to GO platelets
(figure 1.3), affording materials with useful nonlinear optical performance [13-15].
Figure 1.3. Functionalization of the carboxylic acid groups of GO showing the covalent attachment of
porphyrins and fullerenes [14].
1.4.2. At the epoxy group of Graphene Oxide

Reactions of the epoxy group of GO have also been used to stabilize solid-phase
dispersions of the resulting product. As an example, covalent attachment of 3aminopropyltriethoxysilane (APTS) to GO platelets has been studied [16]. The authors
proposed that, silane moieties are grafted via a nucleophilic SN2 displacement reaction
between the epoxide and the amine groups of APTS (figure 1.4).
Figure 1.4. Covalent functionalization of the epoxy groups of GO [17].
1.4.3. Non-covalent functionalization of Graphene Oxide

GO can also exhibit non-covalent binding (via - stacking, cation- or van der Waals
interactions) on the sp2 networks, which are not oxidized or engaged in hydrogen bonding. For
instance, a DNA sensor has been recently manufactured, holding promise as a platform for
sensitive and selective detection of DNA and proteins [18].
1.5. Graphene Oxide applications

GO, an inexpensive material with a low cost method of production, possesses
electronic, optical, thermal, mechanical, antimicrobial and intrinsic bio-compatibility
properties. Consequently, with regard to rapid development of synthesis and functionalization
approaches, GO has shown outstanding potentials in many fields which will be mentioned in
the following.
1.5.1. Transparent conductive films

One of the major areas, where GO can be expected to be used, is in the production of
transparent conductive films. GO films can be deposited on essentially any substrate, and later
converted into a conductor. Such coatings can be used in flexible electronics, solar cells, liquid
crystal devices and chemical sensors [19].
1.5.2. Composites and paper-like materials

GO mixes readily with many polymers, forming nanocomposites and greatly enhances
the properties of original polymer including elastic modulus, tensile strength, electrical
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Faculty of Engineering

Department of Chemical Engineering

Title of the Thesis:

Evaluated and approved by thesis committee: as

Dr. Jamshid Behin

Dr. Laleh Rajabi

Dr. Mojtaba Ahmadi

Dr. Ramin Abiri

Title of the Thesis:

In The Name of God

Chapter 1: General Introduction

1.2.1.Structural features and properties of Graphite Oxide

Graphene Oxide (GO)

1.3.1. Solvent-based exfoliation

1.3.2. Thermal exfoliation

1.3.3. Microwave exfoliation

1.4. Chemical functionalization of Graphene Oxide

1.4.1. At the carboxylic acid group of Graphene Oxide

1.4.2. At the epoxy group of Graphene Oxide

1.4.3. Non-covalent functionalization of Graphene Oxide

1.5. Graphene Oxide applications

1.5.1. Transparent conductive films

1.5.2. Composites and paper-like materials

1.5.3. Energy-related materials

1.5.4. Graphene Oxide sheets at interfaces

1.5.5. Applications in biology and medicine

1.5.6. Graphene Oxide as antibacterial agent

1.6. Antibacterial agents

1.6.1. Definition of antibacterial agents

1.6.2. Mechanisms of antibacterial action

1.6.3. Chlorophyll as a natural antibacterial agent

1.6.3.1. Medical and antimicrobial effect of Chlorophyll and its

1.6.3.3. Chlorophyll reactions

1.7. Escherichia coli (E. coli) as a model bacterium

1.8. Overview of spread plate as a colony counting methodology

1.9. Objective and scope of this research work

Chapter 2: Literature Review

2.1.1.Antimicrobial property of GO and its mechanism against

3.3. Synthesis of GO nanomaterial

3.3.1. Synthesis of Graphite Oxide

3.3.2. Preparation of GO nanosheet suspension

3.4. Preparation of water soluble material

3.4.2. Solvent method

3.5. Functionalized GO nanomaterials syntheses

3.5.1. Synthesis of Graphene Oxide nanomaterial

3.7. Cell viability test

3.8. Characterization techniques

Chapter 4: Results And Discussion

4.2. Preparation of water soluble

4.3. FE-SEM and EDX Analysis of soluble material

4.4. Synthesis of Graphene Oxid nanomaterial

4.5. Synthesis of Graphene Oxid

4.6. Characterization of GO and functionalized GO nanomaterials

4.6.1. FT-IR analysis

4.6.2. UV-Visible analysis

4.6.3. FE-SEM and EDX analysis

4.7. Cell viability tests

4.7.1. Concentration-dependent antibacterial test

4.7.1.1. Concentration-dependent antibacterial tests

4.7.1.2. Concentration-dependent antibacterial tests

4.7.1.3. Concentration-dependent antibacterial tests

4.7.1.4. Concentration-dependent antibacterial tests

4.8. images of E. coli in control sample and treated E. coli with GO

4.9. Antibacterial mechanism