Synthesis, Characterization, Chemical Reduction and

Biological Application of Graphene Oxide

by

Xiguang Gao

A thesis

presented to the University of Waterloo

in fulfillment of the

thesis requirement for the degree of

Master of Science

in

Chemistry - Nanotechnology

Waterloo, Ontario, Canada, 2013

© Xiguang Gao 2013

Author’s Declaration

I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis,

including any required final revisions, as accepted by my examiners.

I understand that my thesis may be made electronically available to the public.

ii
Abstract

As an atomic layer of sp2-hybridized carbon atoms closely packed in a honeycomb

lattice, graphene has been attracting increasing attention since its discovery in 2004 due to its

extraordinary physicochemical properties. Graphene oxide (GO), a non-stoichiometric

graphene derivative with the carbon plane abundantly decorated with hydroxyl, epoxide and

carboxylic groups, can be massively and cost-effectively produced from natural graphite

following Hummers method. GO has greater aqueous solubility than pristine graphene due to

its oxygen-functionalities. Various solution-based chemical methods can be applied to GO,

which has stimulated a new research area called ‘wet chemistry of grahene’. Among them,

chemical reduction of GO provides a facile route for large-scale synthesis of graphene.

With abundant oxygen-functionalities in its structure, GO can potentially act as a

suitable precursor for chemical modifications of graphene through methods used in organic

chemistry. Special attention should be paid to that the hydroxyl groups in GO belong to

tertiary alcohols, and steric hindrance should be considered when performing chemical

modifications. Diethylaminosulfur trifluoride (DAST), a fluorinating reagent, is ineffective in

fluorinating GO due to the steric hindrance of tertiary hydroxyls. However, DAST is effective

in reducing GO. The capability of DAST for GO reduction is close to hydrazine, but the

reduction reaction can be performed at lower temperature for DAST.

As a two-dimensional (2D) nanomaterial with good aqueous solubility, biocompatibility

and excellent intrinsic mechanical properties, GO is particularly useful in preparing 3D hybrid

hydrogel scaffolds for tissue engineering applications.

iii
Acknowledgements

First of all, I would like to sincerely thank my supervisor Prof. Xiaowu (Shirley) Tang for her

guidance and support during my Master study! Discussing with her about the experimental

results benefited my research a lot. Her broad range of knowledge enables her to give me

insightful ideas and suggestions about research. I made steady progress under her supervision.

Second, I want to thank my parents who have brought me up and educated me! They can

always cheer me up whenever I feel down. I gain self-confidence and positive energy from

them.

Third, I want to thank all my colleagues in the Tang lab! Their hard-working and keen

attitudes towards research really encouraged me. They have created a wonderful atmosphere

for me to conduct research. They helped me a lot as well.

I also want to thank the Department of Chemistry at University of Waterloo for accepting me

as a graduate student and providing me with funding! I had such a great time studying and

working here. This experience will be a treasure to both of my life and future career.

Finally, I want to thank all the people who have helped me toward this stage of my life!

iv
Table of Contents

Author’s Declaration ................................................................................................................. ii

Abstract .................................................................................................................................... iii

Acknowledgements .................................................................................................................. iv

List of Figures ........................................................................................................................ viii

List of Tables ............................................................................................................................ xi

List of Equations ..................................................................................................................... xii

List of Abbreviations .............................................................................................................. xiii

Chapter 1 - Introduction ............................................................................................................ 1

1.1 The Discoveries of Graphene as Well as Other Quasi Two Dimensional (2D)

Materials ............................................................................................................................ 1

1.2 Graphene Synthesis...................................................................................................... 2

1.2.1 Scotch Tape Method (Micromechanical Cleavage of Graphite) ....................... 2

1.2.2 Epitaxial Growth on Silicon Carbide (SiC) ....................................................... 4

1.2.3 Chemical Vapor Deposition (CVD) on Transition Metals ................................ 5

1.2.4 Chemical Reduction of Graphene Oxide (GO) ................................................. 7

1.2.5 Sonication-Assisted Exfoliation of Graphite in Organic Solvents .................. 13

1.2.6 Bottom-Up Synthesis ...................................................................................... 15

1.3 Graphene Derivatives - Fluorinated Graphene .......................................................... 17

1.4 Diethylaminosulfur Trifluoride (DAST) as a Useful Fluorinating Reagent for

hydroxyl, Carbonyl/Ketone and Carboxylic Groups ....................................................... 22

Chapter 2 - Research Objectives, Synthetic Methods and Characterization Tools.................. 24

2.1 Research Objectives ................................................................................................... 24

2.2 Synthetic Methods ..................................................................................................... 24

2.3 Characterization Tools ............................................................................................... 24

2.3.1 Fourier Transform Infrared Spectroscopy (FTIR) ........................................... 24

2.3.2 Raman Spectroscopy ....................................................................................... 26

2.3.3 Ultraviolet-Visible Spectroscopy (UV-Vis) ..................................................... 26

v
 2.3.4 Atomic Force Microscopy (AFM)................................................................... 28

2.3.5 Scanning Electron Microscopy (SEM)............................................................ 29

2.3.6 X-ray Photoelectron Spectroscopy (XPS) ....................................................... 30

2.3.7 Four-Probe Method for Measuring Thin Film Conductivity ........................... 31

Chapter 3 - Synthesis, Characterization of Graphene Oxide and Preparation of Free-Standing

Graphene Oixde Thin Films .................................................................................................... 34

3.1 Hummers Method and Modified Hummers Method for Graphene Oxide Synthesis 34

3.1.1 Reaction Mechanisms - Formation of Graphite Intercalation Compounds

(GICs) ....................................................................................................................... 34

3.1.2 Experimental Procedures - Modified Hummers Method ................................ 38

3.2 Graphene Oxide Characterization .............................................................................. 40

3.2.1 FTIR Characterization ..................................................................................... 40

3.2.2 UV-Vis Characterization ................................................................................. 41

3.2.3 AFM Characterization ..................................................................................... 42

3.2.4 Raman Characterization .................................................................................. 47

3.2.5 XPS Characterization ...................................................................................... 48

3.3 Preparation of Free-Standing Graphene Oxide Thin Films (or Papers) ..................... 49

3.3.1 Vacuum Filtration ............................................................................................ 50

3.3.2 Self-Assembly at the Water-Air Interface ....................................................... 51

3.4 Conclusions and Future Aspects ................................................................................ 53

Chapter 4 – Exploring the fluorination of Grpahene Oxide Using DAST - Chemical

Reduction of Graphene Oxide ................................................................................................. 55

4.1 Experimental Procedures and Observances ............................................................... 55

4.2 Characterization ......................................................................................................... 57

4.2.1 XPS Characterization ...................................................................................... 57

4.2.2 FTIR Characterization ..................................................................................... 60

4.2.3 Raman Characterization .................................................................................. 61

4.2.4 Thin Film Conductivity Measured by a Four-Probe Method .......................... 63

4.3 Discussions ................................................................................................................ 65

4.4 Conclusions and Future Aspects ................................................................................ 67

vi
Chapter 5 – Biological Application of Graphene Oxide - Tissue Engineering........................ 68

5.1 Introduction................................................................................................................ 68

5.1.1 Tissue Engineering .......................................................................................... 68

5.1.2 Gelatin Methacrylate (GelMA) Hydrogel as a Scaffold for Tissue Engineering

.................................................................................................................................. 69

5.2 Hybrid Hydrogel of GelMA and Graphene Oxide Through Non-Covalent Interaction

......................................................................................................................................... 69

5.3 Incorporation of Graphene Oxide into GelMA Hydrogel through covalent bonding 75

5.4 Conclusions and Future Aspects ................................................................................ 76

References ............................................................................................................................... 78

Appendix ................................................................................................................................. 87

Publications...................................................................................................................... 87

vii
List of Figures

Figure 1: Schematic of the structure of graphene. ………………………………………………...…….1

Figure 2: Schematic of the micromechanical cleavage of graphite by Scotch-Tape method. …….…….3

Figure 3: AFM image of graphene obtained by Scotch Tape method. The folded region exhibiting a

relative height of 4 Å indicates that it is single layer. …...........................................................3

Figure 4: Photograph and optical microscope image of transferred graphene films onto a glass substrate

and a 285-nm-thick SiO2/Si substrate. ......................................................................................6

Figure 5: Chemical vapor deposition synthesis of ultralarge-area graphene film (30 inches). ………….7

Figure 6: Schematic of the structure of graphite oxide. …………………………………………………8

Figure 7: Scheme showing the chemical route to the synthesis of aqueous graphene solution. …..…….9

Figure 8: Photograph of a 10-μm-thick chemically converted graphene (CCG) film (diameter ~38 mm)

prepared by vacuum filtration of a CCG colloid. ………………………………….………...10

Figure 9: Schematic illustrating the formation of pyrazole structure at the graphene platelet edges by

hydrazine reduction. …………………………………………………………………………11

Figure 10: Schematic showing the possible mechanisms for graphene oxide reduction by HI. ……….12

Figure 11: Schematic showing the sonication-assisted exfoliation of graphite. ……………………….13

Figure 12: TEM images of monolayer and bilayer graphene…………………………………………..14

Figure 13: Reaction schemes and STM images of graphene nanoribbons. ……….…….……………..16

Figure 14: Schematic showing the crystal structures of graphene and graphane. ……………………..18

Figure 15: Schematic for the syntheses of graphene halides (G-Br and G-Cl) using microwave-sparks-

assisted halogenation reactions. ……………………………………………………………19

Figure 16: Models showing the chair-like structure of FG. ……………………………………..……..21

Figure 17: Chemical structure of DAST. ……………………………………………….……………...23

Figure 18: The electromagnetic spectrum. ……………………………………………..………………27

Figure 19: Four-probe measurement of thin film sheet resistance. …………………………………… 32

Figure 20: Photographs and Raman spectra of graphite and H 2SO4-K2S2O8-GIC. ……………………35

Figure 21: Photographs of graphite and H2SO4-KMnO4-GIC. ………………………………………...36

viii
Figure 22: Photographs of graphene oxide aqueous solutions of different concentrations. ……….…..40

Figure 23: FTIR spectrum of graphene oxide. ........................................................................................41

Figure 24: UV-vis spectrum of graphene oxide. .....................................................................................41

Figure 25: AFM images of different resolutions showing graphene oxide sheets on SiO 2/Si

substrates. ……………..…………………………………….…….……………..…………43

Figure 26: Height profiles of AFM images showing the thickness of graphene oxide. ………....……..44

Figure 27: AFM phase and 3D topological images of graphene oxide sheets on SiO2/Si substrates. …46

Figure 28: Raman spectra of graphene oxide and graphite. ……………………………………………47

Figure 29: XPS survey spectrum and C1s spectrum of graphene oxide. ……………………………….48

Figure 30: Photograph of a graphene oxide thin film and SEM image showing the cross-section of the

film. …………………………………………………………………………………...……51

Figure 31: Digital image of a free-standing graphene oxide thin film prepared in this thesis by

self-assembly at the water-air interface and schematic of the film formation mechanism. ..52

Figure 32: Schematic of the experimental setup for the reaction of graphene oxide film with DAST. ..55

Figure 33: Digital images of GO and rGO (50 °C) showing the color change after DAST treatment. ..56

Figure 34: Water contact angle comparison of rGO (50 °C) and GO thin films. ……………………...56

Figure 35: XPS Survey spectra of graphene oxide film and reduced graphene oxide films. ………….57

Figure 36: High resolution XPS C1s spectra of GO and rGO films. …………………………………...58

Figure 37: XPS High resolution F1s spectrum of rGO (50 °C). ………………………………………..59

Figure 38: XPS survey spectrum on the cross-section of rGO (50 °C) film. ………………………......60

Figure 39: FTIR spectra of rGO (50 °C) and GO. ……………………………………………….…….61

Figure 40: Raman spectra of GO and rGO. (Excitation wavelength is 633 nm.) ……………………...62

Figure 41: Schematic of the four probe connection, linear plots of voltage between probe 2 and 3

versus current for GO film, and rGO (50 °C) film. ..............................................................64

Figure 42: Schematic of the SN2 reaction between an alcohol and DAST. ……………………………65

Figure 43: Schematic of the reduction of GO by DAST. ………………………………………………67

Figure 44: Schematic showing the principle of tissue engineering. …………………………………...68

Figure 45: Schematic of the structure of gelatin methacrylate (GelMA). ……………………………..69

Figure 46: Optical, AFM and fluorescence images of GO-GelMA hybrid hydrogels. ………………...70

Figure 47: Mechanical, porosity, and degradation characteristics of GO-GelMA hybrid hydrogels. …71

ix
Figure 48: Raman spectra of GelMA and GO-GelMA before and after degradation. …………………72

Figure 49: Cellular behavior of NIH-3T3 fibroblasts encapsulated in microfabricated GO-GelMA

hybrid hydrogels. ………………………………………………………………………..…73

Figure 50: Fabrication and characterization of cell-laden microconstructs. …………………………...74

Figure 51: Schematic of the surface functionalization of graphene oxide with methacrylate groups,

mechanical properties of GO-GelMA and MeGO-GelMA hydrogels. …………………….75

x
List of Tables

Table 1: Atomic ratio of GO and rGO determined from XPS survey spectra. ……………………...…57

Table 2: Raman D and G positions, intensity ratio of D to G (I D/IG) of GO and rGO. ………………...62

xi
List of Equations

Equation 1: Vibration frequency of a molecule excited by infrared light. ……………………………..25

Equation 2: The relation between the binding energy of a core electron and the kinetic energy of an

emitted photoelectron in XPS. ………………………………………………....…………30

Equation 3: XPS quantitative analysis of chemical composition. ……………………………………..31

Equation 4: Sheet resistance of a thin film measured by the four-probe method. ……………………..32

Equation 5: Simplified equation for calculating the sheet resistance of an ultrathin film with probes

being far away from the sample edges in the four-probe method. ………………………..33

xii
List of Abbreviations

2D: two-dimensional

FET: field effect transistor

AFM: atomic force microscopy

CVD: chemical vapor deposition

GO: graphene oxide

rGO: reduced graphene oxide

CCG: chemically converted graphene

TBA: tetrabutylammonium hydroxide

DSPE-mPEG: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-

glycol)-5000]

TEM: transmission electron microscopy

GNRs: graphene nanoribbons

STM: scanning tunneling microscopy

QHE: quantum hall effect

FG: fluorinated graphene

DAST: diethylaminosulfur trifluoride

FTIR: Fourier transform infrared spectroscopy

UV-Vis: ultraviolet visible spectroscopy

SEM: scanning electron microscopy

XPS: X-ray photoelectron microscopy

GICs: graphite intercalation compounds

XRD: X-ray diffraction

GelMA: gelatin methacrylate

FITC: fluorescein isothiocyanate

MeGO: methacrylic graphene oxide

xiii
 Chapter 1 - Introduction

1.1 The Discoveries of Graphene as Well as Other Quasi Two Dimensional

(2D) Materials

The term graphene was first introduced by Boehm et al. in 1986 and derived from the

combination of the word ‘graphite’ and the suffix ‘ene’ that refers to polycyclic aromatic

hydrocarbons (e.g. anthracene, benzene).1,2 However, free-standing graphene crystal was not

discovered until 2004 when Dr. Andre K. Geim and Dr. Konstantin S. Novoselov from the

University of Manchester successfully peeled off highly ordered pyrolytic graphite (HOPG)

and obtained single layer graphene crystal using the ‘Scotch Tape’ method.3 Afterwards, they

also obtained other atomic 2D crystals including BN, MoS2, NbSe2 and Bi2Sr2CaCu2Ox using

the same technique.4 The researches on graphene and other 2D materials have intensively

expanded ever since.5-8 The Nobel Prize in Physics 2010 was awarded jointly to Andre Geim

and Konstantin Novoselov “for groundbreaking experiments regarding the two-dimensional

material graphene”.9

Fig. 1 Schematic of the structure of graphene.10

Schematic of the structure of graphene is shown in Fig. 1. It can be seen as a single layer

of sp2-hybridized carbon atoms closely packed in a honeycomb crystal lattice. Every carbon

1
atom in graphene is covalently bonded to three adjacent carbon atoms. The large π network

across the molecular chicken wires contributes to its excellent electrical properties. Graphene

is a building block for other carbon-based materials. It can be wrapped up into 0D fullerene,

rolled into 1D carbon nanotubes and stacked into 3D graphite.11

Graphene has stimulated tremendous research interests among other 2D materials since

its advent due to its extraordinary physicochemical properties. Graphene has remarkably high

charge carrier mobility in excess of 15,000 cm2 V−1 s−1 under ambient conditions,11 excellent

thermal conductivity of ~5000 Wm-1K-1 at room temperature,12 Young’s modulus of 1.0 TPa,13

optical transmittance of 97.7%,14 high theoretical specific surface area of 2630 m2g-1. And

graphene is chemically stable under ambient conditions. Graphene has a wide range of

applications including field effect transistors (FETs),15 gas sensors,16 nanocomposite

materials17 and supercapacitors18 due to its excellent physicochemical properties.

1.2 Graphene Synthesis

1.2.1 Scotch Tape Method (Micromechanical Cleavage of Graphite)

Scotch Tape method or micromechanical cleavage of graphite led to the discovery of

graphene in 2004. This method involves repeatedly peeling highly oriented pyrolytic graphite

(HOPG) using a scotch tape, transferring graphene as well as thick graphite flakes onto a Si

substrate with a SiO2 layer of a carefully chosen thickness (300 nm), and hunting graphene

under an optical microscope. Schematic of this method is shown in Fig. 2. An atomic force

microscopy (AFM) image of a graphene crystal produced by this method is shown in Fig. 3.

2
The thickness of graphene was measured to be 0.4 nm by AFM, which is close to its

theoretical value of 0.34 nm.19

Step 1 Step 2

HOPG flake
Repeatedly peel HOPG

Step 3 Step 4
300 nm SiO2/Si
substrate

Transfer graphene as well as thick graphite

Hunt graphene under optical microscope
flakes onto a SiO2/Si substrate

Fig. 2 Schematic of the Scotch-Tape method.19

Fig. 3 AFM image of a graphene crystal obtained by Scotch Tape method. The folded region

exhibiting a relative height of 4 Å indicates it is single layer. (Adapted with permission from

ref. 11. © 2007 Nature Publishing Group.)

3
 Peeling HOPG with an adhesive tape is a commonly used technique to prepare freshly

cleaved surfaces for depositing samples in AFM characterization. The peeled graphite flakes

have a wide range of thicknesses. Breakthrough in getting single layer graphene did not come

until the use of a Si wafer with a carefully chosen thickness of SiO2 layer which makes

graphene visible under an optical microscope. Single layer graphene, few layer graphene and

thicker graphite flakes show different colors on a 300-nm-thick SiO2/Si substrate due to

feeble interference-like contrasts with respect to an empty substrate.4

This method can provide high quality graphene crystal with lateral size up to 100 μm,

however, it is laborious and the yield is very low, rendering this method unsuitable for large

scale production of graphene.

1.2.2 Epitaxial Growth on Silicon Carbide (SiC)

Graphitization of silicon carbide by Si sublimation under high temperature and vacuum

conditions was first reported in the 1960s.20 Berger et al. have refined this technique and

demonstrated that patterned epitaxial graphene grown on single SiC crystal showed electronic

confinement and coherence, which envisages coherent graphene molecular electronics. 21, 22

Lin et al. reported that FETs fabricated on wafer-scale epitaxial graphene exhibited high

cutoff frequency of 100 GHz which exceeded Si/metal-oxide semiconductor FETs,

demonstrating the high potential of epitaxial graphene grown on SiC for electronic

applications.15

Epitaxial growth on SiC has been one of the leading methods for mass-production of

graphene. However, growing large graphene domains and sophisticated control over the

4
thickness of the graphene film remain as major challenges so far. The high growth

temperature (1200 °C~1800 °C), high cost of SiC substrates, ultrahigh vacuum condition

(UHV) and non-transferability of as-grown graphene films to arbitrary substrates are the

disadvantages of this method.

1.2.3 Chemical Vapor Deposition (CVD) on Transition Metals

The syntheses of graphitic materials (e.g. carbon nanotubes) by chemical vapor

deposition on transition metals (e.g. Fe, Co, Ni, Cu, Ru, Pd) have a long history. Different

from using transition metal nanoparticles for growing carbon nanotubes, transition metal thin

films are normally used for growing graphene films. Large-scale patterned growth of few

layer graphene films on thin Ni films were realized by Kim et al.23 The as-grown graphene

films could be easily transferred to arbitrary substrates. The transferred graphene films

showed low sheet resistance of 280 Ω per square at 80% optical transparency. At low

temperatures, monolayer graphene transferred to SiO2 substrates showed electron mobility

greater than 3,700 cm2 V-1 s-1 and exhibited the half-integer quantum Hall effect (QHE),

implying that the quality of CVD-grown graphene on Ni is comparable to graphene obtained

by mechanically cleavage of graphite.

However, as the carbon solubility in Ni is relatively high (1.2 at% at 1000 °C), controlled

growth of exact monolayer graphene is difficult. When carbon species start precipitating out

from the surfaces of Ni films, multilayer graphene or graphite can be formed along with

monolayer graphene. Graphene films grown on Ni usually do not possess uniform thicknesses,

which limits their application in electronics. In order to prevent the formation of multi-layer

5
graphene, fast cooling rate (~10 °C s-1), thin Ni films (<300 nm), and/or extremely low

concentration of carbon source are usually required for growing monolayer graphene.23-24 Cu

has a much lower carbon solubility (less than 0.004 at% at 1000 °C) than Ni, making it a

better candidate for making strict monolayer graphene by CVD. Li et al.25 reported the

large-area synthesis of high-quality and uniform graphene films on copper foils and

concluded that graphene growth on Cu is a surface-catalyzed and self-limiting process rather

than an absorption-precipitation process proposed for Ni. Graphene grown on Cu showed

electron mobility as high as 4,050 cm2 V-1 s-1 at room temperature. Fig. 4a shows the

graphene films transferred on a glass substrate grown by CVD Cu. The area of the film is

~1.2 cm×1.0 cm. Fig 4b shows the optical image of graphene films transferred on a

285-nm-thick SiO2/Si substrate. Graphene of different layers show different light contrasts

with the substrate. The films consist of predominantly single layer graphene with a percentage

of >95%.

(a) (b)

Fig. 4 (a) Graphene films transferred onto a glass substrate, (b) Optical microscope image of

graphene films transferred onto a 285-nm-thick SiO2/Si substrate. (Adapted with permission

from ref. 25. © 2009 AAAS.)

6
 By using CVD on flexible wrapped-up copper foils, Bae et al. synthesized a 30-inch

predominantly monolayer graphene film (Fig. 5).26 The flexibilities of graphene films and

copper foils allowed efficient transfer process using a roll-to-roll method. The scalability of

CVD on Cu for large-scale graphene synthesis was well illustrated in this work.

(a) (b)

Fig. 5 (a) Copper foil wrapping around a 7.5-inch quartz tube was inserted into an 8-inch quartz

reactor, (b) a transparent ultralarge-area graphene film (30 inches) transferred on a 35-inch

polyethylene terephthalate (PET) substrate. (Adapted with permission from ref. 26. © 2010 Nature

Publishing Group.)

CVD on transition metals (Ni, Cu) can produce high-quality graphene and are suitable

for large-scale synthesis. However, high temperature heating (normally 1000 °C), low

pressure growth condition in case Cu is used, and transfer of graphene films to other

substrates are required, making this method not very cost-effective and facile.

1.2.4 Chemical Reduction of Graphene Oxide (GO)

The synthesis of graphite oxide can date back to as early as 1859. British chemist Brodie

first explored the reaction of graphite with potassium chlorate (KClO3) in fuming nitric acid

(HNO3), and synthesized “graphitic acid” (graphite oxide) with a net molecular formula of

C2.19H0.80O1.00 by successive oxidation processes (four reactions).27 Later in 1898,

7
Staudenmaier improved Brodie’s method by adding KClO3 in multiple aliquots in the course

of the reaction and using concentrated sulfuric acid in addition to nitric acid.28 This method

was more convenient compared with Brodie’s multiple reactions and resulted in graphite

oxide with a C/O atomic ratio of 2.89:1. In 1958, Hummers and Offeman developed an

alternate oxidation method to prepare graphite oxide by reacting graphite with potassium

permanganate (KMnO4) in concentrated sulfuric acid and sodium nitrate (NaNO3).29 The

resulting graphite oxide has a C/O ratio of 2.25:1.

The structure of graphite oxide:

The reactions of graphite with oxidants (KClO3 or KMnO4) in concentrated sulfuric acid

and nitric acid are complicated and the precise reaction pathways are unknown so far. The

exact chemical structure of graphite oxide is also unknown as a result. However, there are

some structure models depicting it, e.g. Hofmann model,30 Ruess model,31 Scholz and Boehm

model,32 Lerf and Klinowski model.33 Among them, the Lerf and Klinowski model is the most

widely accepted.

Fig. 6 Schematic of the structure of graphite oxide.

According to the Lerf-Klinowski model, the majority of oxygen-functional groups in

graphene oxide are epoxide and tertiary hydroxyl groups which are located in the middle of

the graphene plane, while small amount of carboxylic and/or carbonyl groups are located on

8
the edges. A schematic of the structure of graphite oxide according to this model is shown in

Fig. 6. Recently Gao et al. showed evidences for the presences of five- and six-

13
membered-ring lactols (not shown in Fig. 6) in graphite oxide by solid state C nuclear

magnetic resonance (NMR) characterization.34

Graphite oxide can be easily dispersed in water and exfoliated into single-layered

graphene oxide by ultrasonication.35 The synthesis of graphene oxide from graphite is

cost-effective and scalable for mass production. While graphene oxide is insulating, its

deoxygenation by chemical reducing agents can restore the conductivity and produce the

so-called reduced graphene oxide (rGO) or chemically converted graphene (CCG). A myriad

of reductants have been developed up to now, including sodium borohydride,36-37

hydrazine,35,38-39 hydroquinone,40 strong base (KOH or NaOH),41 hydriodic acid (HI),42-43

alumina powder,44 L-ascorbic acid,45 vitamin C,46 benzyl alcohol,47 zinc/sulfuric acid

(Zn/H2SO4),48 lithium aluminum hydride (LiAlH4).49 Among them hydrazine (N2H4) and

hydriodic acid (HI) are the most commonly used ones.

Fig. 7 Scheme showing the chemical reduction of graphene oxide to produce aqueous graphene

solution. (Adapted with permission from ref. 38. © 2008 Nature Publishing Group.)

Fig. 7 shows the schematic of chemical reduction of graphene oxide for the production

of graphene aqueous solution. Step 1 is oxidation of graphite (black blocks) to graphite oxide

9
(lighter colored blocks) with larger interlayer distance. Step 2 is exfoliation of graphite oxide

in water by sonication to obtain graphene oxide colloids that are stabilized by electrostatic

repulsion. Step 3 is controlled conversion of graphene oxide colloids to conducting graphene

colloids by hydrazine reduction at pH=10.38

Fig. 8 Photograph of a 10-μm-thick chemically converted graphene (CCG) film (diameter ~38

mm) prepared by vacuum filtration of a CCG colloid. The inset image shows a strip of this

film is bendable. (Adapted with permission from ref. 38. © 2008 Nature Publishing Group.)

Photograph of a chemically converted graphene (CCG) film (thickness ~10 μm, diameter

~38 mm) prepared by vacuum filtration of the resulting graphene colloid is shown in Fig. 8.

The film exhibited shiny metallic luster with flexibility. The conductivity of the film

measured by a four-probe method was 7,200 S m-1. The highest values reported for C/O

atomic ratio and conductivity of rGO by hydrazine reduction are ~9.97 and ~7,200 S m-1,

respectively, while those values of rGO by HI reduction are 15. 27 and 30,400 S m-1,

respectively.38,42

The mechanism for graphene oxide reduction by hydrazine is unknown so far. However,

it is generally accepted that hydrazine reduction leads to the incorporation of a small amount

of nitrogen (1.0 at%~3.0 at%) into the structure of rGO.50 The incorporation of nitrogen is

10
probably through the formation of pyrazoline structure at the edges of graphene platelet which

can evolve into pyrazole structure under thermal annealing, leading to the generation of

aromatic nitrogen doping as shown in Fig. 9.51

Fig. 9 Schematic illustrating the formation of pyrazole structure at the graphene platelet

edges by hydrazine reduction. (Adapted with permission from ref. 51. © 2012 Nature

Publishing Group.)

The precise mechanism for graphene oxide reduction by HI is also unknown. Possible

reduction pathways are shown in Fig. 10. Since iodine ion (I-) is a well-known strong

nucleophile, I- can attack the epoxide and hydroxyl groups in graphene oxide and lead to the

formation of C-I intermediate, in other words, nucleophile substitutions of epoxide and

hydroxyl groups by iodine. However, iodine is eventually eliminated from the C-I

intermediate with the formation of new C=C bonds meanwhile as C-I bond is not

thermodynamically favored. The reduction by HI can be viewed as an iodine-ion-catalyzed

reaction.

11
 Fig. 10 Schematic showing possible mechanism for graphene oxide reduction by HI. (Adapted

with permission from ref. 42. © 2010 Nature Publishing Group.)

Chemical reduction of graphene oxide for graphene synthesis is a solution-based method

which is suitable for mass production. It is particularly advantageous over other methods in

the areas of large-area transparent conductive films (TCFs) for electronics simply by

spin-coating to deposit ultrathin graphene oxide films first followed by chemical reduction,

graphene-metal-oxide composites for supercapacitors and lithium ion batteries, and

graphene-polymer composites.52-54,17 Moreover, free-standing graphene film with thickness in

the range of micrometer can be prepared by vacuum filtration a graphene solution. Last but

not least, it is facile and cost-effective. The temperature needed for synthesizing and reducing

graphene oxide is relatively low (<100 °C) which is in stark contrast to the high temperature

(~1000 °C) needed for the CVD method.

However, the disadvantage of this method is that the electrical property of rGO is not as

good as that of graphene obtained by CVD or scotch tape method. The severe oxidation of

graphite creates many defects in the structure of carbon plane, which cannot be repaired by

12
chemical reduction. The large D band with respect to the G band, the broadening of the G

band and the disappearance of the 2D band in the Raman spectrum of rGO42 reveal that rGO

has many defects (holes or vacancies) existing in its structure and is different from graphene

obtained by CVD or scotch tape method. Chemical reduction of graphene oxide can restore

the π-networks and make it conductive, but cannot repair the defects in graphene oxide.

1.2.5 Sonication-Assisted Exfoliation of Graphite in Organic Solvents

Exfoliation of graphite by sonication in organic solvents with or without surfactants was

first reported by Dai’s group and Coleman’s group in 2008.

Fig. 11 Schematic showing the sonication-assisted exfoliation of graphite. (a) Intercalating

graphite with sulfuric acid molecules (teal spheres), (b) inserting TBA (blue spheres), (c)

sonication of TBA-inserted-sulfuric-acid-intercalated graphite with DSPE-mPEG. A photograph

of an as-produced DSPE-mPEG/DMF solution of graphene sheets is also shown. (Adapted with

permission from ref. 55. © 2008 Nature Publishing Group.)

13
 Dai’s group obtained high-quality graphene sheets by briefly heating commercial

expandable graphite at 1,000 °C to exfoliate it first, reintercalating the exfoliated graphite

with oleum, then inserting tetrabutylammonium hydroxide (TBA) molecules into the

intercalated graphite, finally sonicating the TBA-inserted-oleum-intercalated graphite with

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-5000]

(DSPE-mPEG). Schematic of the whole process is shown in Fig. 11. The as-produced

graphene sheet had a resistance of 10-30 kΩ at room temperature which was 1,000 times

lower than thermally reduced graphene oxide (800 °C in H2 atmosphere) with a resistance of

20 MPa. And the resistance of as-produced graphene sheet showed only a small drop at low

temperature indicating its quality was comparable to the peel-off pristine graphene.55

(a) (b) (c)

Fig. 12 (a) Bright-field and (b) dark-field TEM images of monolayer graphene, (c) bright-field

TEM image of bilayer graphene. Scale bars are all 500 nm. (Adapted with permission from ref.

56. © 2008 Nature Publishing Group.)

Coleman’s group exfoliated graphite to sub-five-layer graphene by sonication of graphite

in organic solvents.56 According to their theory, it is possible to exfoliate graphite to graphene

when the energy cost is balanced by the solvent–graphene interaction for solvents whose

surface energies match that of graphene. Such organic solvents suitable for graphite

14
exfoliation have surface tensions in the region of 40-50 mJ m-2, e.g. benzyl benzoate,

1-methyl-2-pyrrolidinone (NMP), γ-butyrolactone (GBL), N,N- dimethylacetamide (DMA),

1,3-dimethyl-2-imidaz-olidinone (DMEU). Some typical transmission electron microscopy

(TEM) images of graphene flakes obtained by sonication of graphite in NMP are shown in

Fig. 12. A thin graphene film made by vacuum filtration of a graphene NMP solution showed

a conductivity of ~6,500 S m-1 which is close to rGO by hydrazine reduction at pH=10

(conductivity ~7,200 S m-1).

Sonication-assisted direct exfoliation of graphite in organic solvents is advantageous in

making high quality graphene sheets which are much less defective than chemically or

thermally reduced graphene oxide. However, the concentration of as-produced graphene sheet

in organic solvent is very low (~0.01 mg/mL) which limits its applications. Yet removing the

organic solvent or surfactant in case that surfactant is used is annoying. Coleman’s group

pointed out that an air-dried graphene thin film prepared by vacuum filtration of a graphene

NMP solution contained ~11 wt% residual NMP as determined by X-ray photoelectron

spectroscopy (XPS), and this value remained unchanged after a subsequent vacuum annealing

at 400 °C.56

1.2.6 Bottom-Up Synthesis

Cai et al.57 reported the synthesis of atomically precise graphene nanoribbons (GNRs) by

a bottom-up way. This method involved the use of surface-assisted coupling of molecular

precursors into linear polyphenylenes and their subsequent cyclodehydrogenation. The reaction

schemes and some typical scanning tunneling microscopy (STM) images are shown in Fig. 13. The

15
calculated bandgap for an N=7 armchair GNR (Fig. 13a) was 1.6 eV. Moreover, GNR heterojunctions

could be created by heteromolecular coupling as shown in Fig. 13e.

(b)

(a)

(c) (d)

(e)
(f)

Fig. 13 Reaction schemes and STM images of GNRs. (a) Reaction scheme for synthesizing

armchair GNR from precursor monomer 1. (b) STM image of armchair GNRs. (c) Reaction

scheme for synthesizing chevron-type GNRs from monomer 2. (d) STM image of

chevron-type GNRs. (e) Heteromolecular coupling between monomer 2 and 3. (f) STM image

showing the threefold GNR junction. (Adapted with permission from ref. 57. © 2010 Nature

Publishing Group.)

16
 The first step during the bottom-up synthesis of GNRs is the thermal sublimation of monomers on

Au(111) or Ag (111) surfaces which results in some surface-stabilized biradical species, then the

biradical species diffuse across the surface and undergo radical addition to form linear polymer chains.

The second step is formation of aromatic GNRs through surface-assisted cyclodehydrogenation.57 This

method can produce GNRs at a modest temperature (<450 °C), however, a suitable technique

for transferring as-synthesized GNRs onto SiO2 substrates for electronics needs to be

developed. Yet the electrical properties of as-synthesized GNR are open to doubt.

Jiang et al.58 reported the bottom-up synthesis of graphene films at low temperature

(220~250 °C) via a radical reaction. Hexabromobenzene (HBB) radicals produced by

cleavage of C-Br bonds coupled efficiently on Cu (111) to form graphene films. The charge

carrier mobility of as-synthesized graphene evaluated by field effect transistor was

1000~4200 cm2 V-1 s-1. The bottom-up synthesis of graphene film via radical coupling

reaction is a newly-arisen method, and the quality of as-synthesized graphene film regarding

conductivity, thickness uniformity, graphene domain size are not clear.

1.3 Graphene Derivatives - Fluorinated Graphene

Recently there are growing research interests in exploring graphene derivatives. In

addition to graphene oxide which is a nonstoichiometric graphene derivative with mainly

hydroxyl and epoxide groups randomly distributed on the carbon plane, researchers are

interested in stoichiometric graphene derivatives which can be viewed as new

two-dimensional crystals. Such stoichiometric graphene derivatives are expected to possess

17
different electronic properties and may be used as precursors for further chemical

modifications of graphene. Furthermore, researchers are interested in opening the band gap of

graphene for electronics by exploring graphene derivatives.

Elias et al. synthesized graphane (hydrogenated graphene) by reacting graphene with

atomic hydrogen in a plasma.59 Hydrogenation (attaching atomic hydrogen to each of the

carbon atoms in graphene) changed the hybridization way of carbon from sp2 to sp3, which

removed the conducting π-bonds and opened the band gap. Their experimental results showed

that single layer graphene exhibited standard ambipolar field effect with charge carrier

mobility of ~14,000 cm2 V-1 s-1 at room temperature and the half integer quantum Hall effect

at cryogenic temperature, while graphane was insulating with charge carrier mobility

decreasing to ~10 cm2 V-1 s-1 at liquid-helium temperature and did not exhibit the half integer

QHE at cryogenic temperature. Graphane was stable at room temperature and could be

changed back to graphene by annealing in argon at 450 °C indicating that the hydrogenation

process is reversible.

(a) (b)

Fig. 14 Schematic showing the crystal structures of (a) graphene and (b) graphane.

Carbon atoms are blue spheres and hydrogen atoms are red spheres. (Adapted with

permission from ref. 59. © 2009 AAAS.)

18
 Schematic of the crystal structures of graphene and graphane are shown in Fig. 14.

Graphene possesses a planar structure with a C-C bond length of 0.142 nm while graphane

possesses a chair-like structure with a longer C-C bond length of 0.153 nm due to the change

in the hybridization of carbon.

Fig. 15 Schematic for the syntheses of graphene halides (G-Br and G-Cl) using microwave-sparks-

assisted halogenation reactions. (Adapted with permission from ref. 60. © 2012 Nature Publishing

Group.)

Besides hydrogenated graphene, covalently attaching halogen atoms (F, Cl, Br, I) to the

carbon plane have also aroused intense research interests. Zheng et al.60 synthesized chlorine

and bromine modified graphite using microwave-sparks-assisted halogenation reactions, and

obtained monolayer graphene halide by sonication of the resulting chlorinated or brominated

graphite dimethylformamide (DMF). A schematic for the syntheses of graphene halides is

shown in Fig. 15. The chlorinated graphene (or graphene chloride) had 21 at% chlorine while

brominated graphene had 4 at% bromine. They also synthesized laurylamine modified

graphene by using the substitution reaction between a graphene halide (Cl or Br) and

laurylamine, which implied that graphene halides are suitable precursors for performing such

chemical functionalization of graphene.60

19
 Fluorination of graphitic materials has a long history. Graphite fluoride was first reported

in the 1930s.61 Graphite fluoride is mainly used as solid lubricant.62 The compositions of

graphite fluoride can be varied from (C2F)n to (CF)n depending on the reaction conditions.63

(CF)n represents the formula of graphite fluoride with saturate fluorine content in which each

carbon atom is bonded with a fluorine atom. With the advent of graphene, researchers are

interested in synthesizing fluorinated graphene (FG) and exploring its applications in such

areas as electronics, optics, and so on. Robinson et al.64 obtained a partially fluorinated

graphene film of C4F composition (i.e. 25 at% fluorine) by fluorination on one side of the

CVD-grown graphene film with XeF2 gas. The as-prepared C4F film is optically transparent

with a calculated band gap of 2.93 eV. They also showed that the same fluorination method

could be used to fluorinate both sides of the graphene film to form perfluorographene (CF)

which had a calculated band gap of 3.07 eV.

Nair et al.65 synthesized stoichiometric fluorographene or fluorinated graphene (FG) in

which each carbon atom is attached by a fluorine atom by exposing graphene crystals

(obtained by scotch tape method) to XeF2. As a two-dimensional (2D) material in the family

of fluorinated carbon materials, FG is markedly different from other members including

Teflon which is a fluorinated carbon chain (1D) and graphite fluoride (3D). They showed that

FG was a high-quality insulator with resistivity >1012 Ω and had an optical gap of 3 eV. FG

inherited the excellent mechanical strength of its parent graphene with a Young’s modulus of

100 N m-1 (0.3 TPa, 3 times less than graphene) and sustaining strains of 15%. FG was

chemically inert and stable up to 400 °C in air, which was similar to Teflon. FG could be

20
potentially used as an atomically thin insulator or a tunnel barrier in graphene-based

devices.65

(a)

Fig. 16 Models showing the chair-like structure of FG. (a) Ball-and-stick model of FG, big dark

grey balls represent carbon atoms and small light grey balls represent fluorine atoms. (b) 2D

unit cell (C2F2) and translation vectors. (Adapted with permission from ref. 66. © 2010 Wiley.)

Models for the structure of FG are shown in Fig. 16. The structure of FG is similar to

that of graphane. The hybridization way of carbon changes from sp2 in graphene to sp3 in FG,

which leads to the change from planar structure in graphene to chair-like structure in FG.

Zboril et al. reported that quantum-mechanical calculations revealed that FG was the most

thermodynamically stable among five hypothetical graphene derivatives: graphane,

fluorinated graphene, chlorinated graphene, brominated graphene and iodinated graphene.66

Recently, Wang et al.67 demonstrated that FG could be used to enhance adhesion and

proliferation of mesenchymal stem cells (MSCs), and that FG exhibited a neuro-inductive

effect via spontaneous cell polarization. They also showed that large-scale produced and

patterned FG sheets might be a viable platform for tissue-engineering applications.

21
1.4 Diethylaminosulfur Trifluoride (DAST) as a Useful Fluorinating Reagent

for hydroxyl, Carbonyl/Ketone and Carboxylic Groups

Currently fluorination of carbon materials is mainly performed by exposing them to XeF2 gas,

F2 gas or F-based plasma. However, XeF2 is air-sensitive, fluorination with XeF2 needs to be

performed in a glove box; F2 gas is very reactive and dangerous, fluorination with F2 requires

special equipment and great care; the use of F-based plasma is also not very facile. Since graphene

oxide can be synthesized cost-effectively and has many oxygen-containing functionalities in its

structure, converting these functionalities to C-F bonds (deoxyfluorination) through methods used

in organic chemistry could be a viable way to synthesize fluorinated graphene. With this idea in

mind, I did a thorough literature research and found diethylaminosulfur trifluoride (DAST) might

be a suitable reagent for such purposes.

Sulfur tetrafluoride (SF4) was reported to be a useful fluorinating reagent for replacing

oxygen with fluorine in hydroxyl, carbonyl/ketone and carboxylic groups.68 However, SF4 is

gaseous, toxic and corrosive making it hard to handle in organic synthesis. Middleton69 first

reported that aminosulfur fluorides synthesized by substitution(s) of one or two of the fluorine

atoms in SF4 with dialkylamino groups were also useful fluorinating reagents. Aminosulfur

fluorides are liquid and thus easier to handle than gaseous SF4. Middleton showed that

diethylaminosulfur trifluoride (DAST) could convert R-C-OH and R-C=O to R-CF, R-CF2,

respectively, with high yields of ~70-90%.69-70 The chemical structure of DAST is shown in Fig.

17. Lal et al.71 reported that bis(2-methoxyethyl)aminosulfur trifluoride (BAST), a fluorinating

reagent similar to DAST, could convert R-COOH to R-CF3 by two steps, converting R-COOH to

R-COF first, then converting R-COF to R-CF3. The yields for both steps were >90%.

22
 Fig. 17 Chemical structure of DAST.

Since graphene oxide has many hydroxyl groups, minor carbonyl and carboxylic groups,

DAST may be a useful reagent for replacing them with fluorine to synthesize fluorinated

graphene.

23
 Chapter 2 - Research Objectives, Synthetic Methods and

Characterization Tools

2.1 Research Objectives

①Synthesis and characterization of graphene oxide. ②Fluorination of graphene oxide using

DAST to synthesize fluorinated graphene. ③Exploring the biological application of graphene

oxide

2.2 Synthetic Methods

Conc. H2SO4, NaNO3, KMnO4 Ultrasonication

35~40 °C

Graphite Single-layered
Graphite oxide
graphene oxide

2.3 Characterization Tools

2.3.1 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR stands for Fourier Transform InfraRed spectroscopy, an advanced method of

infrared spectroscopy which uses the mathematical process-Fourier transform to convert raw

data to actual spectrum. Infrared spectroscopy is a technique which utilizes the interactions of

24
infrared light with matter to identify unknown materials. Infrared light can be divided into

near-infrared (13000-4000 cm-1), mid-infrared (4000-400 cm-1) and far-infrared (400-10 cm-1).

The mid-infrared region is the most commonly used because the vibrational excitations of

most organic functional groups (e.g. -CH3, -C=C-, O-C=O) and inorganic ions (e.g. CO32-,

SO42-, MnO4-) are induced by mid-infrared light.72

Infrared light imposed on a molecule does not contain enough energy to cause electronic

transitions, but can cause vibrational and rotational changes of the molecule. Possible

vibrational rotational motions of a molecule can be categorized into symmetric/asymmetric

stretching, scissoring (symmetric in-plane bending), rocking (asymmetric in-plane bending),

wagging (out of plane bending) and twisting (out of plane bending). The vibration frequency

of a molecule excited by infrared light can be expressed in equation 1.

1 k
v= √( 𝑚1𝑚2 ) Equation 1
2π
𝑚1 +𝑚2

, where v is the frequency in cm-1, k represents the force constant in N cm-1, m1 and m2 are the

masses of two atoms, respectively.72

By passing infrared light through a sample and measuring the transmittance or

absorbance at each frequency of light, an infrared spectrum is obtained with peaks

corresponding to the vibrational frequencies of functional groups in the sample. The

vibrational characteristics of functional groups are unique. Therefore, functional groups in the

sample can be identified by analyzing the positions and shapes of the peaks in the infrared

spectrum.

25
 Since graphene oxide has many oxygen-functional groups and chemical modification

can lead to structural changes in these functionalities, FTIR will be a useful technique in

analyzing the structure of graphene oxide and structural evolutions resulted by modifications.

2.3.2 Raman Spectroscopy

The main spectroscopies which deal with molecular vibrations are based on processes of

infrared absorption and Raman scattering. They are widely used to provide valuable

information on the chemical structures of substances by analyzing their characteristic spectral

patterns. The phenomenon of inelastic scattering was first observed experimentally in 1928 by

Raman and Krishnan. Since then this phenomenon has been referred to as Raman scattering

and Raman spectroscopy has been developed.

In infrared spectroscopy, an infrared beam covering a range of frequencies (typically

400~4,000 cm-1) is directed onto the sample, and absorption occurs when the frequency of

incident radiation matches that of a molecular vibration. By contrast, in Raman spectroscopy,

a single frequency of radiation (typically 514 nm or 633 nm or 785 nm) is employed and the

radiation scattered from the molecule is detected. Intense Raman scattering occurs when

vibrations cause changes in the polarizability of the electron cloud around the molecule, while

intense infrared absorption occurs when vibrations cause changes in the dipole moment of the

molecule. Therefore, Raman and infrared spectroscopy are complementary and often used

together to give a better view of the molecular structure.73

2.3.3 Ultraviolet-Visible Spectroscopy (UV-Vis)

26
 Color is an important feature of matter. For example, polytetrafluoroethylene is white,

conjugated graphitic materials (e.g. graphite, carbon nanotubes) are black, transition metals

(e.g. Ir, Os) organometallic complexes have various colors depending on the structures of

ligands. Human eyes act as spectrometers in analyzing the light reflected from the surface of a

solid or passing through a liquid when differentiating matter by color. Sunlight or white light

is actually composed of a broad range of radiations in the ultraviolet, visible and infrared

regions of the electromagnetic spectrum. When white light passes through or is reflected by a

colored substance, a portion of the light is absorbed by the substance and the color of the

substance perceived by human eyes is determined by the remaining light which is

complementary to the absorbed light. For example, a substance appears yellow if it absorbs

indigo light from 420 to 430 nm, while a substance appears red if it absorbs green light from

500 to 520 nm.74

Fig. 18 The electromagnetic spectrum.75

The electromagnetic spectrum (Fig. 18) is very broad ranging from short wavelengths

(including cosmic ray, gamma ray) to long wavelengths (including sonic, infrared sonic). The

ultraviolet (ca. 10-400 nm) and visible radiations (ca. 400 nm-800 nm) constitute only a small

27
portion of it. However, ultraviolet radiations less than 200 nm are difficult to handle and

seldom used for structural analysis of matters. The energies of UV-Vis light range from 1.55

eV to 6.20 eV corresponding to wavelengths of 800 nm and 200 nm, respectively. Such

energies are sufficient to cause electronic transitions from low energy orbitals to high energy

orbitals in molecules. When the energy of light matches the gap between two energy levels,

the light is absorbed and electronic transition or promotion occurs. By passing UV-Vis light

through a liquid and detecting the intensity differences between transmitted light and incident

light, a UV-Vis spectrometer can determine the wavelengths at which absorption maxima

occurs which can be used to identify certain chromophores and conduct quantitative analysis

of the amount of molecules based on the Beer-Lambert law.

Under UV-Vis irradiation, π→π* transition in –C=C- bond and n→π* transition in C=O

bond can take place. Since graphene oxide contains many such bonds, UV-Vis spectroscopy

will be useful in monitoring the structural changes during chemical reduction or modification.

2.3.4 Atomic Force Microscopy (AFM)

Atomic force microscopy (AFM) belongs to the big family of scanning probe

microscopies (SPMs). AFM was first described in the literature in 1986. It was created as a

supplement to scanning tunneling microscopy (STM) which can only image conductive

samples in vacuum. AFM can image samples with high resolution regardless of their

conductivities under ambient conditions. The first AFM instrument became available by the

early 1990s.

28
 In AFM, a very sharp stylus probe is used to interact with the surface of interest, probing

the repulsive and attractive forces between the probe and the surface to give high-resolution

topographic imaging of the surface. AFM can be used in contact and non-contact (tapping)

mode depending on the properties of the samples and the information to be exacted from it. In

the former, the probe is in constant contact with the sample, while in the latter, the probe (or

cantilever) is oscillating. AFM is able to image samples in air or fluid environment rather in

high vacuum, rendering it particularly useful in imaging polymeric or biological samples in

their native states.76

AFM is very useful in imaging nano- and micro-sized graphene oxide sheets, providing

valuable information on sizes, shapes and thicknesses of graphene oxide sheets.

2.3.5 Scanning Electron Microscopy (SEM)

Scanning electron microscopy is a type of electron microscopy that images samples by

scanning it with a focused beam of electrons. The sample’s surface topography and

composition are attained by collecting various signals produced by interactions between the

electron beam and atoms of the sample. The first scanning electron microscope was invented

by M. Ardenne in 1937, and the first commercial SEM instrument was developed in 1965 by

Cambridge Scientific Instrument Company.

The most common and important imaging mode of SEM is by detecting secondary

electrons emitted from the k-shell of the specimen atoms by inelastic scattering interactions

with beam electrons. Other imaging modes include backscattered electrons, specimen current,

transmitted electrons, electron-beam-induced current, cathodoluminescence, acoustic

29
thermal-wave microscopy, environmental electron microscopy and imaging with X-rays. The

resolution of SEM is somewhere between 1 nm and 20 nm. SEM can image both conductive

and non-conductive samples. For imaging non-conductive samples with conventional SEM,

coating with conductive materials (e.g. gold, chromium) is required for getting better images.

However, environmental SEM can directly image non-conductive samples and wet samples,

making it particularly useful in biological applications.77

2.3.6 X-ray Photoelectron Spectroscopy (XPS)

X-ray photoelectron spectroscopy, also known as electron spectroscopy for chemical

analysis (ESCA), is a powerful surface chemical analysis technique which provides such

information as the elemental composition of the surface (top 1~10 nm), empirical formula of

pure materials, chemical bonding states of the element in the surface, line-profiling (mapping)

and depth-profiling of chemical composition uniformity. The first commercial monochromatic

XPS instrument came into being in 1969.

Qualitative XPS (element identification) is based on equation 2, where 𝐸𝑏 is the

binding energy of a core electron with reference to the Fermi level, 𝐸𝐹 , ℎ𝜈 is the energy of

the X-ray being used, 𝐸𝑘𝑖𝑛 is the kinetic energy of the electron, 𝛷𝐴 is the work

function of the analyzer. The energy of the X-ray is known and the analyzer work function

is constant, the kinetic energy determines the binding energy and vice versa. Each element has

a unique set of XPS peaks at characteristic binding energies, which provides direct

identification of them.78

𝐸𝑏 = ℎ𝜈 − 𝐸𝑘𝑖𝑛 − 𝛷𝐴 Equation 2

30
 An XPS spectrum is usually given by intensity (counts per second) as a function of the

binding energy. Besides photoelectron core level and valence band peaks, XPS spectra

contain Auger electron peaks, and may also contain satellite peaks and energy loss peaks.

Chemical composition analysis (quantification) can be carried out using the low-resolution

XPS survey spectra with equation 3, where Xi is the molar fraction of element i, Ii or Ij is the

intensity or area of the XPS peak of element i or j, Si or Sj is the relative sensitivity factor

(RSF) of element i or j. In brief, to get the atomic percentage of element i, its XPS signal is

divided by its RSF and normalized over all of the elements detected.78
𝐼𝑖
𝑆𝑖
𝑋𝑖 = 𝐼𝑗 Equation 3
∑𝑛
𝑗=1 ( ) 𝑆𝑗

Chemical bonding states of an element (e.g. C-C, C=C, C-OH, O=C-O) can be obtained

from its high resolution XPS core-level spectrum. However, XPS can detect all other

elements except for hydrogen (atomic number Z=1) and helium (Z=2). The binding energies

of H and He are so small compared with the energy of X-ray thus making the absorption

efficiency very small. Ultraviolet photoelectron spectroscopy (UPS) is designed for detecting

H and He.

Chemical composition and chemical bonding states analyses are very important to the

research of chemical modification and reduction of graphene oxide, therefore XPS is an

indispensable tool.

2.3.7 Four-Probe Method for Measuring Thin Film Conductivity

Four-probe method, also called Kelvin method, is a technique that measures resistance

using separate pairs of current-carrying and voltage-sensing probes to make more accurate

31
measurements than traditional two-probe method. In a sheet resistance measurement, several

resistances need to be considered as shown in Fig. 19a. The probe itself has a probe resistance

Rp. A probe contact resistance Rcp exists in the interface between the probe tip and the thin

film. A spreading resistance Rsp arises when the current flows from the probe tip into the thin

film and spreads out in the thin film. And the thin film to be measured has a sheet resistance

Rs. Schematic and equivalent circuit of the four-probe technique are shown in Fig. 19b and

Fig. 19c, respectively.

Fig. 19 Measurement of thin film sheet resistance by a four-probe method. (Adapted with

permission from ref. 79. © 2011 InTech.)

Two outer probes carry the current and two inner probes sense the voltage. Since the

voltage is measured with a high impedance voltmeter, voltage drops across the parasitic

resistances (Rp, Rcp, Rsp) of the two inner probes are significantly small and can be neglected.

The thin film sheet resistance can be calculated via equation 4:

𝑉
𝑅𝑠 = 𝐹1 · 𝐹2 · 𝐹3 · Equation 4
𝐼

, where Rs is the sheet resistance, V is the voltage between the two inner probes, I is the

current, F1, F2 and F3 are correction factors for collinear probes with equal inter-probe

32
spacing. F1 corrects for finite sample thickness, F2 corrects for finite lateral sample

dimensions, and F3 corrects for placement of the probes with finite distances from the sample

edges. For very thin samples (the thickness is less than half of the inter-probe spacing) with

the probes being far from the sample edges, F2 and F3 are approximately equal to 1.0, and the

equation can be simplified as:

𝜋 𝑉
𝑅𝑠 = 𝑙𝑛 2 · Equation 5
𝐼

The thickness of the thin film can be determined using SEM. Then its bulk conductivity can

be calculated.79,80

The four-probe method can eliminate the measurement errors caused by probe resistance,

contact resistance and spreading resistance. Therefore it is more accurate than the two-probe

method. Conductivity measurement is vital to revealing the quality of reduced graphene oxide.

The four-probe method for measuring thin film conductivity will be very useful for the

research of chemical reduction of graphene oxide.

33
 Chapter 3 - Synthesis, Characterization of Graphene Oxide

and Preparation of Free-Standing Graphene Oixde Thin

Films

3.1 Hummers Method and Modified Hummers Method for Graphene Oxide

Synthesis

3.1.1 Reaction Mechanisms - Formation of Graphite Intercalation Compounds

(GICs)

Modified Hummers Method: Kovtyukhova et al.81 first reported the modified

Hummers method for graphite oxide synthesis in 1999. Modified Hummers method involves

an additional pre-oxidation step compared with Hummers method. In Kovtyukhova’s paper,

he claimed that this pre-oxidation step was necessary to avoid the formation of incompletely

oxidized graphite-core/graphite oxide-shell particles. Modified Hummers method is now

widely used for graphite oxide (or graphene oxide) synthesis, yet it is ambiguous regarding

the differences in yield, size distribution of graphene oxide sheets, chemical structure and

oxidation degree of graphene oxide compared with Hummers method. Typically in modified

Hummers method, graphite is pre-oxidized with conc. H2SO4, K2S2O8 and P2O5, then the

pre-oxidized graphite is further oxidized following Hummers method.

Hummers method: Hummers method for graphite oxide synthesis was first reported in

1958.29 After the discovery of graphene in 2004, people gradually noticed graphite oxide can

be exfoliated into single-layered graphene oxide simply by ultrasonication in water. Now

Hummers method is also widely used for graphene oxide synthesis. Typically in Hummers

34
method, natural graphite flakes (or powders) is oxidized into graphite oxide with conc. H2SO4,

NaNO3 and KMnO4 at 35 °C. Graphite oxide still possesses a laminar structure with an

increased interlayer distance of 0.61 nm~1.2 nm compared with graphite which has an

interlayer distance of 0.34 nm.81

Formation of graphite intercalation compounds (GICs): The first pre-oxidation step in

modified Hummers method involves the formation of graphite intercalation compounds

(GICs) as was reported by Tour’s group.82 They found that conc. H2SO4 does not

spontaneously intercalate into graphite, however, in the presence of oxidants (e.g. KMnO 4,

K2S2O8 or (NH4)2S2O8), conc. H2SO4 can intercalate into graphite and lead to the formation of

graphite intercalation compounds (GICs). By contrast, I found that the intercalation of conc.

H2SO4 and K2S2O8 into graphite to form GIC can be completed in 15-20 minutes with the

help of sonication, while the intercalation needs 6-8 h without sonication.82

(a) (b) G
Graphite GIC

2D

Fig. 20 (a) Photographs and (b) Raman spectra of graphite and H2SO4-K2S2O8-GIC.

As shown in Fig. 20a, the H2SO4-K2S2O8-graphite intercalation compound has lost its

luster and possesses a much larger volume compared with its parent graphite. The volume

expansion is caused by the intercalation of other species (H2SO4-K2S2O8) into the graphite

35
lattice which largely increases the inter-layer distance. Raman spectroscopy was further used

to characterize the GIC and graphite. Raman measurements were performed with a Horiba

Jobin Yvon LabRAM HR 800 Raman spectrometer using a 633 nm excitation laser. The

Raman spectrum of graphite (Fig. 20b, bottom) shows two pronounced peaks: G band at

~1580 cm-1, and 2D band at ~2690 cm-1. The Raman spectrum of H2SO4-K2S2O8-GIC (Fig.

20b, top) shows only one prominent G peak located at 1610 cm-1, which is shifted compared

with that of graphite. Another noteworthy aspect is that the intensity of the G band of GIC is a

lot higher than that of graphite indicating that intercalation of graphite with other species (or

heavy doping) can enhance the G band in the Raman spectrum, which is in accordance with

the literature.2 The Raman spectrum of H2SO4-K2S2O8-GIC after being exposed to air for 8 h

is shown in Fig. 1b, middle. The reappearance of the 2D band indicates the deintercalation of

GIC when exposed to air. This can be explained by that concentrated H2SO4 from GIC

adsorbed water vapors from the air when it is exposed which leads to the deintercalation.

Thus the intercalation of concentrated H2SO4-K2S2O8 with graphite is somewhat reversible.

Graphite GIC

Fig. 21 Photographs of graphite and H2SO4-KMnO4-GIC.

Similarly, another concentrated H2SO4-KMnO4-GIC (graphite flakes 1.0 g, KMnO4 3.0 g,

concentrated H2SO4 10 mL) was prepared with sonication. Again, the intercalation was

36
accomplished in about 15-20 minutes with the help of sonication, large volume expansion and

color change were observed as shown in Fig. 21.

The as-prepared H2SO4-K2S2O8-GIC and H2SO4-KMnO4-GIC may have applications in

producing high quality graphene (much less oxidized compared with graphene oxide

synthesized by Hummers method) via liquid-phase exfoliation with the help of sonication55

and MnO2-graphene hybrid material.83

The chemical processes of making graphene oxide in Hummers method involve

formation of some kind of H2SO4-KMnO4-GIC and meanwhile oxidation on some edges,

defects, cracks, and/or vacancies of graphite sheets (carboxylic and carbonyl groups are most

likely formed at this stage), further hydrolysis of the GIC affords more oxygen-functional

groups attached to the carbon plane (hydroxyl and epoxide groups are most likely formed at

this stage). Once the oxygen-containing groups are attached to the graphene plane, the van der

Waals forces between graphene planes are minimized, therefore, the exfoliation of graphite

(or oxidized graphite) into few-layered graphite or even single-layered graphite (i.e. graphene)

is possible by mechanical stirring which is used throughout the whole reaction. Sonication of

few-layered graphite oxide leads to mass production of mono-layered graphene oxide.84-85

It is worth pointing out that the size of graphite oxide sheet which finally determines the

size of graphene oxide sheet is also largely reduced by the hot gas bubbles produced by

sonication. Su et al.,86 Zhou et al.87 and Zhao et al.88 reported the synthesis of ultra-large

graphene oxide sheets with dimensions of hundreds of micrometers which are much larger

than conventional graphene oxide sheets that have lateral sizes in the range of hundreds of

nanometers to a few micrometers. In their methods the exfoliation of multi-layered graphite

37
oxide into single-layered graphene oxide is realized by just mechanical stirring or mild

sonication for a short time period of 5 minutes while in conventional method sonication for 30

minutes~1 hour is usually used. Therefore, no sonication or mild sonication should be used in

order to get ultra-large graphene oxide sheets with lateral sizes of hundreds of micrometers.

3.1.2 Experimental Procedures - Modified Hummers Method

1. Graphite flakes (3.6 g, Sigma-Aldrich, 100 mesh) were ground with NaCl (30 g) for

20 minutes. Afterwards, copious water was added to dissolve NaCl, the mixture was filtered

and washed several times to remove NaCl. The remaining solid was dried at 80 °C for 1.5 h.

2. Dry graphite powders (~3.0 g) was added into a solution of K2S2O8 (2.5 g), P2O5 (2.5

g) and concentrated H2SO4 (15 mL, 95-98%). The mixture was kept in an 80 °C oil bath for 4

h with stirring. After the dark blue mixture was cooled to room temperature naturally,

deionized water was added, followed by filtration and rinsing with copious water. The solid

was dried at 60 °C for 1.5 h.

3. The pre-oxidized graphite was transferred into a 500 mL round-bottom flask, 69 mL

concentrated H2SO4 was added. The mixture was stirred for 30 minutes and transferred to a

0 °C ice bath. KMnO4 (15 g) was added slowly with stirring to keep the temperature of the

mixture below 20 °C.

4. The mixture was heated for 2 h with stirring in a 35~40 °C water bath. Then it was

carefully diluted with 140 mL deionized water (violent effervescence occurs with an increase

in the temperature to 92~98 °C) and continued stirring for 30 minutes.

38
 5. Afterwards, the flask was removed from the water bath, and the mixture was

transferred to a large beaker. 420 mL deionized water was added, followed by 20 mL 30 wt%

H2O2 (the color of the suspension turned green). The suspension was stirred for 10 minutes,

repeatedly centrifuged at 11,000 rpm for 10 minutes, and washed with 5% HCl for three times,

followed by deionized water twice (the precipitate was collected and the supernatant which

contained large amounts of salts and small light-weighted particles were thrown away each

time).

6. The slurry-like precipitate was re-dispersed in 350 mL Milli-Q water and stirred for 15

minutes. The suspension was treated with bath sonication (operating frequency 33 kHz, power

60 W) for 30 min. Then the suspension was repeatedly centrifuged at 3,000 rpm for 4~5 times

to remove any insoluble particles (the supernatant was collected each time).

7. Afterwards the homogenous brown solution was dialyzed against Milli-Q water for 1

week and stored for future use. The concentration was determined by filtering 8.0 mL stock

solution using a 0.02 μm Anodisc membrane filter (Whatman), drying the resulting film in a

50 °C oven overnight and weighing its mass. The concentration of the solution was about

2.5~3.5 mg/mL using the preparation method described above.

Photographs of as-prepared graphene oxide stock solution and diluted ones are shown in

Fig. 22. Graphene oxide solution is homogeneous with no visible particles, and has a dark

brown to light yellow color depending on its concentration instead of a black color suggesting

that the π-conjugation of graphene has drastically changed after oxidation which leads to

different band structures and transitions under visible light excitation. Graphene oxide

solution is table for infinite time. The synthesis of graphene oxide by modified Hummers

39
method is mass production as illustrated by the left photograph in Fig. 22 which shows 250

mL 2.8 mg/mL graphene oxide solution.

3.4 mg/mL 1.0 mg/mL 0.10 mg/mL

250 mL (2.8 mg/mL)

Fig. 22 Photographs showing (left) large-scale synthesis of graphene oxide and

(right) graphene oxide aqueous solutions of different concentrations.

3.2 Graphene Oxide Characterization

3.2.1 FTIR Characterization

Graphene oxide solid obtained by vacuum filtration was finely ground with KBr, and

then compressed into thin pellets for FTIR characterization. The FTIR spectrum was collected

using a Bruker Tensor 37 FTIR spectrometer.

The characteristic features of the FTIR spectrum of graphene oxide (Fig. 23) are the

strong and broad band at 3424 cm-1 which can be attributed to the O-H stretching of

carboxylic, hydroxyl groups and absorbed water, weak bands at 2928 and 2851 cm-1 which

can be attributed to the C-H symmetric and asymmetric stretching of CH2 groups, 1725 cm-1

attributed to the C=O stretching of ketone, carboxylic and/or ester groups, sharp and middle

strong band at 1628 cm-1 attributed to carboxylic groups or the C=C stretching, 1401 cm-1

attributed to the O-H bending of carboxylic, hydroxyl groups and absorbed water, 1227 cm-1

40
attributed to the C-OH stretching of carboxylic and hydroxyl groups, 1057 cm-1 attributed to

the C-O-C stretching of epoxide and/or ester groups.34,89-90

Fig. 23 FTIR spectrum of graphene oxide.

3.2.2 UV-Vis Characterization

Fig. 24 UV-vis spectrum of graphene oxide.

Diluted graphene oxide aqueous solution was used for UV-vis characterization. The

measurement was performed with a Thermo Scientific GENESYS 10S UV-Vis

spectrophotometer. The characteristic features in the UV-vis spectrum of graphene oxide (Fig.

41
24) are the sharp peak at 231 nm which can be attributed to the π→π* transitions of -C=C-

bonds, the broad and less obvious peak at 294-305 nm which can be attributed to the n→π*

transitions of -C=O bonds.91-92

3.2.3 AFM Characterization

SiO2/Si substrates were sonicated in deionized water for 15 minutes and blown dry with

pure nitrogen gas, followed by the same treatment with acetone. The pre-cleaned substrates

were further cleaned by oxygen plasma in a glove box. GO sheets were deposited on the

pre-treated SiO2/Si substrates by spin-coating of a GO H2O-EtOH dispersion (volume ratio of

H2O to EtOH is 1:9) at 3,000 rpm. As the volatile solvent evaporated away quickly, GO sheets

stuck to the substrates because of van der Waals force. AFM characterization was performed

with a Nanoscope MultiModeTM AFM instrument in the taping (non-contacting) mode at a

scan rate of 1.0 Hz, and a silicon probe with a resonant frequency of 300 kHz was used. Some

typical AFM images are shown in Fig. 25. GO sheets are of irregular shapes with sizes in the

range of hundreds of nanometers to a few micrometers.

42
 (a) (b)

(c) (d)

(e) (f)

Fig. 25 AFM images showing graphene oxide sheets on SiO2/Si substrates at different resolutions.

Resolutions of the images are as follows: (a) 20 μm × 20 μm, (b) 8 μm × 8 μm, (c) 4 μm × 4 μm,

(d) 4 μm × 4 μm, (e) 2 μm × 2 μm, (f) 8 μm × 8 μm.

43
 d = 0.836 nm

× ×

1 μm

1 μm
d = 1.016 nm

× ×

d = 1.021 nm

1 μm

× ×

Fig. 26 (Left) AFM images of graphene oxide sheets and (right) height profiles along the pink

lines indicated in the AFM images showing the thickness of graphene oxide.

44
 Thickness Analysis:

Height profiles along the pink lines indicated in the AFM images are shown in Fig. 26.

The red triangle represents the AFM tip. The height is larger when the red triangle is on the

surface of a GO sheet compared with that when the red triangle is on the substrate. The height

difference represents the thickness of a GO sheet, which is 0.8~1.1 nm indicating the

as-synthesized GO is single-layered according to the literature.35 Compared with pristine

graphene (unoxidized) which has a van der Waals thickness of 0.34 nm, GO is thicker due to

the covalently bonded oxygen-containing groups on both sides of the carbon plane.

Phase imaging in tapping mode AFM measures the phase shift or lag of an oscillating

cantilever between driving signal and AC output signal. Phase images can also reveal the

morphology of the sample. Some typical phase images are shown in Fig. 27 left. These phase

images evidently show the sheet-like structure of GO. Although these 2D GO sheets seem

rigid on the SiO2/Si substrates, they might be flexible in solutions as has been suggested by

Ruoff and co-workers.90 3D topological view images of GO sheets on SiO2/Si substrates were

also obtained (Fig. 27 right). The surfaces of GO sheets are not very flat in these 3D

topological images, which might be explained by that the functional groups (hydroxyl and

epoxide) on the carbon plane are different which leads to variations in the thicknesses.

Another reason might be that the attachment of oxygen-functional groups to the carbon plane

substantially changes the hybridization ways of carbon (from sp2 to sp3), which destroys the

flat structure of conjugate carbon plane.

45
 (a) (b)

(c) (d)

(e) (f)

(g) (h)

Fig. 27 AFM phase and 3D topographical images of graphene oxide sheets on SiO2/Si

substrates. a, c, e, g are phase images, and b, d, f, h are corresponding 3D topographical

images.

46
3.2.4 Raman Characterization

A small piece of graphene oxide film obtained by vacuum filtration or natural graphite

flake (Sigma-Aldrich) was put on a glass slide for Raman Characterization. All Raman

measurements were performed with a Horiba Jobin Yvon LabRAM HR 800 Raman

spectrometer using a 633 nm excitation laser. A ×50 objective was used.

Fig. 28 Raman spectra of graphene oxide and graphite.

In the Raman spectrum of graphite, the small D band at ~1317 cm-1 arises from the first

order of zone-boundary phonons and is present only in defected graphite. The small D band

indicates graphite has few defects. The intensity ratio of D band to G band is widely used to

quantify the amount of defects in graphitic materials (graphite, graphene, carbon nanotubes).

The strong G band at ~1562 cm-1 is due to the doubly generate zone center E2g mode. The 2D

band is due to the second order of the zone-boundary phonons and is double of the D band.

The 2D band of graphite actually consists of two bands, 2D1 of lower intensity at 2625 cm-1,

and 2D2 of higher intensity at 2674 cm-1. By contrast, the 2D band of graphene is a single

sharp peak.93-94

47
 The Raman spectrum of GO shows two pronounced peaks, the D band at ~1330 cm-1,

and the G band at ~1585 cm-1. The broadening of the D band and G band, and the much

higher intensity ratio of D to G than bulk graphite indicates GO has lots of defects. Notably,

no 2D band is observed in GO, suggesting the structure of GO is much different from

graphene or graphite because of the covalent bonding of considerable amount of

oxygen-containing groups.

3.2.5 XPS Characterization

Small pieces of GO solid obtained by vacuum filtration were used for XPS

characterization. The XPS measurements were performed with a Thermo Scientific

ESCALAB 250Xi XPS spectrometer in Prof. Tong Leung’s lab (University of Waterloo).

Dual Al-Kα X-ray (1486.6 eV, 150 W) with flood gun (0.2 mA) was used to solve the

charging issue since GO is insulating. CasaXPS was used for the deconvolution of XPS

peaks.

(a) (b)

Fig. 29 (a) XPS survey spectrum and (b) C1s spectrum of graphene oxide (red line is the

experimental data and black lines are the fitted peaks).

48
 Fig. 29a shows the XPS survey spectrum of as-prepared graphene oxide. Only C

(binding energy 284.5 eV) and O (binding energy 532.0 eV) were detected with the absences

of other elements such as S and Mn which are common contaminants, suggesting the

as-prepared graphene oxide was of high purity. Quantitative analysis based on the XPS survey

spectrum showed that the atomic percentages of C and O were 66.01% and 33.99%,

respectively. (The relative or atomic sensitivity factors (RSF or ASF) of C and O are 0.296

and 0.711, respectively.) The high resolution C1s spectrum of graphene oxide (Fig. 10b) could

be fitted into three main peaks, C=C (sp2)/C–C (sp3) at 284.4 eV, C–OH/C–O–C (hydroxyl

and epoxide) at 286.6 eV, C=O (carbonyl) at 288.3 eV.36,91

3.3 Preparation of Free-Standing Graphene Oxide Thin Films (or Papers)

Free-standing films or papers play an important role in modern technological society.

They can be used as protective layers, filter membranes for separation applications,

components in batteries or supercapacitors.95 Graphene oxide, the oxidized form of graphene,

is very hydrophilic due to the covalently-bonded oxygen-containing groups, which suggests

that graphene oxide is compatible with aqueous-phase processing. Nair et al.96 reported that

submicrometer-thick graphene oxide membranes allow unimpeded permeation of water while

they are completely impermeable to liquid, vapors and gases including helium. These

graphene oxide membranes were made by spin-coating of graphene oxide aqueous solutions

on Cu foils, followed by polymer masking and etching off the underneath copper. Eda et al.97

reported large-area ultrathin (1~5 layer) films of reduced graphene oxide for transparent and

49
flexible electronics. In their method, large-area graphene oxide films on filter membranes

were first made by vacuum filtration, and then transferred onto plastic substrates, followed by

chemical and thermal reduction to recover the conductivity.

While ultrathin graphene oxide films with thicknesses below hundreds of nanometers are

important to separation technology, transparent and flexible electronics, macroscopic

free-standing graphene oxide films with thicknesses from a few micrometers to tens of

micrometers or even higher are expected to find applications in thin film batteries or

supercapacitors, biomedical areas, mechanically strong and stiff materials with lamellar

structures.98-101 Currently there are two main methods for making macroscopic free-standing

graphene oxide thin films, one is vacuum filtration or flow-directed assembly, the other is

self-assembly at the water-air interface.

3.3.1 Vacuum Filtration

Dikin et al.95 first reported the use of vacuum filtration or flow-directed assembly to

prepare free-standing graphene oxide thin film or paper. In a typical experiment, 3.0 mL

graphene oxide solution (concentration 0.93 mg/mL) was filtered using an Anodisc membrane

filter (diameter 25 mm, pore size 0.02 μm, Whatman), and the film was carefully peeled off

the filter using a razor blade.

Fig. 30 shows the digital image of a free-standing graphene oxide film and SEM (Zeiss

LEO 1550) image of its cross-section. The film is flexible and mechanically strong enough to

be handled with a tweezer. The thickness of the resulting film is about 6.5 μm. The SEM

image of the cross-section clearly shows the highly ordered lamellar structure.

50
 (a) (b)

Fig. 30 (a) Photograph of a graphene oxide thin film, and (b) SEM image showing the

cross-section of the film

The mechanism of forming thin films with highly ordered lamellar structure lies in that

graphene oxide sheets first blocked the pores in the filter membrane, and the sheets come

close to each other as the water vaporization on the top of the solution also occurs during the

filtration which makes the solution denser and denser, the interactions between sheets are

stronger as a result, and the sheets choose to inter-tile with each other. The small amount of

water molecules between the sheets can also act as a smoothing component to facilitate the

formation of layered structure. Finally, van der Waals forces and hydrogen bonds hold the

sheets tightly and mechanically strong graphene oxide thin films with highly ordered lamellar

structure are formed as a result.95

Ruoff’s group reported that the as-prepared graphene oxide papers have excellent

mechanical properties with a modulus of ~32 GPa and a tensile strength of ~72.2 MPa, both

of which values are higher than carbon nanotube thin films.95

3.3.2 Self-Assembly at the Water-Air Interface

Chen et al.102 first reported the method of efficiently making graphene oxide thin films

(typically a few micrometers to tens of micrometers) by self-assembly of graphene oxide

51
sheets at the water-air interface. This method is more efficient than the vacuum filtration

method. This method usually takes 1~2 hours to make the films while the vacuum filtration

method usually takes more than 12 hours or even 1~2 days. In a typical experiment, graphene

oxide solutions with concentrations from 1.0 mg/mL to 3.0 mg/mL were put in polystyrene

weighing dishes. The solutions were heated in an 80~90 °C oven for 1~2 hours. Then the

weighing dish was taken out and the small amount of solution under the film was carefully

poured out. Afterwards the weighing dish was put back into the oven to completely dry the

film. After the film is dry, it is easy to peel off the weighing dish since graphene oxide is not

sticky to polystyrene materials. A digital image of a free-standing graphene oxide film

(diameter 64 mm) prepared in this thesis using this method is shown below.

(a) (b)

Fig. 31 (a) Digital image of a free-standing graphene oxide film prepared in this thesis by

self-assembly at the water-air interface, (b) schematic of the film formation mechanism.102

Schematic of the mechanism for this method is shown in Fig. 31b. When a graphene

oxide water solution is heated, water vaporizes and lifts up graphene oxide sheets in the

solution to the water-air interface. Graphene oxide sheets tend to tile on each other at the

interface due to the effect of surface tension. Then van der Waals forces and strong hydrogen

bonds hold the sheets tightly. Mechanically strong graphene oxide film is formed as a result.

52
 Besides time-saving, another advantage of this method is that large-area graphene oxide

film can be achieved via this method while the area of graphene oxide film is usually limited

by the size of filtration apparatus in the vacuum filtration method. However, the mechanical

properties of graphene oxide thin films prepared by this method are not as good as those

prepared by the vacuum filtration method. The modulus and tensile strength of the former

were reported to be ~12.7 GPa, ~67.7 MPa, respectively, both of which are lower than the

reported values for the latter (32 GPa, 72.2 MPa, respectively).102

3.4 Conclusions and Future Aspects

Graphene oxide, a 2D carbon material decorated with abundant oxygen-containing

groups (mainly hydroxyl, epoxide and carboxylic groups), can be massively and

cost-effectively produced by Hummers method. Graphene oxide is a lot more hydrophilic

than pristine graphene due to the oxygen-functionalities. The concentration of graphene oxide

in water is >4 mg/mL. The thickness of graphene oxide is between 0.8 and 1.1 nm. Raman

characterization shows that graphene oxide bears many defects in its structure which are

created by the severe oxidation during the synthesis process. Graphene oxide sheets produced

by Hummers method are of irregular shapes with sizes in the range of hundreds of nanometres

to a few micrometres. Free standing and mechanically strong graphene oxide thin films (or

papers) can be prepared by vacuum filtration of graphene oxide aqueous solution or

self-assembly of graphene oxide sheets at the water-air interface.

53
 Future aspects lie in gaining further insights into the oxidation process in Hummers

method and elucidating the chemical structure of graphene oxide. Using solution-based

chemical methods to decorate graphene sheets with metal or metal oxide nanoparticles for

energy and sensor applications, to prepare graphene-polymer nanocomposites are also future

directions.

54
 Chapter 4 – Exploring the fluorination of Grpahene Oxide Using

DAST - Chemical Reduction of Graphene Oxide

4.1 Experimental Procedures and Observances

20 mL glass vial

1 mL DAST +
1 mL CH2Cl2 or graphene oxide film
CHCl3

Fig. 32 Schematic of the experimental setup for the reaction of graphene oxide film with DAST.

GO thin films (thickness ~6.5 μm) were put in sealed 20 mL glass vials which contain 1

mL DAST (Matrix Scientific) and 1 mL CH2Cl2 or CHCl3 as solvent. Moisture in the vial was

not intentionally removed since small amount of HF produced by the reaction of DAST with

water could facilitate the fluorination reaction.71 Another reason was that the amount of

DAST added for the reaction was excessive, ruling out the scenario that all the DAST reacted

with moisture and rendered the fluorination fail. The vials were kept at 0 °C, room

temperature and 50 °C, respectively, for reduction and fluorination. For reactions at 0 °C and

room temperature, CH2Cl2 was used as solvent and the reaction time was 1 week. For reaction

at 50 °C, CHCl3 was used as solvent and the reaction time was 17 h. After the reactions were

complete, the films were carefully taken out of the vials using a tweezer and soaked in

CH2Cl2 several times, followed by deionized water to wash away by-products adsorbed on the

films. (Direct contact of DAST with water should be avoided in any case since DAST reacts

55
violently with water!) The films were referred to as reduced graphene oxide-rGO (0 °C), rGO

(R.T.) and rGO (50 °C), respectively.

Schematic of the experimental setup for the reaction of graphene oxide film with DAST

is shown in Fig. 32.

Color Change and Hydrophobicity Change of the Film:

Color change from brownish in GO to black in rGO were observed after DAST treatment

(Fig. 33). And rGO (50 °C) film is more lustrous than the other two rGO films.

DAST

GO rGO (50 °C)

Fig. 33 Digital images of GO and rGO (50 °C) showing the color change after DAST treatment.

During the washing of rGO films with water, they were observed to be very hydrophobic.

A comparison of water contact angle between rGO and GO was conducted to verify this. The

digital image (Fig. 34) was taken immediately after one drop of deionized water was placed

on each of the films. Obviously rGO (50 °C) was more hydrophobic than GO.

rGO (50 °C) GO

Fig. 34 Water contact angle comparison of rGO (50 °C) and GO thin films.

56
4.2 Characterization

4.2.1 XPS Characterization

In order to gain insights into the compositional and structural changes of GO by DAST

treatment, XPS characterization was carried out.

Small pieces of rGO films were used for XPS characterization. XPS measurements were

performed with a Thermo Scientific ESCALAB 250Xi XPS spectrometer. Monochromatic

Al-Kα X-ray (1486.6 eV, 150 W) without flood gun was used.

Fig. 35 XPS Survey spectra of graphene oxide film and reduced graphene oxide films.

C (at%) O (at%) F (at%) C/O

rGO (50 °C) 86.71 9.73 3.55 8.91

rGO (R.T.) 81.33 15.20 3.47 5.35

rGO (0 °C) 74.50 21.95 3.54 3.39

GO 66.01 33.99 0 1.94

Table 1 Atomic ratio of GO and rGO determined from XPS survey spectra.

57
 Fig. 35 shows the XPS survey spectra of GO and rGO. Quantitative analysis based on

the XPS survey spectra was performed with CasaXPS, the relative sensitivity factors of C, O,

F are 0.296, 0.711 and 1.000, respectively. The atomic percentages of GO and rGO are listed

in table 1. The fluorine content of all rGO samples is ~3.5 at% and does not change much

with reaction temperatures. However, the oxygen content decreases significantly when the

reaction temperature is increased from 0 °C to 50 °C. The C/O atomic ratio increases from

1.94 in GO to 5.35 in rGO (R.T.) and 8.91 in rGO (50 °C). The C/O ratio of 8.91 in rGO

(50 °C) is slightly higher than the sequential NaBH4 and concentrated H2SO4 reduced GO

(C/O ratio is 8.57)34 and close to the hydrazine reduced GO (C/O ratio is 10.3)35, which

indicates the effective reduction of GO by DAST.

(a) (b)

(c) (d)

Fig. 36 High resolution XPS C1s spectra of GO and rGO films.

58
 High resolution XPS C1s spectra are further used to confirm the reduction and

fluorination. The C1s spectrum of graphene oxide (Fig. 36a) can be fitted into three peaks,

C=C (sp2)/C–C (sp3) at 284.4 eV, C–OH/C–O–C (hydroxyl and epoxide) at 286.6 eV, C=O

(carbonyl) at 288.3 eV.36,91 By contrast, the C1s spectra of rGO can be fitted into five peaks,

C=C (sp2) at 284.4 eV, C–C (sp3) at 285.6 eV, C–OH/C–O–C (hydroxyl and epoxide) at 286.6

eV, C=O (carbonyl) at 288.3 eV, and CF2 at 289.7 eV.36,103 The C=C (sp2) peaks of rGO (R.T.)

and rGO (50 °C) are narrower than that of GO. The C–OH/C–O–C and C=O peaks of rGO

(50 °C) decrease significantly compared with GO suggesting effective reduction.

Fig. 37 XPS High resolution F1s spectrum of rGO (50 °C).

High resolution F1s spectrum of rGO (50 °C) (Fig. 37) can be fitted into a single peak at

686.9 eV corresponding to covalent C-F bond.67,103

However, the middle part of the film is not reacted as justified by the XPS survey

spectrum (Fig. 38) on the cross section of rGO (50 °C) film. XPS characterization on the

cross section of the film was done by shining the X-ray on the cross section and collecting

resulting data. The chemical composition determined from this survey spectrum is: C 76.22

59
at%, O 22.54 at%, F 1.23 at%. The O/C ratio of the cross section of the film is 0.30 which is

much higher than that of the surface of the film which is 0.11. This lends support to that the

middle part of the film is not reacted probably because it cannot be accessed by DAST.

O1s C1s

F1s

Fig. 38 XPS survey spectrum on the cross-section of rGO (50 °C) film.

4.2.2 FTIR Characterization

FTIR was used to investigate the chemical changes of GO caused by DAST treatment.

Small pieces of rGO and GO films were finely ground with KBr and pressed into thin

pellets for FTIR characterization. The FTIR spectra were collected with a Bruker Tensor 37

FTIR spectrometer.

The FTIR spectra of GO and rGO (50 °C) are shown in Fig. 39. After the treatment with

DAST, the band at 1725 cm-1 decreases significantly since most of the C=O groups are

converted into CF2 groups. The new band at 1580 cm-1 may be attributed to the stretching

vibration of isolated C=C bonds formed by elimination of hydroxyl groups. Another new

band at 1200 cm-1 can be attributed to the stretching of covalent C-F bond which is well

60
known for graphite fluoride, fluorinated carbon nanotubes and fluorinated graphene.63,67,104-105

This band is weak since the fluorination is limited as revealed by XPS characterization. The

band at 1100 cm-1 is likely due to the stretching of C-O-C group (epoxide) since the reactivity

of DAST towards epoxide is low according to the literature.70,106 Epoxide might account for

the residual oxygen in rGO (50 °C) as well. The overlapping of C-F stretching with C–O–C

stretching results in a broad band from 1000 cm-1 to 1300 cm-1. The fingerprint region from

1000 to 1750 cm-1 of rGO (50 °C) is less evident than that of GO suggesting reduction of GO.

The persistently strong band at 3442 cm-1 of rGO (50 °C) is largely due to the moisture in the

KBr pellets.

Fig. 39 FTIR spectra of rGO (50 °C) and GO.

4.2.3 Raman Characterization

Small of pieces of GO and rGO films were placed on glass slides for Raman

characterization. All Raman spectra were collected with a Horiba Jobin Yvon LabRAM HR

800 Raman spectrometer using a 633 nm excitation laser and a ×50 objective.

61
 The Raman spectra of GO and rGO are shown in Fig. 40, of which the characteristic

features are the D band at 1330 cm-1, and G band at 1587 cm-1. D and G positions, intensity

ratio of ID to IG (ID/IG) are listed in table 2.

Fig. 40 Raman spectra of GO and rGO. (Excitation wavelength is 633 nm.)

D position G position ID/IG

rGO (50 °C) 1321 1576 1.63

rGO (R.T.) 1322 1580 1.46

rGO (0 °C) 1325 1582 1.24

GO 1330 1587 1.19

Table 2 Raman D and G positions, intensity ratio of D to G (ID/IG) of GO and rGO.

The D band and G band of GO are both shifted to lower wavenumbers after reduction by

DAST. As the G band of graphite is located at 1562 cm-1 (Fig. 28), the G band shift from 1587

cm-1 in GO to 1576 cm-1 in rGO (50 °C) is expected considering that the structure of rGO is

more close to graphite or graphene than GO after chemical reduction (the Raman spectra of

62
graphite, GO and rGO were collected using the same Raman spectrometer under same

conditions in this thesis). Another notable change in the Raman spectra is that the ID/IG ratio

increases much from 1.19 in GO to 1.63 in rGO (50 °C) indicating that more defects are

introduced into the carbon plane and/or the size of conjugating graphitic domains is reduced

after chemical reduction of GO. The increase of ID/IG ratio after chemical reduction by DAST

is in accordance with the literature data using other reducing reagents including hydrazine and

HI-AcOH.42

4.2.4 Thin Film Conductivity Measured by a Four-Probe Method

In order to evaluate the effect of DAST reduction on the electrical conductivity,

four-probe thin film conductivity measurements were carried out. Small rectangular film (size

12 mm × 5 mm) of GO or rGO (50 °C) was stuck to a four probe stand (spacing ~2.5 mm)

using silver paste and mounted into a resistivity measurement system for collecting data.

mV
Slope for Fig. 41a is: 72000 μA
= 7.2 × 107 Ω

π
Sheet resistance is: Rs = ×slope = 4.53 × 7.2 × 107 Ω·□-1 = 3.3 × 108 Ω·□-1
ln2

Thickness of the film is: 6.5 μm

Resistivity: ρ = Rs ×thickness = 3.3 × 108 Ω·□-1 × 6.5 × 10-6 m = 2.1 × 103 Ω·m

Conductivity of GO film is: κ = 1/ρ = 4.8 × 10-4 S·m-1

mV
Slope for Fig. 41b is: 0.08792 μA
= 87.92 Ω

π
Sheet resistance is: Rs = ln2
×slope = 4.53 × 87.92 Ω·□-1 = 398.3 Ω·□-1

Thickness of the film is: 6.5 μm

Resistivity: ρ = Rs ×thickness = 398.3 Ω·□-1 × 6.5 × 10-6 m = 2.6 × 10-3 Ω·m

63
 Conductivity of rGO (50 °C) film is: κ = 1/ρ = 385 S·m-1

(a)
four-probe connection

GO or rGO film
1 2 3 4

(b) (c)

Fig. 41 (a) Schematic of the four probe connection, linear plots of voltage between probe 2 and 3

versus current for (b) GO film, and (c) rGO ( 50 °C) film.

The conductivity of rGO (50 °C) film (385 S·m-1) is ~6 orders of magnitude higher than

that of GO film (4.8 × 10-4 S·m-1) indicating that DAST is an effective reducing reagent which

can render GO reattain its electrical conductivity by chemical reduction. Since it is not a

homogenous reaction and the middle part of the GO film is not reacted, the film sheet

resistance can be even lower if a homogenous reaction is developed. Also the conductivity of

rGO film can be much higher than the value (385 S·m-1) reported here because of the same

reason. In other words, the capability of DAST in recovering GO’s electrical conductivity has

not been fully brought to light to this point. However, the obtained conductivity (385 S·m-1)

of a GO film reduced by immersing in a DAST solution is comparable to that (456 S·m-1) of a

64
GO film with the same thickness (~6.5 μm) reduced by exposing to hydrazine vapor.42 Both

of these values are much lower than the value (7200 S·m-1) reported for a rGO film prepared

by a homogeneous reaction with hydrazine38 because the middle part of the film is not reacted.

Considering that the C/O ratio (8.9) of GO reduced by DAST is close to that (10.3) of GO

reduced by hydrazine and that rGO films prepared using inhomogeneous ways have similar

conductivities, the capability of DAST in recovering GO’s electrical conductivity should be

close. Further reduction experiment using DAST in a homogeneous way needs to be

conducted to justify this.

4.3 Discussions

The fluorine content of ~3.5 at% in all rGO films is quite out of my expectation, and the

underlying mechanism is discussed below. The reaction of alcohols with DAST is

exothermic,69 i.e. low temperature is favoured for such reactions. However, from the

experimental data the fluorine content of all rGO films shows no notable dependence on

reaction temperature. The fluorination of hydroxyl groups with DAST is likely to be via an

SN2 mechanism (Fig. 21) in which steric hindrance plays an important role.71,107

Fig. 42 Schematic of the SN2 reaction between a hydroxyl group and DAST.

65
 The first step of the SN2 reaction is the elimination of one molecule HF from the

reactants and formation of an intermediate [R---O---SF2NR2], the second step is F- attacking

the intermediate from the side opposite to the [O---SF2NR2] group and leaving of the

[O---SF2NR2] group. The second step is governed by steric hindrance and is the limiting step.

Since most hydroxyl groups in GO belong to tertiary alcohols according to the

Lerf-Klinowski model,33 the second step in the reaction scheme shown above is unlikely to

happen due to big steric hindrance, thus making the fluorination of hydroxyl groups by DAST

fail. However, leaving of the [O---SF2NR2] group can happen if enough energy is provided,

e.g. elevating reaction temperature. As a result, elimination of hydroxyl groups without

incorporating fluorine into the graphene structure takes place at high temperature (room

temperature and 50 °C). The fluorine in all rGO samples is likely to be mostly in the form of

CF2 considering that the fluorination of carbonyl groups with DAST is easy to happen at 0 °C,

room temperature and 50 °C.69 The fluorine content (~3.5 at%) is low since the amount of

carbonyl groups in GO available for fluorination is very small according to the

Lerf-Klinowski model. Due to the low reactivity of DAST towards epoxide groups,70,106 these

groups cannot be effectively removed even at 50 °C and account for the residual oxygen in

rGO (50 °C). Based on the discussions above, a schematic of the reduction of graphene oxide

by DAST is shown in Fig. 43.

66
 Fig. 43 Schematic of the reduction of GO by DAST.

4.4 Conclusions and Future Aspects

Special care should be given to that the hydroxyl groups in graphene oxide belong to

tertiary alcohols, and steric hindrance should be considered when performing chemical

modifications of graphene oxide. DAST is not effective for the fluorination of graphene oxide

due to steric hindrance. However, it is very effective for the reduction of graphene oxide to

make electrically conductive graphene. The C/O atomic ratio and conductivity of rGO by

DAST reduction is comparable to rGO by hydrazine reduction. However, currently the

method of using DAST for graphene oxide reduction is only limited to the surface of

graphene oxide films. A solution-phase homogenous reaction route is yet to be developed for

bulk synthesis of rGO.

67
 Chapter 5 – Biological Application of Graphene Oxide -

Tissue Engineering

Note: This chapter is mainly adapted from my two co-authored papers (ref. 110 and ref. 111).

This was a collaboration research with Prof. Ali Khademhosseini (Harvard-MIT Division of

Health Sciences and Technology), and my contributions in this collaboration were graphene

oxide synthesis, AFM, FTIR and Raman characterizations.

5.1 Introduction

5.1.1 Tissue Engineering

Fig. 44 Schematic showing the principle of tissue engineering.108

Tissue engineering involves the design and creation of functional substitutes for

damaged tissues and organs. In principle, cells from a tissue are isolated from a biopsy first,

cultured in a 2D environment for proliferation, then transferred to a 3D scaffold for tissue

development. After that the as-grown tissue is tested for biomedical applications. The

68
desirable properties of a scaffold include biocompatibility, high porosity and proper pore size

for accommodation of a large number of cells and transportation of nutrients and metabolites,

large surface area to volume ratio to interact with cells, mechanical integrity to support a great

many cells, surface properties that encourage cellular responses (adhesion, growth,

proliferation, etc.), and biodegradability for neo-tissue growth.109

5.1.2 Gelatin Methacrylate (GelMA) Hydrogel as a Scaffold for Tissue Engineering

Fig. 45 Schematic of the structure of gelatin methacrylate (GelMA).

Gelatin methacrylate (GelMA) is one of the most widely used scaffolds for tissue

engineering. Schematic of the structure of GelMA is shown in Fig. 45. GelMA is a polymer

with the gelatin backbone surface-modified with methacrylate groups. The gelatin in GelMA

is a denatured protein which has good binding with cells and affords good cellular responses.

GelMA can be cross-linked via vinyl groups to form a hydrogel under UV light irradiation

with an appropriate initiator.

5.2 Hybrid Hydrogel of GelMA and Graphene Oxide Through Non-Covalent

Interaction

69
 Graphene oxide (GO) was incorporated into GelMA through non-covalent interaction for

the creation of cell-laden GO-based hydrogels. Cellular responses in such a 3D hybrid

scaffold were investigated.

Fig. 46 (a) Optical images of GelMA and GO-GelMA hydrogels. (b-c) AFM images of GO and

GelMA coated GO. Insets show the height profiles along the white lines. (d) Fluorescence image

showing GO sheets coated with FITC-conjugated GelMA. (Adapted with permission from ref. 110.)

Optical images of as-prepared GelMA and GO-GelMA hydrogel pellets are shown in Fig.

46a. The homogeneous brown colour of GO-GelMA pellet indicated the homogeneous

dispersion of GO in the hybrid hydrogel. AFM characterization (Fig. 46b and Fig. 46c)

showed that uncoated GO had a thickness of 1.6 ± 0.1 nm indicating it was sub-bilayer since

single layer GO has a thickness of 0.8~1.2 nm. By contrast, GelMA coated GO was thicker

with a typical thickness of 3.9 ± 0.1 nm. Fluorescence image (Fig. 46d) of GO coated with

fluorescein-isothiocyanate-(FITC)-labeled GelMA showed planar structures with a

homogeneous green colour indicating the successful incorporation of GO into GelMA. The

size of such a sheet-like structure was >100 μm, which was much larger than that of a single

70
GO sheet (hundreds of nanometres to a few micrometres). This could be explained by that the

GelMA-coated GO sheets were cross-linked to form larger planar structures.

Fig. 47 Mechanical, porosity, and degradation characteristics of GelMA and GO-GelMA hydrogels.

(a) Compressive moduli, (b-c) SEM images of GelMA and GO-GelMA hydrogels before degradation.

(d) Degradation profiles of GelMA and GO-GelMA hydrogels when exposed to collagenase. (e-f)

SEM images of GelMA and GO-GelMA after degradation with collagenase for 24 h. In the inset of

(f), yellow arrow indicates a folded GO sheet. (Adapted with permission from ref. 110.)

The mechanical, porosity and degradation characteristics of GO-GelMA hydrogels are

shown in Fig. 47. The compressive modulus for 5% GelMA was ranging from 5 to 9 kPa,

while it had a wider range (4~24 kPa) for GO incorporated GelMA. Incorporation of GO into

GelMA did not change the favourable porous structure of GelMA as shown in the SEM

images of GelMA and GO-GelMA (Fig. 47b-c). Also incorporation of GO did not change the

degradation trend of GelMA hydrogel as shown in Fig. 47d. However, after 24 hours of

collagenase digestion, SEM characterization (Fig. 47e-f) showed that degraded GelMA and

GO-GelMA had different morphologies. Degraded GelMA still possessed an ordered structure

71
with increased pore size while degraded GO-GelMA had a collapsed and disordered structure.

The arrow in the inset of Fig. 47f points to a wrinkled sheet-like structure which is likely to be

remaining GO sheet.

Fig. 48 Raman spectra of GelMA and GO-GelMA before and after degradation.

(Adapted with permission from ref. 110.)

Raman characterization was carried out to investigate whether collagenase digestion

could cause structural changes to GO. As shown in Fig. 48, the Raman spectra of GO-GelMA

before and after degradation showed similar D band (1330 cm-1) and G band (1580 cm-1),

which are characteristic bands of GO. No obvious changes in the positions of D and G bands

or D to G ratio were observed. Therefore, collagenase digestion did not affect the structure of

GO. In other words, GO could not be degraded.

Maintaining normal cellular behaviour in a 3D microenvironment is an important

criterion for a scaffold in tissue engineering. Different cell-laden hydrogel microstructures

containing NIH-3T3 fibroblasts were fabricated using established microfabrication methods.

Fig. 49 shows the fluorescence images of cells in GelMA and GO-GelMA hydrogel

microarrays, star-shaped microstructure and microchannel. The fibroblasts encapsulated in

72
microarrays of GO-GelMA (Fig. 49b) and GelMA (Fig. 49c) hydrogel displayed similar

spreading pattern and morphology, and cells in star-shaped GO-GelMA hydrogel (Fig. 49d)

exhibited uniform elongation and spreading. These results demonstrated that GO is

biocompatible and that cells maintain normal behaviours in 3D GO-GelMA hydrogel

microenvironments.

d e

Fig. 49 Cellular behaviors of NIH-3T3 fibroblasts in 3D GelMA and GO-GelMA hydrogel

microenvironments. Fluorescence images of cells in GO-GelMA hydrogel (a-b) microarrays, (d)

star-shaped microstructure and (e) microchannel are shown. (Adapted with permission from ref.

110.)

Creating multi-layer constructs is important to mimicking stratified native tissues such as

skins and blood vessels. A Multi-layer structure could be fabricated using the method

illustrated in Fig. 50a. Fig. 50b-d show the white light and fluorescence images of as-prepared

different bilayer structures. The Live/Dead assay where the green colour represented live cells

73
and the red colour represented dead cells showed that the number of dead cells in the

GO-GelMA layer was less than that in the pure GelMA layer, which indicated the protection

role of GO. This might be explained by that GO could absorb free harmful radicals for cell

growth which were produced in the UV-light induced hydrogel formation process.

Fig. 50 (a) Fabrication and (b-d) characterization of multi-layer cell-laden microconstructs.

(Adapted with permission from ref. 110.)

In summary, tuneable mechanical strength without deterioration in the porosity and

degradation property were attained by incorporation of GO into the GelMA hydrogel.

Introducing the nanoscale planar structure of GO into the polymeric GelMA matrices could

encourage cellular responses such as adhesion, spreading, proliferation and so on due to the

74
strong interactions between cells and nanomaterials. GO could protect cells from free harmful

radicals produced in the UV-light induced hydrogel formation.

5.3 Incorporation of Graphene Oxide into GelMA Hydrogel through covalent

bonding

(a)

(b) (c)

Fig. 51 (a) Schematic of the surface functionalization of graphene oxide (GO) with methacrylate

groups via silanization to prepare methacrylic graphene oxide (MeGO). (b) Elastic moduli and

(c) ultimate stress values of GO-GelMA and MeGO-GelMA hydrogels. (Adapted with

permission from ref. 111.)

Graphene oxide was chemically modified with methacrylate groups first to prepare

methacrylic graphene oxide (MeGO) by treating GO with 3-(trimethoxysilyl)propyl

75
methacrylate through a silanization reaction (Fig. 51a). Then the MeGO-GelMA hydrogel was

fabricated by UV light irradiation with an appropriate initiator. Modification of GO with

methacrylate groups increased its solubility in 8 wt% GelMA. The maximum solubility of

MeGO in 8 wt% GelMA is 3.0 mg/mL compared with 0.8 mg/mL for GO.

The mechanical properties of GO-GelMA and MeGO-GelMA hydrogels were evaluated.

Elastic moduli (Fig. 51b) and ultimate stress values (Fig. 51c) of MeGO-GelMA hydrogel

were similar to those of GO-GelMA hydrogel up to 1.6 mg/mL. However, the elastic modulus

and ultimate stress of GO-GelMA hydrogel at 3.0 mg/mL decreased significantly compared

with MeGO-GelMA hydrogel. This was in agreement with that a large number of GO

agglomerates in the GelMA polymer solution at 3.0 mg/mL due to the limited solubility of

GO prevented proper hydrogel formation. As a result, these agglomerates within the

hydrogels acted as structural defects and led to deterioration in the mechanical strength.

In summary, covalent incorporation of GO into GelMA hydrogel were achieved by

chemical modification of GO with methacrylate groups. Covalent incorporation of GO into

GelMA hydrogel affords higher mechanical strength at high concentration of GO than the

non-covalent incorporation. Four times increase in elastic modulus and six times increase in

ultimate stress were seen for the covalent incorporation than the non-covalent incorporation.

5.4 Conclusions and Future Aspects

To conclude, as a two-dimensional flexible sheet-like macromolecule, graphene oxide

can be incorporated into GelMA hydrogel either by covalent or non-covalent methods to

76
manipulate the mechanical properties of the resulting hybrid hydrogel. Graphene

oxide-GelMA hybrid hydrogel preserves the favourable porosity of GelMA hydrogel and

exhibits enhanced cell proliferation due to the strong interactions between nanomaterials and

cells. Graphene oxide has been demonstrated to be biocompatible and capable of protecting

cells from harmful radicals produced in the process of UV-light induced hydrogel formation.

Covalent incorporation of graphene oxide into GelMA hydrogel affords higher mechanical

strength than the non-covalent incorporation at high concentration of GO.

Future aspects lie in incorporating conductive graphene instead of insulating graphene

oxide into hydrogel for specific tissue engineering applications. Both the high conductivity

and excellent mechanical strength of graphene can be exploited to make tissues which require

both of these two properties. Since the influences of nanodiamonds (zero-dimension), carbon

nanotubes (one-dimension) and graphene (two-dimension) on the hydrogel properties are not

the same with each of them has pros and cons, incorporation of multi-components of carbon

nanomaterials into hydrogels can also be a viable choice for making tissues with the desired

properties.

77
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Appendix

Publications

1. X. Gao and X. S. Tang. Effective reduction of graphene oxide by a fluorinating reagent:

diethylaminosulfur trifluoride (DAST). Carbon, 2013 (to be submitted).

2. S. R. Shin, B. A.-G.-Bolagh, T. T. Dang, S. N. Topkaya, X. Gao, S. Y. Yang, S. M.

Jung, J. H. Oh, M. R. Dokmeci, X. S. Tang and A. Khademhosseini. Cell-laden

microengineered and mechanically tunable hybrid hydrogels of gelatin and graphene

oxide. Adv. Mater., 2013 (in press).

3. C. Cha, S. R. Shin, X. Gao, N. Annabi, M. R. Dokmeci, X. S. Tang and A.

Khademhosseini. Controlling mechanical properties of cell-laden hydrogel by covalent

incorporation of graphene. Small, 2013 (just accepted).

87