Carbon-Based Materials: Preparation, Functionalization and Applications

N° d’ordre: 41205 THESE DE DOCTORAT présentée à L’UNIVERSITE DE LILLE 1 Ecole Doctoral Régionale Sciences Pour l’Ingénieur Lille Nord-de-France

pour obtenir le grade de DOCTEUR EN SCIENCES

Dans la spécialité:

Micro et Nano Technologies, Acoustique et Télécommunications

par Qi WANG

CARBON-BASED MATERIALS: PREPARATION, FUNCTIONALIZATION AND APPLICATIONS

Directeur de thèse:

Dr. Rabah BOUKHERROUB

Co-directrice de thèse:

Prof. Sabine SZUNERITS

Soutenue le 5 Decembre 2013 devant le jury composé de :

Prof. Zineb MEKHALIF Rapporteur Université de Namur Dr. Claire-Marie PRADIER Rapporteur Université Pierre et Marie Curie Prof. Thierry DJENIZIAN Examinateur Aix-Marseille Université Prof. Tuami LASRI Examinateur Université Lille 1

ABSTRACT

Graphene and its derivatives have attracted tremendous research interest over the years due to their exceptional physical and chemical properties. For the integration of graphene into electrochemical devices, it is essential to have a simple, reproducible and controllable technique to produce high quality graphene sheets on large surfaces. In this respect, the use of chemically derived reduced graphene oxide (rGO) rather than CVD graphene is a promising approach. In this thesis, we have developed simple, environmentally friendly, and controllable approaches for the chemical reduction of graphene oxide to rGO and the simultaneous functionalization of the resulting rGO matrix with the used reducing agents. These techniques are based on the use of tyrosine, 4-aminophenyl boronic acid (APBA), alkynyl-modified dopamine, and diamond nanoparticles (ND) as reducing agents. The robustness of the developed derivatization schemes was evaluated by the post-functionalization of alkynyl- dopamine/rGO with thiolated molecules via a photochemical “click” reaction. The resulting rGO matrices were characterized by a variety of different techniques, including XPS, AFM, SEM, FTIR, Raman, UV-Vis, and electrochemical measurements. The rGO matrices, deposited on glassy carbon (GC) electrodes, have been further used for electrochemical based applications for nonenzymatic detection of hydrogen peroxide, glucose, and simultaneously L-dopa and carbidopa. Furthermore, rGO/NDs nanocomposites have been successfully used as electrode in supercapacitors and exhibited a specific capacitance of 186 F g-1 and excellent long term stability.

Key words: graphene oxide (GO), reduced graphene oxide (rGO), reduction, functionalization, electrochemical sensing, biosensing, supercapacitor.

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RÉSUMÉ

Le graphène et ses dérivées ont suscité un grand intérêt au fil des années en raison de leurs propriétés physiques et chimiques exceptionnelles. Pour l'intégration du graphène dans des dispositifs électrochimiques, il est essentiel d'avoir une technique simple, reproductible et contrôlable afin de produire des feuillets de graphène de bonne qualité sur des grandes surfaces. Dans cette optique, l'utilisation d'oxyde de graphène réduit chimiquement (rGO) plutôt que le graphène produit par la technique CVD représente une alternative très prometteuse. Dans cette thèse, nous avons développé différentes approches simples, respectueuses de l'environnement, et contrôlables pour la réduction chimique de l'oxyde de graphène en rGO ainsi que la fonctionnalisation simultanée de la matrice de rGO formée par les agents de réduction utilisés. Les divers agents réducteurs utilisés sont : la tyrosine, l'acide 4- aminophénylboronique (APBA), la dopamine portant une fonction alcyne, et nanoparticules de diamant (ND). La robustesse des techniques de réduction développées a été évaluée par la post-fonctionnalisation de la fonction alcyne du nanocomposite dopamine/rGO avec des molécules portant une fonction thiol en utilisant la réaction "click" photochimique. Les matrices de rGO ainsi préparées ont été caractérisées par différentes méthodes telles que : XPS, AFM, MEB, FTIR, Raman, UV-Vis et mesures électrochimiques. Les matrices de rGO, déposées sur des électrodes de carbone vitreux (GC), ont ensuite été utilisées pour des applications électrochimiques pour la détection du peroxyde d'hydrogène (en absence d’enzyme), du glucose, et de la L-dopa et carbidopa simultanément. Finalement, les nanocomposites de rGO/NDs ont été utilisés avec succès comme électrodes dans des supercondensateurs, et ont montré une capacité spécifique de 186 F g-1 et une excellente stabilité.

Mots clés: oxyde de graphène (GO), oxyde de graphène réduit (rGO), réduction, fonctionnalisation, détection électrochimique, biocapteurs, supercondensateur.

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ACKNOWLEDGEMENTS

This research work was carried out in the Nanobiointerfaces group at the Interdisciplinary Research Institute (IRI). I would like to express my deepest gratitude to all those people who have helped and encouraged me in the successful completion of my doctoral study. First and foremost, I would like to give my most sincere thanks to my supervisors, Dr. Rabah Boukherroub and Prof. Sabine Szunerits, for their guidance, kindness, stimulating discussion, continued advice permanent support over past three years, as well as the time and patience they spent in reading and correcting the manuscript of this dissertation. With their enormous help and supervisions, I have obtained a lot of precious research experience such as scientific thinking, knowledge in chemistry and materials, as well as laboratory techniques. It was truly a great pleasure and privilege to study under their mentorships. My special thanks go to Prof. Musen Li from Shandong University. Prof. Musen Li is such a virtuous, kind and generous mentor who provided me with guidance, encouragement and support for my study and my career plans. I respect and appreciate him very much. I would like to express my thanks to Prof. Zineb Mekhalif and Dr. Claire-Marie Pradier for accepting to be the reviewers of the manuscript of my thesis and the committee members of my defense. I’m so grateful for their time and precious opinions. I would like to sincerely thank Prof. Thierry Djenizian and Prof. Tuami Lasri for accepting to be the committee members of my defense. I truly appreciate your time and your helpful comments. Also I would like to thank Prof. Thierry Djenizian and Miss Nareerat Plylahan for their generous cooperation, helpful discussions and valuable suggestions. My heartfelt thanks go to all my colleagues for all the help, discussions, and sharing. To Alex, it’s such a pleasure to work with you in the same office for three years. Thanks for all those generous help and valuable advice. To Dr. Yannick Coffinier, thanks for all the kind help in IEMN. Also many thanks go to Guo hui, Palan, Nadia, Manu, Stefka, Magalie, Lionel, Nazek, Amer, Wang Qian… It is truly my honor to work with all of you. I would like also to thank my friends, Hong bin, Qi Haoling, Yang Dapeng, Gong Jinlin,

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ACKNOWLEDGEMENTS

Yang Chang, Zeng Tao, Peng Hui, Zhang Yao, Peng Zhaoxia, Wen Guoguang, Lin Jun, Wang Xiebin, Guo Xiaodong, Zhou Di, Wei Wei… for their friendship and big supports. In particular, I wish to thank the China Scholarship Council (CSC), which offered me the opportunity to pursue my PhD study in France. Finally, my parents are always there for me with their supports and unconditional love throughout, for which my mere expression of gratitude does never suffice.

WANG Qi Villeneuve d’ascq, France 16th October, 2013

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TABLE OF CONTENTS

ABSTRACT ...... I RÉSUMÉ ...... II ACKNOWLEDGEMENTS ...... III TABLE OF CONTENTS ...... i ACRONYM ...... vii OBJECTIVES...... - 1 - CHAPTER 1 INTRODUCTION ...... - 3 - 1.1. Introduction of Graphene ...... - 3 -

1.2. Properties of graphene ...... - 5 -

1.2.1. Surface properties ...... - 5 -

1.2.2. Electronic and electrochemical properties ...... - 6 -

1.2.3. Optical properties ...... - 8 -

1.2.4. Mechanical properties ...... - 9 -

1.2.5. Thermal properties ...... - 10 -

1.3. Synthesis of graphene ...... - 10 -

1.3.1. Chemical Vapor Deposition (CVD) ...... - 11 -

1.3.2. Epitaxial graphene growth on SiC ...... - 13 -

1.3.3. Mechanical exfoliation of graphite ...... - 14 -

1.3.4. Chemically converted graphene ...... - 15 -

1.3.4.1. Synthesis of graphene oxide sheets (GO) ...... - 16 - 1.3.4.2. Reduction of graphene oxide (GO) ...... - 17 - 1.3.5. Unzipping CNTs and other methods ...... - 18 -

1.3.6. Summary ...... - 19 -

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TABLE OF CONTENTS

1.4. Characterization of graphene ...... - 20 -

1.4.1. Microscopic techniques ...... - 21 -

1.4.2. Spectroscopic techniques ...... - 22 -

1.5. Functionalization of graphene ...... - 25 -

1.5.1. Structural advantages of graphene and GO for chemical functionalization ..- 25 -

1.5.2. Functionalization of graphene by covalent bonding ...... - 27 -

1.5.3. Functionalization of graphene by non-covalent bonding ...... - 32 -

1.5.4. Functionalization of graphene with nanoparticles ...... - 38 -

1.6. Applications of graphene and its derivatives ...... - 41 -

1.6.1. Graphene-based biosensors ...... - 41 -

1.6.1.1.Graphene-based electrochemical biosensors for small biomolecules………………………………………………..……………... - 42 - 1.6.1.2. Graphene-based enzymatic biosensors ...... - 44 - 1.6.1.3. Graphene-based nanoelectronic devices ...... - 45 - 1.6.2. Graphene as a supercapacitor electrode ...... - 48 -

1.7. Conclusion and future prospects ...... - 50 -

1.8. References ...... - 51 -

CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) ...... - 75 - 2.1. Graphene reduced by hydrazine ...... - 76 -

2.1.1. Structural characterization of GO and rGO by hydrazine hydrate ...... - 77 -

2.1.2. Surface analysis of GO and rGO by hydrazine hydrate ...... - 81 -

2.1.3. Electrical properties of GO and rGO by hydrazine hydrate ...... - 83 -

2.1.3.1. Resistance of GO and rGO ...... - 83 - 2.1.3.2. Electrochemical properties of rGO by hydrazine ...... - 84 - 2.2. Environmentally friendly reduction approaches of graphene oxide ...... - 86 -

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TABLE OF CONTENTS

2.2.1. Reduction and functionalization of GO by alkynyl-dopamine (rGO/ Alkynyl- dopamine) and post-functionalization ...... - 88 -

2.2.1.1. Formation of rGO/Alkynyl-dopamine ...... - 88 - 2.2.1.2. Post-functionalization of rGO/alkynyl-dopamine ...... - 92 - 2.2.2. Reduction of GO using tyrosine...... - 97 -

2.2.3. Preparation of rGO-Au NPs/Tyr ...... - 100 -

2.2.4. Reduction and functionalization of graphene oxide with aminophenylboronic acid for the formation of rGO/APBA ...... - 103 -

2.3. Diamond particles (NDs) for the redcution of GO and the formation of an rGO/NDs composite material ...... - 107 -

2.4. Conclusion ...... - 115 -

2.5. References ...... - 117 -

CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS ...... - 123 - 3.1. Voltammetric detection of L-dopa and carbidopa ...... - 124 -

3.1.1. Introduction ...... - 124 -

3.1.2. Electrochemical behavior of L-dopa and carbidopa on rGO modified GC electrode ...... - 125 -

3.1.3. Electrochemical quantification of L-dopa and carbidopa ...... - 127 -

3.1.4. Conclusion ...... - 130 -

3.2. Electrochemical detection of hydrogen peroxide (H2O2) using tyrosine reduced GO (rGO/Tyr) ...... - 131 -

3.2.1. Introduction ...... - 131 -

3.2.2. rGO/Tyrosine for nonenzymatic amperometric H2O2 detection ...... - 133 -

3.2.3. Conclusion ...... - 136 -

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TABLE OF CONTENTS

3.3. Electrochemical detection of sugars using 4-aminophenylboronic acid modified reduced graphene oxide (rGO/APBA) interfaces ...... - 136 -

3.3.1. Introduction ...... - 136 -

3.3.2. Electrochemical sugar sensing ...... - 137 -

3.3.3. Conclusion ...... - 141 -

3.4. Summary ...... - 142 -

3.5. References ...... - 143 -

CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES ...... - 151 - 4.1. Introduction of rGO based supercapacitors ...... - 155 -

4.2. Evaluation method of the supercapacitor behavior of rGO/NDs ...... - 156 -

4.3. Supercapacitor behavior of rGO/NDs ...... - 157 -

4.4. Conclusion ...... - 160 -

4.5. References ...... - 162 -

CHAPTER 5 CONCLUSION AND PERSPECTIVES ...... - 165 - APPENDIX ...... - 169 - EXPERIMENTAL PART ...... - 169 - 6.1. Materials ...... - 169 -

6.1.1. Chemicals ...... - 169 -

6.1.2. Synthesis of pro-2-yn-1-yl-5-((3,4-dihydroxyphenetyl)amino)-5-oxopentanoate (alkynyl-terminated dopamine) ...... - 170 -

6.2. Preparation of reduced graphene oxide (rGO) ...... - 170 -

6.2.1. Preparation of graphene oxide ...... - 170 -

6.2.2. Reduction of graphene oxide with hydrazine hydrate ...... - 171 -

6.2.3. Reduction of graphene oxide using tyrosine ...... - 171 -

6.2.4. Preparation of rGO/Tyr/Au NPs ...... - 171 -

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TABLE OF CONTENTS

6.2.5. Preparation of reduced graphene oxide (rGO) modified with 4- aminophenylboronic acid (rGO/APBA) ...... - 172 -

6.2.6. Preparation of graphene/alkynyl-terminated dopamine (rGO/alkynyl-dopamine) ...... - 172 -

6.2.7. Preparation of rGO/NDs nanocomposites...... - 172 -

6.3. Post-functionalization: Thiol-yne reaction on rGO/alkynyl-dopamine ...... - 173 -

6.4. Preparation of graphene coated GC electrodes ...... - 173 -

6.5. Determination of sugar content in apple juice (colorimetric approach) ...... - 174 -

6.6. Instrumentation ...... - 174 -

6.6.1. X-ray photoelectron spectroscopy ...... - 174 -

6.6.2. FTIR spectroscopy ...... - 175 -

6.6.3. Raman spectroscopy ...... - 175 -

6.6.4. UV-Vis measurements ...... - 175 -

6.6.5. Scanning electron microscopy (SEM) ...... - 176 -

6.6.6. Transmission electron microscopy (TEM) ...... - 176 -

6.6.7. Atomic force spectroscopy (AFM) ...... - 176 -

6.6.8. Zeta potential and size ...... - 176 -

6.6.9. Electrochemical measurements ...... - 177 -

PUBLICATIONS ...... - 179 -

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TABLE OF CONTENTS

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ACRONYMS

AA - Ascorbic acid

AFM - Atomic force microscope

APBA - 4-Aminophenyl boronic acid Au NPs - Gold nanoparticles BASD - Bead-assisted sonic disintegration process BDD - Boron-doped diamond BDD NWs - Boron-doped diamond nanowires CNTs - Carbon nanotubes CV - Cyclic voltammetry CVD - Chemical vapor deposition DA - Dopamine DET - Direct electron transfer DMF - Dimethylformamide DMSO - Dimethylsulfoxide DPV - Differential pulse voltammograms EDLC - Electrical double layer capacitor FGSs - Functionalized graphene sheets

FTIR - Fourier transform infrared spectroscopy GC - Glassy carbon GO - Graphene oxide GOD - Glucose oxidase GQDs - Graphene quantum dots HOPG - Highly oriented pyrolytic graphite ITO - Indium tin oxide HPLC - High performance liquid chromatography HS-Fc - 6-(ferrocenyl)-hexanethiol HS-PF - 1H,1H,2H,2H-perfluorodecanethiol

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ACRONYMS

LOD - Limit of detection MWCNT - Multiwalled carbon nanotube

NDs - Diamond nanoparticles NDs-OH - Hydroxylated-nanodiamond particles

NDs-NH2 - Amine-terminated nanodiamonds

NMP - N-Methyl-2-pyrrolidone PBS - Phosphate buffered saline PVDF - Polyvinylidene fluoride

rGO - Reduced graphene oxide SEM - Scanning electron microscopy SWCNT - Single-walled carbon nanotube TEM - Transmission electron microscopy TTF - Tetrathiafulvalene

Tyr - Tyrosine UA - Uric acid

UV-Vis - Ultraviolet-visible spectroscopy

XPS - X-ray photoelectron spectroscopy

XRD - X-ray diffraction

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OBJECTIVES

In recent years, the research on rGO composites has been popular and extensive. Apart from the excellent physicochemical properties of graphene itself (such as large specific surface area, ambipolar field effect, high electronic and thermal conductivity, strong mechanical strength, and so on), the presence of functional groups and structural defects (edge plane like-sites/defects) confers to rGO some unique properties over pristine graphene (such as the possession of active species, favorable electron transfer, and electrocatalytic activity), and enhances the competitive strength of graphene in many fields such as sensors and energy storage and conversion. In the course of the next decade, it is expected that there will be an endless stream of reports on new surface functionalization strategies as well as on mild and environmentally friendly reduction and exfoliation approaches. After a general overview on graphene and graphene-based nanocomposite, preparation, characterization, and their potential applications (Chapter 1), we will describe the preparation of rGO based nanocomposites from GO by novel chemical reduction and functionalization methods. One main goal was indeed to develop environmentally friendly approaches that could represent interesting alternatives to the reduction of GO with hydrazine. The second interest was the possibility to simultaneously reduce and functionalize GO in a one step process. Tyrosine, alkynyl-dopamine, 4-aminophenyl boronic acid, and diamond nanoparticles were investigated in this work for simultaneous reduction and functionalization of GO. Besides, we functionalized rGO with gold nanoparticles by using tyrosine as an effective dual reducing agent for both GO and gold salts. The robustness of the developed derivatization schemes was evaluated by the post-functionalization of alkynyl-dopamine/rGO with thiolated molecules via a photochemical “click” reaction (Chapter 2). rGO nanocomposites with unique features showed remarkable performances in electrochemical sensing and biosensing. We used rGO for electrochemical detection of two catecholamines: L-dopa and carbidopa and explored the nonenzymatic detection of H2O2 using rGO/Tyrosine modified glassy carbon electrodes. Furthermore, we investigated a novel strategy for the development of an enzyme-free sugar sensor based on the interaction of

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OBJECTIVES different sugars with aminophenylboronic acid modified rGO (Chapter 3). Furthermore, the intercalation of nanodiamonds particles and its oxidative strength towards the reduction of GO was investigated. The promising supercapacitance behavior of rGO/nanodiamond particles composites has been explored, which expands the potential applications in the field of supercapacitors (Chapter 4).

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CHAPTER 1 INTRODUCTION

CHAPTER 1

INTRODUCTION

Carbon-based materials play a significant role in today’s science and technology [1]. Carbon is a surprisingly versatile element, able to hybridize in three different states, sp1, sp2 and sp3. The changes in local bonding of carbon atoms account for the existence of extremely diverse allotropic phases, starting with the well-known allotropes of amorphous carbon [2, 3], diamond [4-7] and graphite [8-10], and continues on with discovery of new forms including fullerenes [8, 11, 12], graphene [13-18], and more complex structures such as carbon nanotubes [19-21] (Figure 1.1). Individual carbon materials offer a wide range of useful physical and chemical properties, and thus fuel an enormous research interest in applications with an interdisciplinary approach spanning from applied physics, materials science, biology, mechanics, electronics and engineering. Development of current materials advances in their applications and discovery of new forms of carbon are the themes addressed by the frontier research in these fields. In this thesis, one attractive carbon allotropes, graphene, has been investigated and its composite materials have been applied for biosensing and energy storage.

Figure 1.1: Structure of some representative carbon allotropes.

1.1. Introduction of Graphene Since the demonstration of the accessible isolation of graphene by mechanical exfoliation of graphite in 2004 by Geim, Novoselov and co-workers, tremendous attention has

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CHAPTER 1 INTRODUCTION been devoted to this material from both the scientific and engineering communities [22]. Graphene is the name given to a one-atom-thick planar sheet comprising a sp2 hybridized carbon network with a carbon-carbon distance of 1.42 Å and an interlayer spacing of 3.4 Å. Its honeycomb-like lattice is composed of two equivalent sub-lattices of carbon atoms bonded together with σ bonds, as shown in Figure 1.2 A [23]. Each carbon atom in the lattice has a π orbital that contributes to a delocalized network of electrons. Its extended two-dimensional (2D) honeycomb lattice is the basic building block of graphitic materials of all other dimensionalities (Figure 1.2 B). It can be stacked into 3D graphite, rolled into 1D nanotubes or 0D fullerenes [14].

(A) (B)

Figure 1.2: (A) Schematics of the crystal structure, Brillouin zone and dispersion spectrum of graphene [23]. (B) Graphene: the parent of all graphitic forms [14].

Unlike other carbon forms, graphene as a two-dimensional material makes itself the first example of such a thing in the real world. More than 70 years ago, Landau and Peierls argued that strictly 2D crystals were too thermodynamically unstable to exist [14, 24, 25]. The argument was then supported by Mermin and Wagner with their theory that the long-range interactions of crystal in d ≤ 2 dimensional systems would be undermined by the surface fluctuations [26]. But these verdicts didn’t stop the scientists from exploring the graphene layer. Wallace predicted the electronic structure of graphene in 1947 [10], and McClure deduced the corresponding wave function equation in 1956 [27]. The name “graphene” was

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CHAPTER 1 INTRODUCTION first mentioned in 1987 by Mouras and co-workers to describe the graphite layers that had various compounds inserted between them forming the so-called “Graphite Intercalation Compounds” or GIC’s [28]. A big breakthrough occurred in 2004, when A. K. Geim and K. S. Novoselov isolated single-layer graphene by micro-mechanical alleviation of graphite using scotch tape [22]. By virtue of this, they were awarded the Nobel Prize in Physics in 2010. In 2007, the studies of Meyer et al. by transmission electron microscopy (TEM) revealed that the suspended graphene sheets are not perfectly flat and exhibit intrinsic microscopic roughening and nano-scale fluctuations. And the fact that the microscopic roughness is reproducible for different positions on single layer graphene but becomes notably smaller for bilayer graphene and disappears for thicker membranes, proves that the corrugations are intrinsic to graphene membranes. In order to achieve the thermodynamic stability, monolayer graphene tends to decrease the surface energy and forms corrugations, which only requires out-of-plane deformations (below 1 nm) involving a significant elastic strain [29]. The “thinnest” known material graphene with a long range π-conjugation exhibits a high specific surface area [14, 24, 25], extraordinary electronic properties and electron transport capabilities [16, 30, 31], strong mechanical strength [32], unprecedented pliability and impermeability [32, 33], remarkable optical transparency, excellent thermal and electrical conductivities [34]. These features have made graphene and graphene derivatives ideal for diverse applications such as electronic devices [35], energy storage and conversion (supercapacitors [36, 37], batteries [38], fuel cells [39], solar cells [40]), sensors [41-44], and biomaterials [45, 46].

1.2. Properties of graphene 1.2.1. Surface properties The theoretical surface area of single-layer graphene is reported to be ~2630 m2 g-1, surpassing that of graphite (~10 m2 g-1), and two times larger than that of single-walled carbon nanotubes (SWCNTs) (~1315 m2 g-1) [16]. Surface areas of different few layers graphene samples have been measured by the Brunauer-Emmett-Teller (BET) method and are in the range of 270-1550 m2 g-1. Large surface area is an essential characteristic of an electrode material, particularly in sensing devices, and energy production and storage. For example, the

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CHAPTER 1 INTRODUCTION high surface area of electrically conductive graphene sheets can give rise to fast electron transfer and high densities of attached analyte molecules. This in turn can facilitate high sensitivity and device miniaturization. Moreover, high surface area provides an excellent platform for the chemical functionalization.

1.2.2. Electronic and electrochemical properties Isolated graphene crystallites exhibit exceptional electronic properties. The electrical conductivity of graphene has been calculated to be ~64 mS cm-1, which is approximately 60 times more than that of SWCNTs [47]. Its conductivity remains stable over a wide range of temperature and even in liquid-helium, a temperature which is essential for reliability within many applications [14]. It has been demonstrated that pristine graphene is a zero-gap 2D semi-metal with a small overlap between valence and conductance bands (Figure 1.3), and charge carriers move with little scattering under ambient conditions [22, 48, 49]. It reveals itself in a pronounced ambipolar electric field effect at room temperature with the concentration of charge carriers up to 1013 cm-2 and mobilities of ~10 000 cm2V-1s-1, when a gate voltage is applied [22].

Figure 1.3: Band structure of graphene. The conductance band touches the valence band at the K and K’ points [49].

Besides, suspended graphene shows an ultra-high low-temperature mobility approaching 200 000 cm2V-1s-1 for carrier densities below 5 × 109 cm-2 [50]. One of the most interesting aspects is its highly unusual nature of charge carriers, which behave as mass-less relativistic

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CHAPTER 1 INTRODUCTION particles (Dirac fermions) [16, 22]. This triggers the observation of the anomalous integer quantum Hall effect (QHE) at room temperature, with the effective speed of light as its Fermi

6 -1 velocity vF ≈ 10 ms [51]. Graphene has also received considerable interest from the electrochemical community, where graphene has been reported to be beneficial for various applications ranging from sensing [44, 52] to energy storage and generation [36, 53], and carbon-based molecular electronics [54]. Direct electron transfer (DET) is the fundamental process in electrochemical reactions. Study of electrochemical responses of graphene towards different redox systems requires knowledge of the electrochemical aspects of graphene. Key insights into the electrochemical reactivity of pristine graphene have been provided by Brownson et al. [55]. Surprisingly, pristine graphene was found to be not as beneficial for electrochemical experiments as believed. High quality graphene, with low density of defects across the basal plane surface of the graphene sheet and low oxygen content, exhibits in fact slow electron transfer kinetics. A large proportion of edge plane-like sites/defects across the basal surface are found to be favorable for DET [56]. It has been proposed that the presence of oxygen containing groups at the edges or surface of graphene sheets can greatly influence the electrochemical performance of graphene in terms of heterogeneous electron transfer [53]. Thus, it becomes popular to explore the electrochemical properties of chemically converted (reduced or functionalized) graphene oxide (rGO) at electrode surface. Due to its favorable electron mobility and unique surface, chemically derived rGO can facilitate the electron transfer (ET) and accommodate the active

3+/2+ 3- species at electrode surfaces [41, 57]. Several redox species such as Ru(NH3)6 , Fe(CN)6 /4-, Fe3+/2+ and dopamine are used to probe the electrochemical properties of reduced graphene oxide films (rGSFs) by using cyclic voltammetry technique (Figure 1.4). The rGSFs demonstrate fast electron-transfer kinetics and possess excellent electrocatalytic activity towards oxygen reduction and certain biomolecules [58]. As an example of accommodating the active species, Zuo et al. [59] demonstrated that rGO-based electrodes support the efficient electrical wiring of the redox centers of several heme-containing metalloproteins (cytochrome c, myoglobin, and horseradish peroxidase) to the electrode. In addition, it has been shown that rGO exhibits high electrochemical capacitance with excellent cycle

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CHAPTER 1 INTRODUCTION performance and hence has potential application in ultracapacitors [36, 37]. Shao et al. [57] reported that rGO displays much higher electrochemical capacitance and cycling durability than carbon nanotubes (CNTs). The specific capacitance was found to be ~165 and ~86 F/g for rGO and CNTs, respectively.

Figure 1.4: Cyclic voltammograms of four kinds of redox systems at unmodified GC (dashed 3+/2+ line) and rGSF/GC (solid line) electrodes deoxygenated with argon: a) 1.0 mM Ru(NH3)6 3-/4- 3+/2+ in 1M KCl, b) 1.0 mM Fe(CN)6 in 1M KCl, c) 1.0 mM Fe in 0.1M HClO4, d) 1 mM -1 dopamine in 1M HClO4. Data shown are for the second scan. Scan rate, 100mVs [58].

1.2.3. Optical properties Graphene's unique optical properties produce an unexpectedly high opacity for an atomic monolayer suspended in vacuum, with the measured white light absorbance of 2.3% and a negligible reflectance (<0.1%), and this absorbance increased linearly with the layer numbers from 1 to 5 (Figure 1.5) [48, 60]. In addition, the optical transition can be modified by changing the Fermi energy considerably through the electrical gating [61]. Another property of graphene is its photoluminescence (PL). It is possible to make graphene luminescent by

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CHAPTER 1 INTRODUCTION inducing a suitable band gap. Two routes have been proposed. The first method involves cutting graphene into nanoribbons and quantum dots. The second one is the physical or chemical treatment with different gases to reduce the connectivity of the π electron network [62, 63]. The exceptional electrical properties in conjunction with optical properties have fueled lot of interests in novel photonic and optoelectronics devices [64-66]. Numerous applications using graphene as a promising candidate have been suggested, including photodetectors, touch screens, light emitting devices, photovoltaics, transparent conductors, terahertz devices and optical limiters.

Figure 1.5: Looking through one-atom-thick crystal. (A) Photograph of a 50-µm aperture partially covered by graphene and its bilayer. The line scan profile shows the intensity of transmitted white light along the yellow line. (B) Transmittance spectrum of single-layer graphene (open circles). The red line is the transmittance T= (1+0.5πα)–2 expected for two- dimensional Dirac fermions, whereas the green curve takes into account a nonlinearity and triangular warping of graphene’s electronic spectrum. (Inset) Transmittance of white light as a function of the number of graphene layers (squares) [60].

1.2.4. Mechanical properties Graphene has been reported to be the strongest material by testing the intrinsic breaking strength and elastic properties of free-standing monolayer graphene membranes by nanoindentation in an atomic force microscope (AFM) [32]. Measurements have shown that graphene has a breaking strength of 42N m-1 over 100 times greater than a hypothetical steel film of the same thickness [67], with a tensile modulus (stiffness) of 1 TPa (150,000,000 psi).

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CHAPTER 1 INTRODUCTION

The strain on graphene may change electronic band structure, which indicates that the energy band gap can be tuned by introduction of controlled strain [68]. The elastic deformation of functionalized graphene sheets (FGSs), or chemically reduced graphene oxide has also been studied by AFM. After repeatedly folding and unfolding of the FGSs multiple times, the folding lines were found to appear at the same locations, which can be attributed to the pre-existing kinks or defect lines in the FGSs [69].

1.2.5. Thermal properties Thermal management is one of the key factors for better performance and reliability of electronic devices. Considerable amount of heat generated during the device operation needs to be dissipated. Carbon allotropes such as graphite, diamond, and carbon nanotubes have shown higher thermal conductivity due to strong C-C covalent bonds and phonon scattering [70]. Earlier, carbon nanotubes are known for the highest thermal conductivity with room temperature values ~3000 W/mK for MWCNT [71] and 3500 W/mK for single walled CNT [72]. However, a large thermal contact resistance is the main issue with CNTs based semiconductors. Recently, the highest room temperature thermal conductivity up to 5000 W/mK for single-layer suspended graphene (defect free) has been reported [34], whereas the thermal conductivity decreases to ~600 W/mK when graphene is supported on amorphous silica, a case similar to practical application. This reduction is attributed to the leaking of phonons across the graphene–silica interface and strong interface-scattering [73]. Nevertheless, this value is still about 2 times and 50 times higher than copper and silicon, respectively, which are widely used in the electronics today [48].

1.3. Synthesis of graphene Till now, tremendous efforts have been made to develop synthetic methods for graphene and its derivatives, not only to achieve high yield of production, but also for easy processing of the material [48]. Like carbon nanotubes and other nanomaterials, the vital challenge in synthesis and processing of bulk-quantity of graphene sheets is aggregation. Unless well separated from each other, one-atom-thick planar sheet graphene tends to form irreversible agglomerates or even restack to form graphite through Van der Waals interactions. The

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CHAPTER 1 INTRODUCTION prevention of aggregation is essential for graphene sheets because most of their unique properties are only associated with individual sheets [70]. The number of synthetic routes can be generally classified as bottom-up and top-down approaches [48]. The bottom-up approach involves the direct synthesis of graphene from carbon sources using chemical vapor deposition (CVD) [74-76] or through graphitization of carbon-containing substrates (like SiC) [77], solvothermal reaction [78], and organic synthesis [47]. Different from bottom-up approaches, the top-down paths use graphite as starting material, and include mechanical exfoliation (the scotch tape method [22]) and liquid-phase exfoliation [79-84]. The latter features high yield, solution-based processability and ease of implementation. The liquid phase exfoliation has been achieved by means of intercalation, chemical functionalization, and/or sonication of bulk graphite [48].

1.3.1. Chemical Vapor Deposition (CVD) Chemical vapor deposition (CVD) is a well-established and inexpensive technique used to grow large-area, single and few-layer graphene sheets. Recently, Bae and coworkers reported a roll-to-roll production of 30-inch graphene films by using the CVD approach (Figure 1.6) [85].

Figure 1.6: (A) Schematic of the roll-based production of graphene films grown on a copper foil. The process includes adhesion of polymer supports, copper etching (rinsing) and dry transfer-printing on a target substrate. (B) Roll-to-roll transfer of graphene films from a thermal release tape to a PET film at 120°C. (C) A transparent ultralarge-area graphene film transferred on a 35 inch PET sheet. (D) An assembled graphene/PET touch panel showing outstanding flexibility [70, 85].

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CHAPTER 1 INTRODUCTION

The underlying principle of CVD is to decompose a carbon feedstock (usually a hydrocarbon) with the help of heat in order to provide a source of carbon. The carbon can then rearrange to form sp2 carbon species, which is commonly accomplished over a catalyst. In the case of graphene, the most successful catalysts thus far used are metals. However, unconventional routes using non-metallic substrates have also emerged [86]. Graphene CVD was first reported in around 2008 , using Ni [74, 87-89] and Cu [75] substrates as catalytic deposition surfaces, which was followed by a variety of transition metal substrates [76, 90-93]. The mechanism of graphene growth on substrates (Ni and Cu) was investigated by Li et al. [94] by using carbon isotope labeling (Figure1.7). It is reported that CVD growth of graphene on Ni occurs by a carbon segregation or precipitation process, whereas graphene on Cu grows by a surface adsorption process.

Figure 1.7: Schematic diagrams of the possible distribution of C isotopes in graphene films based on different growth mechanisms for sequential input of C isotopes. (a) Graphene with randomly mixed isotopes such as might occur from surface segregation and/or precipitation. The example (c) on Ni, (b) Graphene with separated isotopes such as might occur by surface adsorption. The example (d) on Cu [94].

The CVD process of substrates with medium-high carbon solubility (>0.1 atomic %) such as Ni and Co involves dissolving carbon and hydrocarbon decomposed onto the metal substrate, followed by carbon precipitation on the substrate by cooling down the metal [74, 89]. In contrast, the graphene growth on low carbon solubility (<0.001 atomic%) substrate

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CHAPTER 1 INTRODUCTION like Cu mainly happens on the surface through the following process [75, 94]: First, the catalytic decomposition of CH4 occurs on Cu to form CxHy upon exposure of Cu to CH4 and hydrogen. Then the nuclei start to form as a result of local supersaturation of CxHy and grow

12 13 to graphene islands until the graphene covers the full Cu surface. If CH4 and CH4 are both used as carbon source during the CVD process on Cu or Ni and are fed into the chamber sequentially, 12C- and 13C- are uniformly distributed on the Ni surface, while their spatial distribution on Cu follows the precursor time sequence. This suggests that graphene growth on Ni is through dissolution-precipitation mechanism, while graphene growth on Cu is a surface process [94]. CVD graphene growth on a wide variety of other transition metal surfaces even over dielectrics has been investigated. In particular, on Ru(0001), graphene grows epitaxially across the surface over large lateral distances [76]. The structure of graphene growth on Ir(111) exhibits a Moiré pattern due to the lattice mismatch between the Ir and graphene atomic distances [90]. The weak interaction between the metal substrate and the graphene for the Pt(111) system leads to the formation of many rotational domains [91]. Thermal CVD can also yield graphene over non-metallic catalysts. For example, sapphire was used as substrate with propane as the carbon feedstock at 1350-1650°C [95]. Moreover, to fabricate a graphene device with an atomically uniform gate dielectric providing a uniform electric field, plasma- enhanced CVD (PECVD) is a promising candidate. This technique is suitable for mass production of 2D graphene sheets, because of its simplicity (low-temperature manipulation) and compatibility with traditional semiconductor processes [96, 97].

1.3.2. Epitaxial graphene growth on SiC Silicon carbide is a common material used for high power electronics. It has been demonstrated that graphitic layers can be grown either on the silicon or carbon faces of a SiC wafer by sublimating Si atoms, thus leaving a graphitized surface [98]. Nowadays, hexagonal α-SiC (6H-SiC and 4H-SiC) is widely used to synthesize high-quality graphene with crystallites approaching hundreds of micrometers in size [99]. It has the advantage of being very clean, because the epitaxially matching support crystal provides the carbon itself and no metal is involved. There are potentially several ways to exploit the growth of graphene on SiC,

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CHAPTER 1 INTRODUCTION including the growth of thin SiC on Si, which although requires further development [100]. However, there are two major drawbacks of this approach: the high cost of the SiC wafers and the high temperature (over 1000ºC) required for the growth. This may make the use of graphene on SiC limited to some niche applications, such as high-frequency transistors [101] and devices in metrological resistance standards [102].

1.3.3. Mechanical exfoliation of graphite Graphite can be seen as stacked layers of many graphene sheets, bonded together by weak van der Waals forces. Thus, in principle, it is possible to produce graphene from a high purity graphite sheet, if these bonds can be broken. Mechanical exfoliation and cleavage of graphite is also known as the “scotch tape” or the “peel-off” process, which entails the repeated peeling of small islands of graphite (usually highly oriented pyrolytic graphite (HOPG) or natural graphite) using scotch tape. During the preparation of graphene, an adhesive surface of the tape with graphite crystal is rubbed against another adhesive surface. The number of layers forming a flake can be controlled to a limited degree through the number of repeated peeling steps. The obtained flakes can be released in acetone and transferred to different surfaces like Si wafers for further studies or device fabrication. This method allows the preparation of few layers of graphene up to 10µm in size. While the mechanical exfoliation of graphene used by Novoselov and Geim et al. [22] led to numerous exciting discoveries of graphene electronic and mechanical properties, such approach is limited by the low production of graphene samples. This approach can be mostly used only for fundamental studies. The graphene sheets contain impurities from the adhesive tape [86]. Mechanical exfoliation of graphite can also be performed in liquid-phase with the target of reducing the agglomeration in graphite appreciably. Ultrasonication treatments are usually used for the graphite exposed in solvents. There are two approaches of ultrasonic exfoliation of graphite. One approach is to utilize the similar surface energy of some organic solvents (such as dimethylformamide (DMF), N-Methyl-2-pyrrolidone (NMP) or ethanol [23, 79, 103]) and graphene that facilitates the exfoliation. This route results in a monolayer graphene yield of around 1 wt.%, but can be as high as 12 wt.% [79]. Another approach is based on the incorporation of small molecules between the layers of graphite or by non-covalently

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CHAPTER 1 INTRODUCTION attaching molecules or polymers onto the sheets, forcing the graphene layers to split apart from each other. This is often done by introducing ultrasonic solvents (including sulphuric acid, nitric acid, acetic acid and hydrogen peroxide [104-106]) between the graphene layers and then quickly heating the samples in an air or formic gas at ~1000°C or microwave irradiation. The rapid evaporation of the intercalators yields exfoliated graphite flakes, which can be then separated into single graphene sheets via ultrasonication. This route can produce a suspension of which 90% are single layer graphene sheets [105].

1.3.4. Chemically converted graphene At present, chemical conversion of graphite to graphene oxide has emerged to be one of the most developed methods to obtain higher yields of single-layered graphene. The technique consists of the initial oxidation of graphite to graphite oxide, followed by the subsequent mechanical/chemical or thermal exfoliation of graphite oxide to graphene oxide (GO) sheets, and their reduction to graphene [62, 107-111] (Figure 1.8).

Figure 1.8: The oxidation-exfoliation-reduction process used to generate individual sheets of chemical converted graphene from graphite [112].

This method is both versatile and scalable, offers the greatest ease for functionalization

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CHAPTER 1 INTRODUCTION and is adaptable to a wide variety of applications. We have adopted this technique throughout the thesis to produce various graphene sheets and explore their multifarious applications.

1.3.4.1. Synthesis of graphene oxide sheets (GO) Graphene oxide is the basic material for the preparation of individual graphene sheets in bulk quantities. Exfoliation of graphite oxide under vigorous conditions results in the production of graphene oxide (GO) sheets. The presence of oxygen-containing functional groups on GO sheets confers a hydrophilic character to GO, whereas graphene is a hydrophobic material. Therefore, GO can be easily produced and dispersed in aqueous media and in polar organic solvents. The starting material employed for the synthesis of GO is natural graphite. Natural graphite is first oxidized to graphite oxide. Then GO is obtained by exfoliation of aqueous suspension of graphite oxide via ultrasonication. The history of graphite oxide can be traced back to some of the earliest studies about the intercalation and exfoliation of graphite with sulfuric and nitric acids in 1840s [113, 114]. Then in 1859, graphite oxide was first prepared by Brodie by a repeated treatment of graphite with an oxidation mixture consisting of potassium chlorate (KClO3) and fuming nitric acid (HNO3) [115]. This method was modified by Staudenmaier in 1898 by adding KClO3 in multiple aliquots over the course of the reaction rather than in a single step addition as in Brodie’s experiment [116]. Another modification of Brodie’s method was reported by Jeong and co-workers, in which graphite was oxidized with a mixture of fuming HNO3 and sodium chloride oxide (NaClO2) at room temperature without subsequent aging typically used in the conventional ‘Brodie’s method [117]. In 1958, Hummers and Offeman described the most efficient method for the preparation of graphite oxide. In this method, graphite was oxidized with a mixture of sodium nitrate

(NaNO3) in concentrated sulphuric acid (H2SO4) and potassium permanganate (KMnO4) [118]. In current studies related to GO synthesis, the Hummers and Offeman method is improved or modified by getting rid of NaNO3 and simplifying the procedure [119]. Reaction between

KMnO4 and H2SO4 generates dimanganese heptoxide (Mn2O7) (Equations 7.1–7.2), which acts as an effective oxidizing agent for the oxidation of graphite to graphite oxide [109, 120].

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CHAPTER 1 INTRODUCTION

+ + + - KMnO4 + 3 H2SO4 → K + MnO3 + H3O + 3 HSO4 (Eq. 7.1)

+ - MnO3 + MnO4 → Mn2O7 (Eq. 7.2)

In the Hummers’ method, a treatment with hydrogen peroxide (H2O2) has often been performed to reduce the residual permanganate and manganese dioxide to colorless soluble manganese sulfate [118, 119]. Upon treatment with peroxide, the resulting suspension turned bright yellow. To reduce the residues, the product was then separated and washed several times to obtain a yellow-brown graphite oxide which is dispersed in water to prepare GO aqueous suspension with the aid of ultrasonication.

Oxidation of graphite can also be carried out by using benzoyl peroxide ([C6H5C(O)]2O2) at elevated temperature, which is an inexpensive and simple method [121]. However, this process is quite dangerous as peroxide may explode when heated in a closed container.

1.3.4.2. Reduction of graphene oxide (GO) The reduction process is among the most important reactions of graphene oxide (GO), to date, because of the similarities between reduced graphene oxide (rGO) and pristine graphene. Although GO itself is electrically insulating due to its disrupted sp2 bonding network, the graphitic π-network can be substantially restored by thermal annealing or through treatment with chemical reducing agents, a number of which have been explored [13, 62, 69, 107, 108, 110, 111]. Commonly used reducing agents include hydrazine monohydrate [13, 82, 111, 122], sodium borohydride [123, 124], hydroquinone [125, 126], strong alkaline solution [127], sulfur-containing compounds [128-131], amines [132, 133], and so on. Among the reducing agents described above, hydrazine monohydrate is the most widely used, mainly due to its strong reduction activity to eliminate most oxygen-containing functional groups of GO and its ability to yield stable rGO aqueous dispersions [82]. However, with hydrazine as the reducing agent, its residual trace may strongly decrease the performance of rGO-based devices. In addition, hydrazine is a highly toxic and potentially explosive chemical. To avoid using hydrazine, many environmentally friendly and highly-efficient reductants have been developed and used for the reduction of GO [134], including vitamin C

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CHAPTER 1 INTRODUCTION

[67, 135, 136], amino acid [67], reducing sugar [137], alcohols [138], hydroiodic acid [139], reducing metal powder [140, 141], sodium citrate [142], tea [143], lysozyme[144], dopamine [131, 145], and so on. For example, the reducing ability of L-ascorbic acid was found to be close to that of hydrazine [136]. The average conductivity of the rGO (reduced for 48 h using L-ascorbic acid) was 800 s m–1, comparable to the conductivity of GO reduced by using hydrazine, indicating that the electronic conjugation was re-established after reduction. Dextran [146] has been shown to be an efficient multifunctional reducing agent. More recently, Salas and co-workers reported on the reduction of GO via bacterial respiration [147]. Chemical reduction is certainly the most common method for reducing GO, but other methods have also been explored for the reduction of GO. Thermal reduction (solvothermal and hydrothermal) has been implemented in solution, where moderate temperatures (125– 300°C) are used to deoxygenate aqueous or organic GO dispersions [148-152]. Solvothermal reduction, with either direct or microwave-assisted heating, makes use of relatively ‘nontoxic’ organic solvents with high boiling points, such as N,N-dimethylformamide or N,N- dimethylacetamide. In the hydrothermal approach, the physicochemical properties of water can be tuned with temperature and pressure, offering a ‘greener’ chemistry alternative to organic solvents. A notable effect of this method is the structural damage caused to the platelets by the release of carbon dioxide, which can further affect the properties of the product, compared to a chemically-reduced sample. Electrochemical reduction has been successfully used to remove the oxygen functional groups of GO [57, 153-156]. This clean method has been investigated for the reduction of both GO dispersions and GO films pre-deposited onto the working electrode. The reduction process can be readily controlled by the reduction peak using cyclic voltammetry. However, this method mostly yields the final product as a solid film on the surface of the working electrode, which is less suitable for applications that require the manipulation and processing of well-dispersed materials. Other methods based on photoirradiation [157, 158], bacterial respiration [147], pulsed laser irradiation [159] and electron beam generated plasmas produced in methane/argon mixtures [160] were used to deoxygenate GO as well. 1.3.5. Unzipping CNTs and other methods

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CHAPTER 1 INTRODUCTION

A very recent method of graphene synthesis has used multi-walled carbon nanotubes (MWCNT) as the starting material [161]. The process is popularly known as ‘unzipping of CNTs’. Three groups, working independently on this issue, reported their findings in almost the same time period. In the earliest of them, it was claimed that MWCNTs can be opened up longitudinally by using intercalation of Li and ammonia, followed by exfoliation in acid and abrupt heating [162]. The product, among nanoribbons and partially opened MWCNTs, also contained graphene flakes. In another study [163], graphene nanoribbons were produced by plasma etching of MWCNTs, partially embedded in a polymer film. The etching treatment basically opened up the MWCNTs to form graphene. In a different approach [164], MWCNTs were unzipped by a multi-step chemical treatment, including exfoliation by concentrated H2SO4, KMnO4 and H2O2, stepwise oxidation using KMnO4 and finally reduction in NH4OH and hydrazine monohydrate (N2H4·H2O) solution. This new process route of unzipping MWCNTs to produce graphene creates possibilities of synthesizing graphene in a substrate-free manner.

Besides, new processing routes are constantly emerging [161]. Highly oriented pyrolytic graphite (HOPG) has been cleaved by using a micro-cantilever (such as an AFM tip) to form very thin graphitic sheets [165]. In another approach, conducting nanocarbon films (thickness ∼1 nm) and membranes were produced through a complex processing route based on molecular self-assembly, electron irradiation and pyrolysis [166, 167].

1.3.6. Summary Table 1.1 summarizes and compares the typical synthetic methods for graphene and its derivatives from the processing, yield, properties and applications [18]. The market of graphene applications is essentially driven by the progress in the production of graphene with appropriate properties for the next decade or at least until each of the graphene’s many potential applications meets its own requirements. In this thesis, the approaches to obtain various graphene sheets focus on producing chemically converted graphene through reduction and functionalization of GO. Table 1.1 Comparison of different synthesis methods for graphene

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CHAPTER 1 INTRODUCTION

Methods Conditions Yield and properties Applications Ref.

Top-down approaches

Research, “Scotch-tape” method Sample size: 10 µm in size. Low yield. [22] prototyping

Liquid exfoliation with organic Monolayer graphene yield 7-12 wt.%. Film Mechanical Coating, [79] solvent (NMP, DMF, etc.) conductivity: ~6500 Sm-1 exfoliation composites, inks, Single-layer yield: 90% after purification. Size: Liqud incorporation with transparent [104- ~250nm. Resistance of single sheet with 100 H2SO4, HNO3, H2O2, etc. conductive layers 106] nm in width: 10-20kΩ

Folded graphene structures. Incomplete [13, Reduction agent: hydrazine removal of oxygen-containing groups. Sheet 82] -1 resistance of graphene paper: ~7200 Sm Coatings,

Reduction agent: Vitamin C Film conductivity: ~7700 Sm-1 paint/ink, [135] Chemically Reduction via bacteria composites, converted Sheet resistance decreased to 104 Sm-1 [147] respiration transparent graphene Microwave-assisted reduction Formation of 1-8 layers of rGO with size up to conductive layers, from GO [168] with hydrazine a few micrometers energy storage,

Electrochemical reduction Sheet size: 500×700 nm bioapplications [57]

Thermal reduction of GO Sheet resistance: 6-8 kΩsq-1 [137]

photoirradiation Sheet size: 1µm. Sheet resistance: 9.5 kΩsq-1 [157]

Bottom-up approaches

Photonics, [74, nanoelectronics, 85, Substrate: Ni, Cu, Ru, etc. Sheet size of up to a few tens of micrometers. transparent CVD 88, 2 -1 1 Carbon sources: CH4, etc Charge carrier mobility: 10000 cm V s- conductive layers, 94, sensors, 169] bioapplications

High-frequency [77, Epitaxial Substrate: SiC Grain size: up to 50 µm long, 1 µm wide. transistors and 98, growth Temperature : 1280°C Charge carrier mobility: ~10000 cm2V-1s-1 other electronic 99] devices

1.4. Characterization of graphene

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CHAPTER 1 INTRODUCTION

With new access to 2D crystallites, scientists are eager to confirm results long predicted by theory. Thus, characterization of graphene forms an important body of graphene research. For practical applications and quality control, it is important to identify the number of layers, and the structure, especially for the purity of sample in terms of absence or presence of defects, which can affect the electrical properties. Many approaches from subjective microscopic observations to accurate spectroscopic analysis offer their own advantages. And multiple complementary approaches are often used together to achieve robust and verifiable characterization [170].

1.4.1. Microscopic techniques Among the microscopic techniques, atomic force microscopy (AFM) is one of the foremost methods used in definite identification of single-layer graphene. It has been reported in the literature that the thickness of single-layer graphene is in the range of 0.34-1.2 nm [82, 136, 171]. Transmission electron microscopy (TEM) can also accurately identify the thickness of a graphene sheet. As reported by Hernandez et al. [79], the appearance of stable and transparent graphene sheets in the TEM analysis indicates the presence of single-layer graphene, and the edges of the suspended film always fold back, allowing for a cross-sectional view of the film. With high resolution TEM (HRTEM), the investigation of the edges provides a precise way to measure the number of layers at multiple locations on the film [89]. Additionally, TEM is a suitable tool to explore the composites of graphene/nanoparticles [172-175]. Scanning electron microscopy (SEM) is commonly used as well to observe the morphology of a graphene sheet; the reflected light contrast generated can be also used to determine the number of layers due to the corresponding secondary electron intensities [176, 177]. Figure 1.9 shows microscopic observations of graphene using AFM, TEM and SEM. Besides, scanning tunneling microscopy (STM) has been used to scan the electronic topography of graphene [178].

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CHAPTER 1 INTRODUCTION

Figure 1.9: (A) AFM image (a) and the thickness (b) of a monolayer graphene sheet deposited on mica [179]. (B) TEM image (c) of graphene nanosheets, resembling crumpled silk. The featureless regions indicated by the arrows are monolayer graphene. High- magnification TEM of Co3O4/graphene composite (d) [172, 180]. (C) SEM image of a monolayer graphene sheet (e), and aggregated reduced GO sheets (f) [122, 181].

1.4.2. Spectroscopic techniques There are several spectroscopic techniques to analysis graphene quality, including Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (SSNMR), X-ray diffraction (XRD), and so on. Raman spectroscopy provides a quick and simple approach to determine the number of graphene layers, the stacking order, as well as the density of defects and impurities [182-185]. As shown in Figure 1.10 (A), the fingerprints of graphene under Raman spectroscopy are D, G and 2D (G’) bands at about 1350 cm-1, 1580 cm-1, and 2700 cm-1, respectively, with a smaller D’ band near 1620 cm-1 [183]. The G band is introduced by in-plane optical vibrations

2 of the sp -bonded carbon atoms (degenerate zone center E2g mode), whereas the 2D band arises from second-order zone boundary phonons. The D band is attributed to first-order zone boundary phonons, which is induced by defects in the graphene lattice (especially in chemically converted graphene), and is absent from defect-free graphene. The intensity ratio of the G and D band can be used to characterize the number of defects in a graphene sample.

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CHAPTER 1 INTRODUCTION

The line shape of the 2D peak, as well as its intensity relative to the G peak, can be used to characterize the number of layers of graphene. Single-layer graphene is characterized by a very sharp, symmetric, Lorentzian 2D peak with intensity greater than twice the G peak. As the number of layers increases the 2D peak becomes broader, less symmetric and decreases in intensity [182].

Figure 1.10: (A) Raman spectroscopy of pristine (upper left) and at an edge (lower left) of a monolayer graphene showing D, G, D’ and 2D (G’) peak heights and 2D peaks of 1 through 4 layers and bulk graphite (right), showing the broadened FWHM and multipeak fits for multilayers [170, 183]. (B) The C1s XPS spectra of GO and reduced GO by hydrazine [181].

For analyzing the structure of graphene, X-ray photoelectron spectroscopy (XPS) is a powerful surface analysis technique, which could reach core level electrons, and yields the composition and some bonding information from the binding energy, averaged over a large area [170]. This is useful for instance for doping [186, 187] or functionalizing graphene, or in determining the degree of reduction of GO [109, 111, 123, 181, 188]. In the case of GO and the reduced exfoliated GO [181], the C1s XPS spectrum of GO (Figure 1.10 (B)) clearly indicates a considerable degree of oxidation with four components that correspond to carbon atoms of different functional groups: the non-oxygenated ring C, the C in C–O bonds, the carbonyl C, and the carboxylate carbon (O–C=O). Although the C1s XPS spectrum of the reduced exfoliated GO also exhibits these same oxygen functionalities, their peak intensities are much smaller than those in GO, while the peak of C-C becomes predominant. Besides, for characterization of functional groups in chemically derived graphene, FTIR

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CHAPTER 1 INTRODUCTION spectroscopy and NMR are also recognized as useful tools. In the case of graphene oxide, FTIR spectrum (Figure 1.11 (A)) shows the presence of hydroxyl (broad peak at 3050-3800 cm-1), carbonyl (1750-1850 cm-1), carboxyl (1650-1750 cm-1), C=C (1500-1600 cm-1), and ether or epoxide (1000-1280 cm-1) groups [189-191]. Meanwhile, the 13C NMR spectrum of GO (Figure 1.11 (B)) exhibits three main peaks: the peak around 60 ppm assigned to carbon atoms bonding to the epoxy group, the peak around 70 ppm corresponding to the hydroxyl group connected to the carbon atoms, and the peak around 130 ppm ascribed to the graphitic sp2 carbon [192-194]. In addition, in the high-resolution 13C NMR spectrum, three small peaks are also found at about 101, 167 and 191 ppm, which are tentatively assigned to lactol, the ester carbonyl, and the ketone groups, respectively [192].

Figure 1.11: (A) FTIR spectra of GO and rGO/ZnS nanocomposite [195]. (B) Solid-state 13C magic-angle spinning (MAS) NMR spectra of GO [192].

Independently, UV-Vis spectroscopy (Figure 1.12 (A)) can provide a general idea about graphene formation and the number of layers [82, 196, 197]. The strictly two-dimensional graphene exhibits an absorption peak at around 262 nm, while a single-layer of GO shows absorption at around 228 nm in the UV-Vis spectrum. This is attributed to the π-π* transitions of aromatic C-C bonds. And the transparency of GO is much higher than that of graphene. This decrease in transmittance originates from the recovery of sp2 carbons from chemically derived graphene after reduction. This is an indication of the restoration of electronic conjugation in reduced graphene. In addition, the transparency of stacked graphene is much lower than that of the monolayer graphene. Sun et al. [198] have reported that monolayer

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CHAPTER 1 INTRODUCTION graphene shows transmittance of 97.1% at a wavelength of 550 nm, while for bilayer graphene is 94.3%, which shows linear enhancement in the ultraviolet absorption. Although XRD is not a perfect tool for identifying single-layer graphene, it is very informative. Pristine graphite exhibits a basal reflection (002) peak at 2θ = 26.6° (d spacing = 0.335 nm) in the XRD pattern. Upon oxidation of pristine graphite, the 002 reflection peak shifts to a lower angle at 2θ = 11.2° (d spacing = 0.79 nm). This increase in d spacing is due to the intercalation of water molecules and generation of oxygen functionality in the interlayer spacing of graphite. When graphite oxide is completely exfoliated to a single layer of GO, a straight line with no apparent diffraction peak in the XRD pattern is obtained (Figure 1.12 (B)). The XRD pattern of single-layer graphene is exactly the same to that of single-layer GO, indicating that the periodic structure of graphite has been eliminated and that it has been completely exfoliated into individual sheets [197, 199].

Figure 1.12: (A) UV-Vis absorption spectra of GO and rGO suspensions (left); UV–Vis absorption spectra of monolayer graphene and bilayer graphene (right) (peaks are labeled with the wavelength of maximum absorption and the value of maximum absorption). The UV transmittance (T, %) is measured at 550 nm [197, 198, 200]. (B) X-ray diffraction patterns of pristine graphite, graphite oxide, and graphene [199].

1.5. Functionalization of graphene 1.5.1. Structural advantages of graphene and GO for chemical functionalization Despite the great potential applications, it is worth mentioning that graphene itself possesses zero band gap as well as inertness to reaction, which weakens the competitive strength of graphene in the field of semiconductors and sensors [201]. Band gap opening of graphene by doping, intercalation, and striping would be useful for functional nanoelectronic

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CHAPTER 1 INTRODUCTION devices [202]. This is one of the reasons for the huge increase in the number of research projects aimed at functionalization of graphene, including reactions of graphene and its derivatives with organic and inorganic molecules, chemical modification of the large graphene surface, and the general description of various covalent and non-covalent interactions with graphene [63, 189, 203-205].

Figure 1.13: (A) Structural model of GO [192]. (B) Digital pictures of as-prepared graphene oxide dispersed in water and 13 organic solvents through bath ultrasonication (1 h). Top: dispersions immediately after sonication. Bottom: dispersions 3 weeks after sonication. The yellow colour of the o-xylene sample is due to the solvent itself [206].

Graphene oxide (GO), a single-layer of graphite oxide, has been considered widely as a precursor for synthesis and functionalization of the processable graphene via either chemical or thermal processes. With respect to its structure, there have been several models proposed over the years [109, 192]. One difference from an ideal graphene sheet, which consists of only trigonally bonded sp2 carbon atoms, is the fact that the GO sheet consists of a hexagonal ring- based carbon network having both (largely) sp2-hybridized carbon atoms and (partly) sp3- hybridized carbon bearing oxygen functional groups, mostly in the form of hydroxyl and epoxy groups on the basal plane, with smaller amounts of carboxyl, carbonyl, phenol, ketone, lactone, and quinone at the sheet edges (Figure 1.13 (A)) [65, 194].

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CHAPTER 1 INTRODUCTION

Enriched with oxygen-containing functional groups, GO shows a range of solubility in water and organic solvents as shown in Figure 1.13 (B) [206]. The presence of polar oxygenated (such as carbonyl and carboxyl) groups, located at the edge of the sheets, renders GO strongly hydrophilic and can swell and disperse easily in many solvents. The negatively charged oxygen species also help in addition to disperse graphene oxide as a single sheet by providing electrostatic repulsion and solvation [62, 109, 207]. This is important for processing and further derivatization. The resulting GO-stable dispersion can be subsequently deposited on various substrates by means of common methods such as drop-casting, spraying, spinning coating, or forming Langmuir-Blodgett films [84]. These can be used as excellent electrode materials. One drawback about GO is that the oxygen groups on the basal plane disrupts the π-conjugation, conferring insulating properties to GO. However, the conductivity of graphene can be restored through either the reduction of GO to remove several oxygen-containing groups, or the utilization of these oxygenated functional groups to serve as sites for chemical modification or functionalization of GO, which in turn can be employed to immobilize various electroactive species for the design of sensitive electrochemical systems. And sometimes the versatile reduction methods to generate rGO makes the functionalization happen simultaneously [130, 208]. Therefore, the chemical composition of GO, which can be chemically, thermally, or electrochemically engineered, allows the tunability of its physicochemical properties [65, 194, 209, 210]. The conjugated graphene sheet can be readily functionalized via covalent C-C coupling or non-covalent π-π stacking reactions and the immobilization of nanoparticles as well. These chemical functionalization strategies will be demonstrated in the following three sections.

1.5.2. Functionalization of graphene by covalent bonding The covalent functionalization of graphene and its derivatives can take place at the edges of the sheets and/or on the basal plane of graphene and is associated with re-hybridization of one or more sp2 carbon atoms of the carbon network into sp3 configuration, which accompanies a simultaneous loss of electronic conjugation [211]. According to Loh et al., the chemical reactivity of geometrically strained regions of graphene lattices is much higher in comparison with other regions [212], which is attributed to the easier displacement of electron

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CHAPTER 1 INTRODUCTION density. Indeed, it is the nature of graphene edges that determines the optical, magnetic, electrical and electrochemical properties of the material. Graphene edges can manifest either ‘zig-zag’ structure or ‘arm-chair’ configuration. Each carbon atom of the ‘zig-zag’ edges has an unpaired electron, which is active to combine with other reactants and is expected to display higher reactivity than ‘arm-chair’ edges [213]. The formation of aromatic sextets is perturbed in the zig-zag edges, leading to thermodynamic instability. The carbon atoms of the arm-chair edge side are more stable (less reactive) because of a triple covalent bond between the two open edge carbon atoms of each edge of the hexagonal ring [214, 215]. There is one challenge for covalent functionalization linked to the chemical inertness of the basal plane of graphene. The lack of chemical reactivity can be traced to its homogeneity and high delocalized electron structure [50]. Chemical reactions usually occur at locations having labile bonds, highly localized orbitals, dangling bonds (radicals) or localized charges, none of which are present in graphene planar composed of sp2 hybridized aromatic carbon with equilibrium geometry. The formation of a covalent bond on the basal plane of the graphene sheet necessitates the breaking of sp2 bonds and formation of sp3 bonds, which are tetrahedral in geometry [216] (Figure 1.14 (A)). This transformation creates a geometric distortion expending over multiple lattice positions. At the site adjacent to the point of covalent bonding, an unpaired electron is generated, enhancing the reactivity of the site leading to a chain reaction from the point of initial attack and an unzipping of the conjugated structure [212] [47].

Figure 1.14: (A) Typical lattice distortion resulting from a single covalent adsorbate on graphene. Atoms are colored according to their out of plane height to better visualize the surrounding distortion [217]. (B) Schematic structure of a double-sided decoration of graphene with functional groups (4-(2-(pyridine-4-yl)vinlyl)phenyl [212].

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CHAPTER 1 INTRODUCTION

A unique opportunity for organic reactions on graphene is the possibility of double-sided functionalization at exfoliated and well-separated graphene nanosheets (Figure 1.14 (B)). Figure 1.15 shows a selection of currently available covalent functionalization strategies of GO including reduction, acylation, esterification, amidation, nucleophilic substitution, electrophilic substitution and others. Among the chemical reactions, amidation [45, 218, 219] and esterification [220, 221] are the most common approaches used for linking molecular moieties onto oxygenated groups at the edges of GO. Haddon and co-workers were the first to show that the carboxylic acid groups of GO can be converted into acyl chlorides by the use of thionyl chloride (SOCl2) in N, N-dimethylformamide (DMF), playing the role of a catalyst in this reaction [222] (reaction III in Figure 1.15). Acyl chlorides are excellent leaving groups and can be used to prepare carboxylic acid derivatives, including acid anhydrides, esters and amides (reaction IV in Figure 1.15). In the work of Haddon’s group, the SOCl2-treated GO was further acylated with octadecylamine (ODA) and modified with long alkyl chains. The resulting GO product showed a solubility of 0.5 mg/mL in tetrahydrofuran (THF), carbon tetrachloride (CCl4) and 1,2-dichloroethane [222]. By the acylation method, the carboxylic groups can be linked to amine-functionalized molecules. Extending on this functionalization, Liu et al. have reported the first example of GO functionalized with amine-terminated poly

(ethylene glycol) (PEG-NH2) via carbodiimide-catalyzed amide bond formation. PEG is a biocompatible superhydrophilic polymer, thus GO-PEG conjugate is highly dispersible in water, as well as in several biological solutions such as serum or cell medium. This characteristic makes GO-PEG conjugate an important candidate for the high-efficient delivery in biological systems of hydrophobic drugs, such as camptothecin analogue named SN38, which can be immobilized on the surface of graphene via van der Waals interactions. The resulting GO-PEG-SN38 complex exhibited excellent water solubility while maintaining its high cancer cell killing potency similar to that of the free drug in organic solvents. The efficiency of GO-PEG-SN38 was far higher than that of irinotecan, an approved water-soluble SN38 prodrug used for the treatment of colon cancer [45].

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CHAPTER 1 INTRODUCTION

Figure 1.15: Covalent functionalization strategies of GO and graphene: I. reduction of GO into rGO, II. Functionalization of rGO via diazonium reaction, III. SOCl2 activation of carboxylic acid groups of GO into chloro acid groups, IV. Amination of the chloro acid groups of GO, V. Nucleophilic ring opening reaction of the epoxy groups with amine- terminated molecules, VI. Esterification reaction of the COOH groups of GO, VII. Reaction of the epoxy groups with sodium azide, VIII. Reduction of the azide function resulting in amino-functionalized GO, IX. ‘Click’ chemistry of the azide groups with molecules bearing an alkynyl group, X. Treatment with organic isocyanates leading to the derivatization of edge carboxyl and surface hydroxyl groups via the formation of amides and carbamate esters.

As an example of esterification of GO carboxyl functional groups (reaction VI in Figure 1.15), Xu et al. [221] have synthesized β-cyclodextrin functionalized GO with high bio- recognition capability by nucleophilic addition reaction between thionyl chloride modified

GO through SOCl2 and the hydroxyl groups of β-cyclodextrin (β-CD). The particular structures of β-CD suggest that this molecule should be able to accommodate small guest molecules (tetraphenyl porphyrin (TPP) in this particular case) that fit spatially within the cavities formed by the annular structure. The β-CD-modified GO with bound TPP showed excellent electrocatalytic activity in the reduction and oxidation of haemoglobin with a detection limit of 5 nM.

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CHAPTER 1 INTRODUCTION

GO contains some epoxy groups on its basal plane, which are prone to nucleophilic substitutions (reaction V in Figure 1.15). Thus through the ring-opening reaction between the epoxy groups of GO and amines (R-NH2), the surface of GO can be modified in situ with octadecylamine (ODA) in dichlorobenzene, giving black GO-ODA nanosheets nicely dispersed in organic solvents [223]. The reaction is not limited to primary aliphatic amines [126, 218, 223-229], but aromatic amines, amino acids and amine-terminated biomolecules have also been used as nucleophiles for the ring-opening reaction of epoxy-groups on graphene [126, 197, 224, 225, 227, 230, 231]. For small-chain primary amines (CnH2n+1NH2, n = 2, 4, 8, 12), grafting is completed at room temperature, while for long-chain aliphatic amines (CnH2n+1NH2, n = 18) heating at reflux for 24 h is required. All of these GO derivatives are more easily dispersed in organic solvents. X-ray diffraction (XRD) analysis showed that the interlayer distance of the amine-intercalated GO derivatives depends on the amine chain length and their orientation relative to the layers [225, 230]. According to Bourlino [225], the tilted orientation is more likely to be due to the hydrophilic nature of GO. In the case of amino acids, XRD suggested that the amino acid molecules adopt a flat orientation in the interlayer zone of GO. Another extension of this concept involves the stabilization of GO by amine-terminated ionic liquids (IL) [231]. The ionic-liquid functionalized GO could be dispersed easily in water, DMF and DMSO due to the enhanced solubility and electrostatic intersheet repulsion provided by the ionic liquid units. Stankovich et al. [18, 232] used a series of isocyanate (RNCO) compounds to modify GO (reaction X in Figure 1.15). They have demonstrated that such chemical treatment dramatically alters the exfoliation behavior of graphite oxide and allows for the complete exfoliation of GO into individual chemically derivatized graphene oxide to be achieved in organic solvents. The treatment of GO with organic isocyanates leads to the derivatization of both the edge carbonyl and the surface hydroxyl groups via formation of amides or carbamate esters (reaction X in Figure 1.15) [232]. The resulting isocyanate-modified GO can be readily dispersed in DMF and is very useful in the preparation of polymer composites [18]. Recently, Salvio and co-workers [233] developed an elegant method to transfer GO sheets onto silicon oxide surfaces. GO was first reacted with sodium azide (NaN3) and then further reduced with lithium aluminum hydride (LiAlH4) to form amino groups on both sides

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CHAPTER 1 INTRODUCTION of the GO nanosheets (reaction VII and VIII in Figure 1.15). These groups react through microcontact printing with an isothiocyanate monolayer on a silicon oxide substrate to form covalent bonds. The graphene sheets that are not covalently bound to the surface can be exfoliated by ultrasonication, leaving single-layer sheets attached to the substrate. During the process, the material with amino groups, after the reduction step with LiAlH4, is conductive with a resistivity approximately seven times larger than that of unprocessed graphite. This implies that after reduction of the GO, the conjugated sp2 network is largely restored.

Moreover, the azide-functional groups present on the GO-N3 composite can be used for a different kind of functionalization through Cu(I) catalyzed ‘click’ chemistry approach with alkynes (R- ≡) (reaction IX in Figure 1.15) [233, 234]. Besides, one popular electrophilic substitution reaction with graphene is the spontaneous grafting of aryldiazonium salts (reaction II in Figure 1.15) [235-239]. The addition of an aryl radical, the electrophile, to graphene leads to the creation of a carbon-carbon bond between the graphene and the aryl moiety, and consequently accompanied by the formation of a sp3 centre in the pile of sp2 hybridized carbons in the graphene honeycomb lattice. The unique characteristics of aryldiazonium salts are the very strong electron-withdrawing effect of the diazonium moiety and the high stability of dinitrogen as a leaving group. Both factors account for the relative ease of reducing aryldiazonium salts. The reduction of aryldiazonium salts proceeds through a concerted mechanism, in which electron transfer and dinitrogen loss are concomitant. This approach to link organic thin films onto graphene has been first shown by Tour and co-workers [238], where hydrazine-reduced and sodium dodecylbenzenesulfonate (SDBS) wrapped GO was treated with aryl diazonium salts (reaction I and II in Figure 1.15). The functionalized graphene is highly dispersible in DMF solvents.

1.5.3. Functionalization of graphene by non-covalent bonding Non-covalent functionalization is a well-known technique for the modification of carbon-based nanomaterials and has been extensively used for the modification of the sp2 network of carbon nanotubes [240]. Current research shows that the same chemistry can be applied for graphene oxide and graphene. Non-covalent interactions are primarily based on van der Waals forces and π-π stacking of aromatic molecules on the graphene plane [241, 242]

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CHAPTER 1 INTRODUCTION and/or by electrostatic interactions [243], requiring the physical adsorption of suitable molecules on the graphene surface. This functionalization is usually achieved by polymer wrapping, adsorption of surfactants or small aromatic molecules, and interaction with porphyrins or biomolecules such as deoxyribonucleic acid (DNA) and peptides [197]. The advantage of this surface functionalization strategy is that, unlike covalent functionalization, it does not disrupt the extended π-conjugation of GO and graphene, which creates defects on the graphene sheets. Numerous schemes have been reported by now to form such hybrid materials and some of them will be described in more detail below. Non-covalent modification of GO and graphene has been achieved using a variety of polymers such as poly(sodium-4-styrenesulfonate) (PSS) [18], sulfonated polyaniline (SPANI)

[244], poly(N-isopropylacryl-amide) (PNIPAM) [245], amine-terminated polystyrene (NH2- PS) and poly(methyl methacrylate) (PMMA) [243], or copolymers such as polypyrrole/poly(styrenesulfonic acid g-pyrrole) [246] and coil-rod-coil conjugated triblock copolymers composed of two hydrophilic polyethylene glycol coils and one lipophilic π- conjugated oligophenyl ethynylene [247]. Tailor-made nanocomposites exploiting the superlative properties of both components can show enhanced performance for various applications ranging from flexible packaging and printable electronics to thermoplastics. Bai et al. [244] reported that graphene functionalized by the conjugated polymer, sulfonated polyaniline (SPANI), exhibited excellent water dispersity (>1 mg/mL) and enhanced electrocatalytic properties for ascorbic acid. The interactions are mainly due to the strong π-π interaction between the backbones of SPANI and the graphene basal planes. Zhang et al. [246] showed that the modification of rGO with a conducting copolymer (polypyrrole/ poly(styrenesulfonic acid g-pyrrole)) resulted in a matrix with excellent electrocatalytic activity for hydrogen peroxide and uric acid. More recently, sodium dodecyl benzene sulfonate (SDBS) modified graphene has been prepared by Chang et al. [248] and Zeng et al. [249]. The SDBS-wrapped graphene has high transparency to visible light. This clearly shows the interest of graphene-polymer hybrids not only for electrical and electrochemical applications, but also as an interesting alterative to optical elements. Pyrene and its derivatives have also shown strong affinity to the basal plane of graphene via π-π stacking interactions and this approach has been applied to stabilize graphene in

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CHAPTER 1 INTRODUCTION aqueous solutions [250-254]. Chen et al. [253] proposed a novel optical biosensor to detect concanavaline A (ConA) using pyrene-conjugated maltose assembled graphene based on fluorescence resonance energy transfer (FRET). When pyrene-maltose was assembled onto the graphene surface by means of π-stacking interactions, its fluorescence was quenched thus the graphene acts as a ‘nano-quencher’ of the pyrene rings due to FRET. In the presence of ConA, competitive binding of ConA destroyed the π-stacking interaction between pyrene and graphene, thereby causing the fluorescence recovery (Figure 1.16).

Figure 1.16: (a) Self-assembled mannose-pyrene and fluorescence recovery on the graphene surface. a. Mannose-pyrene was quenched when assembled onto the graphene due to π-π stacking interaction; b. the fluorescence recovers while ConA combines with altose-pyrene, which causes the pyrene to migrate far away from the graphene surface. (b) Fluorescence spectra of maltose-pyrene (1 M) in the presence of various concentrations of graphene in 20 mM PBS buffer (pH 7). Inset: fluorescence intensity versus concentration of graphene; excitation: 340 nm, emission: 447 nm. (c) Fluorescence recovery by addition of different concentrations of ConA (0, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1, 5 µM) in 10 mM PBS, maltose-pyrene (1 µM), graphene 0.15 mg ml–1 [253].

In addition, Wang et al. modified hydrophobic CVD grown graphene with pyrene butanoic acid succidymidyl ester (PBASE) to improve the hydrophilic character of graphene, which then allowed the deposition of poly(3,4-ethylenedioxythiophene) (PEDOT), as anode for applications in photovoltaic devices. The noncovalent modification of graphene improved

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CHAPTER 1 INTRODUCTION the power conversion efficiency to 1.71%, which corresponds to 55.2% of the power conversion efficiency of a control device based on indium tin oxide (ITO), paving the way for the substitution of ITO in optical devices with low-cost graphene [255]. In principle, donor type and acceptor type aromatic molecules can be π-π stacked on the graphene surface, respectively, to tune the electron density of graphene. For example, a donor–acceptor complex can be created on graphene using pyrene-1-sulfonic acid sodium salt (PyS) as the electronic donor, and the disodium salt of 3,4,9,10-perylenetetracarboxylic diimide bisbenzenesulfonic acid (PDI) as the electronic acceptor [256]. Both PyS and PDI have large planar aromatic structures that strongly anchor them onto the hydrophobic surface of graphene sheets via π-π interactions. The negative charges in both molecules act as stabilizing species to maintain a strong static repulsion force between the negatively charged graphene sheets in solution, giving rise to high monolayer yield. Remarkably, the composite of PDI with graphene sheets resulted in a significant increase in conductivity for graphene– PDI (13.9 S cm-1) compared to pristine reduced graphene (3.0 S cm-1), whereas a 30% decrease in conductivity was observed for graphene-PyS (1.9 S cm-1). Thermal annealing resulted in a further reduction of the rGO sheets and the conductivity of both composites increased dramatically to >1100 S cm-1 at 1000°C, about twice as high as that of pristine graphene, indicating an improved π-conjugation upon ‘doping’ of graphene sheets with nongraphene molecules. The authors of [130, 208, 234] and others [145] have shown more recently that some aromatic molecules (e.g. dopamine, tetrathiafulvalene (TTF) and its derivatives) allow, in parallel to noncovalent functionalization of graphene via π-π stacking interaction, the reduction of GO to rGO. Indeed, in order to obtain functionalized graphene from GO, reduction is an essential step. The most commonly used reduction agents are hydrazine monohydrate and sodium borohydride [15, 16]. Hydrazine is hazardous to human health and environment and precautions must be taken when large quantities of this strong reducing agent are used. In order to overcome this limitation, milder and possible environmentally friendly methods for the efficient reduction of GO to rGO have been proposed including, in addition to others microwave induced reduction, photochemical reduction, solvent-assisted thermal reduction, and so on [83, 127, 138, 141, 152, 257-259]. Xu and co-workers have

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CHAPTER 1 INTRODUCTION reported on dopamine-induced reduction and subsequent functionalization of GO sheets (Figure 1.17) [145].

Figure 1.17: Schematic illustration of the preparation of polydopamine (PDA) capped rGO and subsequent reactions [145].

The reduction was carried out at pH 8.5 and elevated temperature, resulting in the reduction of GO to rGO and self-polymerization of the amine-groups of dopamine, which adsorb onto rGO by π-π stacking interaction. The polydopamine adlayer containing catechol groups is a versatile platform for further modification and functionalization of rGO with additional organic layers (Figure 1.17). At weak alkaline pH, the oxidized quinine in the form of catechol groups can react with thiolated and amine-terminated molecules (in this case polyethyleneglycol) via Michael addition and Michael addition/Schiff base reaction. Our group has demonstrated that dopamine polymerization can be prevented by performing the reaction at ambient temperature under sonication (Figure 1.18) [26]. The use of an azide-functionalized dopamine allows in addition the incorporation of azide function to the graphene/dopamine hybrid material, which can be further reacted in a copper(I)-catalyzed 1,3-dipolar cycloaddition to ‘click’ alkynyl-terminated molecules to the interface [260-263].

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CHAPTER 1 INTRODUCTION

Figure 1.18: Schematic illustration of the preparation of azido-dopamine capped graphene nanosheets and subsequent ‘click’ chemistry [234].

Next to dopamine, tetrathiafulvalene (TTF) has shown remarkable features when integrated into rGO. As with dopamine, the simple mixing of GO with TTF at neutral pH and room temperature results in the reduction of GO and the formation of rGO/TTF composite, easily dispersible in organic solvents [130, 264]. From cyclic voltammetric experiments, the amount of TTF units in the graphene matrix, drop casted onto a solid interface, was determined as Г=8.9 × 1014 molecules cm-2, being higher than TTF doped graphene (0.6 ×1012 molecules cm-2) [265] and in the same order as graphene sheets modified with nitrophenyl groups (10 ×1014 molecules cm-2) [235]. Thanks to multistable oxidation states of the TTF unit (TTF0, TTF•+, TTF2+), it can be expelled and reintegrated into the graphene network, forming one of the first reported graphene-based chemical switches (Figure 1.19). Azide-terminated TTF can be incorporated in a similar manner with the advantage that the azide groups can be further reacted with various molecules bearing alkynyl groups. Our group has successfully “clicked” mannose to this nanocomposite material. The TTF-mannose units could be released efficiently from the graphene matrix by chemical oxidation of TTF-

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CHAPTER 1 INTRODUCTION

2+ mannose surface units to TTF -mannose, using Fe(ClO4)3 or the electron-deficient tetracationic cyclophane cyclobis(paraquat-p-phenylene) (CBPQT4+) [264].

Figure 1.19: (a) Schematic illustration of the preparation of rGO/TTF nanocomposites and the chemical release and reintegration of TTF units. (b) Cyclic voltammograms of ITO interface modified with graphene/TTF nanosheets before (black), after chemical oxidation in

10 mM Fe(ClO4)3 (grey) and after immersion in 10 mM TTF (red); solution: 0.1 M KCl, scan rate 100 mV/s. (c) Change of anodic (black) and cathodic (grey) peak currents of graphene/TTF composite films deposited on ITO when immersed sequentially into 10 mM

Fe(ClO4)3 and 10 mM TTF solution. Each cycle consists of the following steps: (i) cyclic voltammetry measurement in 0.1 M KCl, scan rate 100 mV/s;(ii) keeping the electrode in

Fe(ClO4)3 aqueous solution for 30 min; (iii) rinsing with water, (iv) cyclic voltammetry measurement in 0.1 M KCl, scan rate 100 mV/s, (v) immersion of the electrode in an acetonitrile solution of 10 mM TTF for 30 min, (vi) rinsing with water and (vii) cyclic voltammetry measurement in 0.1 M KCl, scan rate 100 mV/s [130].

1.5.4. Functionalization of graphene with nanoparticles Composite materials derived from fine dispersions of metallic nanoparticles or oxides on carbon nanotubes have been extensively studied and applied in catalytic or optoelectronic applications, supercapacitors, fuel cells, batteries, etc. [266, 267]. Due to the increased interest

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CHAPTER 1 INTRODUCTION in graphene arising from its exceptional electrical and mechanical properties, the immobilization of nanoparticles on graphene sheets has become the focus of many researchers [201]. For GO-based nanocomposites, the unique properties of GO nanosheets make them particularly useful as the nanoparticle support. This is because the high surface area is essential for the dispersion of the nanoparticles in order to maintain their chemical activities. The GO-supporting materials not only maximize the availability of the nanosized surface area for electron transfer, but also provide better mass transport of the reactants to the chemically active sites. On the other hand, the functionalization of GO with nanoparticles has made the realization of nanoscale composite electrodes possible, and the nanocomposites present unusual advantages over macroelectrodes, including excellent catalytic activity, enhancement of mass transport, high effective surface area, and control over the electrode microenvironment. In addition, the combination of GO with nanoparticles may prove capable of contributing additional performance in some functional applications [194]. The various nanoparticles used to functionalize graphene are of different chemical nature, including noble metal nanoparticles [23, 63, 169, 268-272], metal oxide nanoparticles [75, 174, 273-281], quantum dots [81, 282, 283], and other nanoparticles [284-289]. These functional graphene nanocomposites can be prepared using different physical or chemical approaches: physical attachment approach [273], in situ chemical reduction process [23, 63, 268, 278], electrochemical synthetic processes [269, 275, 276], impregnation processes [169], self-assembled approach [279], ultrasonic spray pyrolysis [277], and so on. By means of these approaches, GO-based nanocomposites with noble metal nanoparticles (Pt [23, 63, 169, 268-272], Au [270, 290], Ru [271, 272]), and oxide nanoparticles (TiO2 [75, 273, 274], ZnO [275-277], SnO2 [174, 277-279], Cu2O [276], MnO2

[279], M3O4 [280], NiO [279], and SiO2 [279, 281]) have all been reported recently and used in a variety of applications ranging from catalytic systems to fuel cells, sensors, supercapacitors, and storage batteries. For example, Vinodgopal et al. [290] reported on the ultrasound-induced reduction of GO and HAuCl4 in 2% aqueous solution of poly(ethylene glycol) to synthesize Au-graphene nanocomposites. Both simultaneous and sequential reduction procedures can be adopted to obtain well-dispersed reduced Au NPs-rGO composites (Figure 1.20(A)).

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CHAPTER 1 INTRODUCTION

Figure 1.20: (A) TEM micrograph of the rGO-Au composite at low resolution on a holey carbon grid. (a) Sequentially reduced sample. (b) Simultaneously reduced sample [290]. (B)

Schematic illustration of the synthesis and the structure of SnO2/GNS [174].

The Raman spectra of the RGO-Au composites showed a distinct surface enhancement of the graphene Raman bands upon increasing the surface coverage of gold nanoparticles. Dong et al. [272] reported a simple approach for the deposition of Pt and Pt−Ru nanoparticles onto surfaces of GO nanosheets by ethylene glycol reduction. They systematically investigated the effect of GO as a catalyst support on the electrocatalytic activity of Pt and Pt−Ru nanoparticles for both methanol and ethanol oxidation used in fuel cell applications. In comparison to carbon black as a catalyst support, GO effectively enhanced the electrocatalytic activity of Pt and Pt−Ru nanoparticles for the oxidation of methanol and ethanol into CO2. By using an exfoliation-reassembling method, Paek et al. [174] prepared SnO2/GO nanoporous electrodes with three-dimensionally delaminated flexible structures. They were used for the enhancement of cyclic performance and lithium storage capacity. From their experimental results, it appears that the dispersed GO nanosheets in the ethylene glycol solution were reassembled and homogeneously distributed between the loosely packed SnO2 nanoparticles.

The resulting SnO2/GO nanoporous electrodes, which exhibited a reversible capacity of 810 mAh/g, were able to limit the volume expansion upon lithium insertion, and this resulted in superior cyclic performances (Figure 1.20(B)). In addition, quantum dots (QDs), which are attractive in biological labeling, solar cells,

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CHAPTER 1 INTRODUCTION and light-emitting devices [291-293], have also been used to functionalize GO [81, 282, 283]. GO nanoplatelets decorated with QDs do promote direct charge transport and efficient charge transfer to QDs, thus increasing their efficiency in solar cells and other devices. For instance, Chang et al. [282] have successfully prepared QDs-sensitized rGO nanocomposites by the in situ growth of QDs on non-covalently functionalized rGO. These QDs-sensitized rGO photoelectrodes were then used as an efficient platform for photoelectrochemical applications. Other nanoparticles, such as Prussian blue [284, 285], metal hydroxides [286, 287], and polyoxometalates [288, 289], have also been used in the functionalization of GO-based electrodes. For example, Chen et al. [287] reported a facile soft chemical approach for the fabrication of rGO−Co(OH)2 nanocomposites in a water−isopropanol system. They demonstrated that the electrochemical performance of Co(OH)2 was significantly improved after deposition on rGO sheets.

1.6. Applications of graphene and its derivatives Graphene has attracted tremendous research interest due to extensive applications in the field of nanoelectronics and transistors [70, 294-297], biosciences/biotechnologies (biosensors [41, 57, 298, 299], drug delivery [45, 46, 300], etc.), energy storage and conversion (supercapacitors [36, 37, 301-303], batteries [38, 304, 305], fuel cells [306-308], solar cells

[309, 310], H2 storage [311, 312]), and polymer nanocomposites [313-315]. However, in some cases, the use of pristine graphene is problematic due to its tendency toward aggregation and difficulty of processing [212]. Chemically converted graphene, on the other hand, has the advantage to overcome this problem, which is the enhanced processability afforded by the presence of functional groups on its surface. Thus the use of functionalized reduced graphene oxide (rGO) in the aforementioned areas has attracted extensive research interest. In the current section, we will highlight the applications of rGO nanocomposites as biosensors and supercapacitors.

1.6.1. Graphene-based biosensors Generally, biosensors comprise a selective interface in close proximity to or integrated with a transducer, which relays information about interactions between the surface of the

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CHAPTER 1 INTRODUCTION electrode and the analyte either directly or through a mediator (Figure 1.21 (A)) [316, 317]. Biosensors can be categorized depending on the transducing mechanism: (i) resonant biosensors, (ii) optical-detection biosensors, (iii) thermal-detection biosensors, (iv) ion- sensitive FET biosensors, and (v) electrochemical biosensors [318]. Among these, electrochemical biosensors possess advantages over the others, because their electrodes can sense materials present within the host without damaging the system [318].

Figure 1.21: (A) Schematic presentation of a biosensor [316]. (B) Schematic presentation of graphene based sensor [194].

The unique physicochemical properties of graphene have made it as a potential candidate for applications in biosensing. For instance, the high surface area of electrically conductive graphene sheets can give rise to high densities of attached analyte molecules. This in turn can facilitate high sensitivity and device miniaturization. The facile electron transfer between graphene and redox species opens up opportunities for sensing strategies based on direct electron transfer rather than mediation [194]. The proposed graphene-modified electrodes are expected to be useful in simple and effective electrochemical sensors and biofuel cells. Graphene-based biosensors (Figure 1.21 (B)) include electrochemical biosensors for small biomolecules [52, 246, 319-321], enzymatic biosensors [57, 171, 218, 249, 299, 316, 322- 328], and nanoelectronic devices [43, 52, 54, 329-335].

1.6.1.1. Graphene-based electrochemical biosensors for small biomolecules Graphene-based electrochemical sensors exhibit excellent electron transfer promoting ability and excellent catalytic behavior toward small biomolecules such as hydrogen peroxide

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CHAPTER 1 INTRODUCTION

(H2O2), dopamine (DA), ascorbic acid (AA), uric acid (UA) and some monosaccharides (fructose, glucose, mannose) etc.) [52, 246, 319-321].

H2O2 is not only a general enzymatic product of oxidases and a substrate of peroxidases, which is important in biological processes and biosensor development, but also is an essential mediator in food, pharmaceutical, clinical, industrial, and environmental analyses [52]. The key point in developing electrodes for detecting H2O2 is to decrease the oxidation/reduction overpotentials. Zhou et al. [52] studied the electrochemical behavior of H2O2 on rGO modified electrode, which shows a remarkable increase in electron transfer rate compared with graphite/GC and bare GC electrodes (Figure 1.22 (A)). These can be attributed to the high density of edge-plane-like defective sites on graphene, which might provide many active sites for electron transfer to biological species [336]. Zhang et al. [246] designed PSSA-g-

PPY functionalized graphene for the electrochemical detection of H2O2. The biosensor showed wide linear range, low detection limit, good reproducibility and long-term stability. The biosensor could successfully detect hypoxanthine in fish samples. In situ selective and quantitative extracellular detection of H2O2 was first realized by Guo et al. [319]. Dopamine (DA), an important neurotransmitter, plays a significant role in the central nervous, renal, hormonal, and cardiovascular systems [320], and its detection has gained significant attention. For a traditional solid electrode, DA and its coexisting species ascorbic acid (AA) and uric acid (UA) have an overlapping voltammetric response, resulting in rather poor selectivity and sensitivity of DA. Shang et al. [320] used multilayer graphene nanoflake films (MGNFs) for the selective determination of DA, AA and UA in 50 mM PBS solution containing 1 mM AA, 0.1 mM DA, 0.1 mM UA, and their ternary mixture. The MGNFs demonstrated good ET kinetics and allowed well-resolved simultaneous discrimination of DA, AA and UA at a detection limit of 0.17 μM (Figure 1.22 (B)). Zhou et al. [52] investigated the biosensing efficiency of rGO. The electroanalytical performances towards the detection of DA, AA and UA were much better compared to bare GC or graphite/GC electrode. Wang et al. [321] reported that rGO exhibited high selectivity for sensing dopamine with a linear range of 5μM – 200μM, and a better performance than multi-walled carbon nanotubes. They attributed it to the high conductivity, high surface area and π-π stacking interaction between dopamine and graphene surface.

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CHAPTER 1 INTRODUCTION

Figure 1.22: (A) Background-subtracted CVs (50 mV/s) on (a) graphene/GC, (b) graphite/GC, and (c) GC electrodes in 4 mM H2O2+0.1 M PBS (pH 7.0) [52]. (B) Cyclic voltammetry of (a) graphene nanoflake/GCE and (b) bare GCE in solutions containing 1mM AA, 0.1mM DA and 0.1mM UA [320].

1.6.1.2. Graphene-based enzymatic biosensors Direct electrochemistry of enzymes involves direct electron transfer (DET) between the electrode and the active center of the enzymes without the participation of mediators or other reagents [57, 337]. New mediator-free (or reagentless) biosensors, enzymatic bioreactors, and biomedical devices are designed to employ DET by immobilizing enzymes on conducting substrates. However, the redox centers of the biomolecules are usually embedded deep in their large three dimensional structures. Recent research has shown that graphene can enhance DET between enzymes and electrodes. Strong performances of rGO nanocomposites have been reported with low detection limits and high accuracies. Graphene-based enzymatic biosensors can detect glucose [171, 299, 322], horseradish peroxidase (HRP) [218, 249],

Cytochrome c (Cyt-c) [323, 324], β-nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) [57, 325], hemoglobin [326, 327], cholesterol [316, 328], etc. For example, in order to close monitor blood glucose levels in human body for the diagnosis of diabetes mellitus, various groups have shown that with glucose oxidase (GOD) as an enzyme model, graphene-based glucose biosensors exhibit good sensitivity, selectivity and reproducibility [171, 299, 322], on the basis of the high electrocatalytic activity of graphene toward H2O2 and the excellent performance for direct electrochemistry of GOD. Shan et al. [299] reported the first graphene-based glucose biosensor with graphene/ polyethylenimine/GOD-functionalized ionic liquid nanocomposites modified electrode which

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CHAPTER 1 INTRODUCTION exhibits wide linear glucose response (2 to 14 mM, R=0.994), good reproducibility (relative standard deviation of the current response to 6 mM glucose at -0.5 V was 3.2% for 10 successive measurements), and high stability (response current +4.9% after 1 week). Wu et al. [171] investigated GOD integrated rGO. The assembled GOD could electrocatalyze the reduction of dissolved oxygen and the detection of rGO-GOD towards glucose has a low detection limit (10 ± 2 μM). Graphene/metal nanoparticles (NPs) based biosensors have also been developed. GOD/rGO/Pt NPs/chitosan based nanocomposite film has been used for the detection of glucose [338]. Introduction of Pt NPs increased the sensor’s sensitivity, allowing a detection limit of 0.6 μM glucose. The biosensor possessed good reproducibility and long- term stability with negligible interference signals from AA and UA. Apart from GOD-based biosensors, similar sensitivities and stability improvements have been found for electrochemical biosensors based on other enzymes. Zeng et al. developed an

SDBS-rGO/HRP modified electrode for the detection of H2O2. It displayed high electrocatalytic activity to H2O2 with high sensitivity, wide linear range, low detection limit and fast amperometric response. The current signal was proportional to the concentration of

H2O2 closely related to the activity of the HRP. The HRP/rGO electrode remained stable after 30 measurements without significant loss of sensitivity (>90%) [249].

1.6.1.3. Graphene-based nanoelectronic devices Interest in graphene arises from its potential applications in nano-electronic devices, which include graphene-based fabrication for the detection of biomolecules [41, 52, 331, 339, 340], heavy metal ions [329, 330, 341, 342], and gas/water vapor [43, 54, 332-335]. Sensitive, selective, rapid, and cost-effective analysis of biomolecules is important in clinical diagnosis and treatment [316]. Zhou et al. [52] reported an electrochemical DNA sensor based on rGO. As shown in Figure 1.23, the current signals of the four free bases of DNA (i.e., guanine (G), adenine (A), thymine (T), and cytosine (C)) on the rGO/GC electrode are all separated efficiently, indicating that rGO can simultaneously detect four free bases, but neither graphite nor bare glassy carbon can. This is attributed to the antifouling properties and the high electron transfer kinetics for bases oxidation on rGO/GC electrode which results from high density of edge-plane-like defective sites and oxygen-containing functional groups

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CHAPTER 1 INTRODUCTION on rGO that provide many active sites. The lattter are beneficial for accelerating electron transfer between the electrode and species in solution.

Figure 1.23: Differential pulse voltammograms (DPV) for (A) at the GC electrode for G, A, T, and C, respectively; (B) at the graphite/GC electrode for G, A, T, and C, respectively; (C) at the rGO/GC electrode for G, A, T, and C, respectively; (D) for a mixture of G, A, T, and C at rGO/GC, graphite/GC, and GC electrodes; (E) for ssDNA at rGO/GC, graphite/GC, and GC electrodes; (F) for dsDNA at rGO/GC, graphite/GC, and GC electrodes. Concentrations for different species (A−F): G, A, T, C, ssDNA or dsDNA: 10 μg mL−1. Electrolyte: 0.1 M pH 7.0 PBS [52].

In addition, rGO/GC electrode is also able to efficiently separate all four DNA bases in both single-stranded DNA (ssDNA) and double-stranded DNA (ds-DNA), which are more difficult to oxidize than free bases, at physiological pH without the need of a pre-hydrolysis step, which allows to detect a single-nucleotide polymorphism (SNP) site for short oligomers with a particular sequence at the rGO/GC electrode without any hybridization or labeling processes. Besides, the research trend showed that the detection of bacteria by graphene based biosensor has attracted a significant interest to the researchers [331, 339, 343, 344]. Very recently, a facile, sensitive and reliable impedimetric immunosensor graphene electrode was developed for the selective detection of marine pathogenic SRB [331]. Metal ions, such as lead, cadmium, silver, mercury and arsenic have severe environmental and medical effects and so require careful monitoring. Therefore, the development of a sensitive and selective detection method would benefit to both the environmental and food chemists. Li et al. [329] reported that Nafion-graphene composite film based electrochemical sensors not only exhibits improved sensitivity for metal ion (Pb2+ and Cd2+) detection, but also alleviates the interferences due to the synergistic effect of graphene nanosheets and Nafion. Wen et al. [330] developed a fluorescence sensor of Ag+

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CHAPTER 1 INTRODUCTION ions based on target-induced conformational changes of a cytosine-rich oligonucleotide. Graphene also has promising application in gas sensors owing to its 2D structure with extremely high surface area. Gas sensing by graphene generally involves the adsorption and desorption of gaseous molecules (which act as electron donors or acceptors) on the graphene surface, leading to change in conductance in the graphene. The high sensitivity of graphene towards different gaseous molecules has led to its use in hydrogen (H2), carbon monoxide

(CO), ammonia (NH3), hydrogen sulfide (H2S), chlorine (Cl2), nitrogen dioxide (NO2), oxygen (O2), iodine vapor, and ethanol gas sensors [43, 54, 332-335]. In summary, Table 1.2 exhibits some typical graphene and its derivatives based biosensors as mentioned in this section. Table 1.2 Various types of graphene-based biosensors

Detected Sensor type Sensor materials Detection limit Ref. target

MnO2/rGO 0.8 μM [345] H2O2 Ag NPs/rGO 31.3 μM [346] rGO by hydrazine DA AA UA 0.17 μM DA [52] Electrochemical sensors - rGO by hydrazine DA [321] (linear range: 5μM-200μM)

Silanized graphene DA 0.01 μM [347]

GOD/rGO 10 ± 2 μM [171] GOD/rGO/PtNPs/chitosan Glucose 0.6 μM [338] GOD/ Ag NPs/rGO 180 μM [348] Enzymatic biosensors HRP /SDBS-rGO 100 nM [249] H2O2 HRP/Au NPs/rGO 1 μM [349] NADH/rGO NADH 0.1 μM [350] Epitaxial graphene 1 μM [351] Biomolecules rGO DNA 200 nM [352] sensors PANIw/Graphene 3.25×10-13 M [353] graphene Cd2+ 200 μg/L [354] Heavy metal ions rGO Ag+ 5 nM [330] sensors Nanoelectronic rGO Ag+ and Hg2+ 20 and 5.7 nM [355]

devices NO2 NH3 ＜5ppm (NO2 NH3) rGO by Hydrazine [356] DNT 28 ppb DNT

Mechanically exfoliated NH3, CO, Gas sensors 1ppb [54] graphene ethanol

Less than 0.1 Torr of H2O rGO H2O [357] vapor

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1.6.2. Graphene as a supercapacitor electrode A supercapacitor, also called ultracapacitor or electrochemical capacitor, is a type of electrochemical energy storage device for storing and releasing energy rapidly and reversibly [358]. In comparison to conventional batteries, a high performance supercapacitor has advantages of high energy density ((1-10 Whkg-1, determined by its capacitance and voltage), high power density (103-105 Whkg-1, determined by its voltage and internal resistance), fast charge propagation and charge-discharge processes (with seconds), long cycling life (>100 000 cycles), as well as low maintenance [308, 359]. Thus, supercapacitors act as perfect complements for batteries or fuel cells, and their cooperation is considered to be promising power supplies for versatile applications in portable electronics, memory back-up systems and environmentally friendly hybrid cars, where extremely fast charging is a valuable feature [56, 308, 359, 360]. Supercapacitors can be classified by their storage mechanisms into two groups: the formation of an electrical double layer at the interface of the electrode and the electrolyte (electrical double layer capacitor (EDLC), and the electrode through fast Faradaic redox reactions (pseudo-capacitor). In fact, these two storage mechanisms often occur simultaneously, and are inseparable in real systems. The key point to enhancing specific capacitance is to enlarge the specific surface area and control the pore size, layer stacking and the distribution of the electrode material. Graphene based materials have shown immense theoretical and practical advantages as supercapacitors, and thus have been widely studied. For investigating the capacitance behavior of graphene itself, Du et al. [361] mass- produced graphene nanosheets with a narrow mesopore distribution of ∼4 nm from natural graphite via oxidation and rapid heating processes, and found the GNSs to maintain a stable specific capacitance of 150 F/g under the specific current of 0.1 A/g for 500 cycles of charge/discharge. Wang et al. [303] reported that a single layered graphene (produced via GO) has a maximum specific capacitance of 205 F/g with a power density of 10 kW kg−1 at an energy density of 28.5 W h kg−1, and an excellent cycling ability with∼90% specific capacitance remaining after 1200 cycles. However, it is evident that the general specific capacitance of graphene is not as high as expected; the electrical double layer capacitance performance of graphene is limited by the aggregation and poor interaction between graphene and electrolyte [197, 308]. Thus it is

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CHAPTER 1 INTRODUCTION notable that many researchers have turned to the incorporation and fabrication of graphene based hybrid materials in the pursuit for improved capacitance performance. Wang et al. [362] reported a novel high performance electrode material based on fibrillar polyaniline (PANI) doped graphene oxide sheets with a mass ratio of PANI/graphene 100:1, which exhibited a high specific capacitance of 531 F/g , obtained by charge-discharge analysis. Further work by Yan et al. [363] reporting that a graphene/PANI composite (synthesized using in situ polymerization) could result in a high specific capacitance of 1046 F/g, as compared to 115 F/g for pure PANI, 463 F/g for SWCNT/PANI, and 500 F/g for MWCNT/PANI. Additionally, the energy density of the graphene/PANI composite could reach 39 Whkg−1 at a power density of 70 kWkg-1. It is apparent that graphene/PANI functionalization offers a highly conductive support material where the well-dispersed deposition of nanoscale PANI particles is attributable to the graphene nanosheets large surface area and flexibility.

Further work has indicated the potential of graphene as the counterparts in a capacitor material. Graphene–ZnO composite film prepared by Zhang et al. [364] exhibits enhanced capacitance behavior/values with better reversible charging/discharging ability by comparison to pure graphene and pure ZnO electrodes. One explanation for this, in addition to graphene’s excellent physicochemical properties, has been ascribed to the increase in lattice defect density exhibited by graphene (such as the fracture of graphene layers and the ratio of edge to basal carbon surface), where it is believed that increased edge plane increases the capacitance of a material [361]. However, Stoller et al. [36] found the weight specific capacitance of chemically modified graphene to be up to 135 F/g, stated that on chemically modified graphene electrodes the supercapacitor performance does not depend on a rigid porous structure to deliver its large surface area but on graphene’s flexibility, where graphene sheets adjust their position depending on the electrolyte used. Many reports indicated the importance of rational design and synthesis of graphene- based nanocomposites for high-performance supercapacitors. Wang et al. [286] have directly grown nickel (II) hydroxide nano-crystals on graphene sheets, finding that the resultant material exhibits a high specific capacitance of∼1335 F/g at a charge/discharge density of 2.8 A/g and ∼953 F/g at 45.7 A/g respectively. For comparison, a physical mixture of pre- synthesized nickel (II) hydroxide nanoplates and graphene exhibits lower specific capacitance, highlighting the importance of direct growth of these nanomaterials on graphene to enhance

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CHAPTER 1 INTRODUCTION the intimate interactions and charge transport between the active nanomaterials and the conducting underlying network [286].

1.7. Conclusion and future prospects In this chapter, the preparation, properties, functionalization and applications of graphene-based materials have been reviewed. It has become evident that the exceptional properties of graphene made it compelling for various engineering applications. Much effort has been devoted to exploring the fundamental physics and chemistry of graphene. However, graphene as a new material still faces many challenges ranging from synthesis and characterization to final device fabrication. The exceptional properties were observed in the defect-free pristine graphene prepared by graphite exfoliation using scotch tape method. However, this technique is not appropriate for any large scale device manufacturing. The chemical exfoliation of graphite into GO followed by thermal or chemical reduction, has offered a cost-effective production route of reduced graphene oxide on a large scale. However, the chemical or thermal modification lowers the electrical and mechanical properties, along with its chemical reactivity leading to lack of control in functionalization. The controlled oxidation/reduction and functionalization are very important in tuning the material properties such as band gap, electrical conductivity, and mechanical properties. Therefore, controlled modification of graphite, GO, and rGO is crucial in expanding the applications of graphene- based materials. In addition, the large surface area of low density GO and rGO in mass production may possess handling difficulty, which can lead to a health risk due to inhaling and handling toxic reducing chemicals. The health risk associated with graphene and their derivatives needs to be evaluated through the investigation of the toxicity and biocompatibility of these novel carbon structures and their derivatives.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

The top-down preparation of graphene using graphite as a starting material is currently considered one of the most cost-effective method for the synthesis of this two-dimensional sp2-bonded carbon material [1]. In a first step, exfoliated graphene oxide (GO) is obtained from graphite by the use of strong acids and ultrasonication. The graphitic nature of the resulting GO nanosheets is, however, highly compromised by the presence of a large amount of surface oxygen functionalities, resulting in a loss of conductivity. Reductive treatment by chemical [2] or thermal [3] means results in a partial recovery of the graphitic character by removing the majority of oxygen functionalities of GO and reestablishing to a certain extent the sp2 network. The product is thus referred to as chemically converted graphene or reduced graphene oxide (rGO). Hydrazine hydrate (H2N-NH2·H2O) has been found to be an efficient reducing agent in producing thin graphene-like sheets [2, 4]. However, due to its toxic nature, many environmentally friendly and high- efficient reductants have been developed and used for the reduction of GO, including vitamin C, amino acids, reducing sugars, alcohols, hydroiodic acid, reducing metal powder, sodium citrate, tea, lysozyme, dopamine, etc. [5]. Our group has recently reported on the possibility for simultaneous reduction and functionalization of GO using tetrathiafulvalene (TTF) [6, 7] and dopamine derivatives [8, 9]. To widen the scope of environmentally friendly reduction approaches of GO, four different strategies have been more deeply investigated in this thesis (Figure 2.1).

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

Figure 2.1: Schematic representation of the four approaches investigated in this work for the reduction and functionalization of GO.

Before discussing some of the developed approaches for the reduction of GO to rGO, a more detailed characterization of hydrazine reduced GO will be given, as this material is mostly used as a reference to other rGO nanosheets.

2.1. Graphene reduced by hydrazine An important property of graphene oxide (GO), brought by the presence of hydrophilic groups on its skeleton, is its easy exfoliation in aqueous media. Consequently, GO readily forms stable colloidal suspensions of thin sheets in water [10]. In this work, GO nanosheets were produced from natural graphite powder by an improved Hummer and Offeman method

(graphite, KMnO4, H2SO4/H3PO4, excluding the NaNO3) [11]. A homogeneous yellow brown suspension of GO sheets in water is achieved by ultrasonication for 3 h (Figure 2.2a). After reaction of GO with hydrazine hydrate for 24 h at 100°C, the yellow-brown color turned black and the reduced sheets aggregate and precipitate (Figure 2.2b). This precipitation is

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) presumably due to the removal of most of the polar oxygen-containing groups. The precipitate is separated by centrifugation and suspended in DMF to give a homogeneous black suspension (Figure 2.2c).

(a) (b) (c)

Figure 2.2: Photographs of an aqueous solution of GO before (a) and after reduction with hydrazine at 100°C for 24 h (b), reduced graphene (rGO) suspended in DMF (c).

2.1.1. Structural characterization of GO and rGO by hydrazine hydrate Oxidized and exfoliated from graphite, graphene oxide possesses various oxygen- containing functional groups, and its layered structure gets distorted by a large proportion of sp3 C-C bonds. The reduction process by hydrazine removes considerable oxygen atoms, reduces the sp3/sp2 C-C ratio, and restores much of the flatness and thus conductivity, although the final product is not the same as pristine graphene and still contains plenty of carbon-oxygen bonds. Stankovich et al. [2] proposed that hydrazine-based reduction is most effective on epoxide-functions present on GO (Figure 2.3) [12]. The carboxylic acid groups mostly present on the edges of GO remain intact during this process. X-ray photoelectron spectroscopy (XPS) was used to follow the chemical changes that occurred upon GO reduction using hydrazine. XPS survey spectrum of the as-prepared GO shows bands at around 285 and 530 eV due to C1s and O1s, respectively, in accordance with the chemical composition of GO. The C1s core level XPS spectrum of GO nanosheets is displayed in Figure 2.4A and can be deconvoluted into four components with binding energies at about 283.8, 284.7, 286.7 and 287.9 eV assigned to sp2-hybridized carbon, C-H/C- C, C-O and O-C=O species, respectively. The C/O ratio of GO is 1.73, comparable to reported data in the literature [13]. After hydrazine reduction, the C/O ratio increased to 6.8. This ratio is a relative value and as a comparasion to other GO and rGO matrices, and can not be used as

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) an absolute value. XPS analysis of the resulting precipitate indicates significant changes in the high resolution spectrum of C1s core level (Figure 2.4B). The C1s exhibits bands at 284.0 (sp2-hybridized carbon), 284.8 (C-H/C-C), 286.2 (C-O/C-N) and 289.9 eV (O-C=O). The intensity of the band at 284.0 eV increased significantly compared to GO, suggesting that the sp2 network has been restored during the process. The presence of C-N functions is due to nitrogen incorporation during the reduction process as reported by Stankovich et al. [2].

Figure 2.3: (a) Oxidation of graphite to graphene oxide (GO) and reduction to reduced graphene oxide (rGO). (b) A proposed reaction pathway for epoxy reduction by hydrazine [2].

Hydrazine reduced GO C1s

1. sp2 C 2. C-C/C-H 1 3. C-O/C-N 4. C=O 2 4 3 (A)

GO Intensity (a.u.) Intensity 3 1. sp2 C 2 2. C-C/C-H 3. C-O 4. C=O 4 1 (B)

296 292 288 284 280 Binding energy / eV Figure 2.4: High resolution XPS C1s core-level spectra of (A) graphene oxide (GO) and (B) hydrazine reduced graphene oxide (rGO).

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) The success of GO reduction was also examined by Fourier transform infrared (FTIR) spectroscopy. Figure 2.5 shows the FTIR transmission spectra of exfoliated GO before and after reduction with hydrazine.

(B)

(A)

Transmission (a.u.) Transmission

C-O

C=O

C-O

O-H C=C

1000 1500 2000 2500 3000 3500 -1 Wavenumber (cm ) Figure 2.5: Transmission FTIR spectra of GO before (A) and after (B) reduction with hydrazine at 100°C for 24 h.

The chemically exfoliated GO contains a variety of functional groups such as hydroxyl (C-OH), epoxide (C-O-C), carbonyl (C=O), and carboxyl (COOH) groups usually present at the defects and edges of the sheets. The spectrum of the exfoliated GO displays a broad and strong band at 3400 cm-1 assigned to the vibration of hydroxyl groups and/or adsorbed water molecules. Furthermore, bands due to C=O (-COOH) vibration, OH deformation, and C-O (alkoxy) and C-O (epoxy) stretching modes are visible at 1744, 1407, 1230 and 1087 cm-1, respectively. A band at 1626 cm-1 assigned to C=C stretching modes is also present in the FTIR spectrum of the initial GO. After reaction with hydrazine at 100°C for 24 h, most of the vibrations due to oxygen-related functional groups disappeared. The OH vibration at 3400 cm-2 becomes much smaller compared to GO. The FTIR spectrum is dominated by a broad band at 1550 cm-1 due to C=C stretching modes, suggesting that the aromatic network has been restored upon reaction with hydrazine. A broad band at 1188 cm-1 is also present and due to C-O vibrations from alkoxy groups remaining on the graphene sheets, which suggests that hydrazine took part in ring-opening reaction with epoxides [12], while the carboxylic acid

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) groups were unlikely to be reduced by hydrazine and therefore remained intact after the reduction process. The GO reduction was further confirmed by UV-Vis spectroscopy. Figure 2.6 displays UV-Vis absorption spectra of GO before and after reduction with hydrazine. The as-prepared GO displays two characteristics bands: a strong absorption band with a maximum at 228 nm and a shoulder at around 300 nm corresponding to π-π* transitions of aromatic C=C bond and n-π* transitions of C=O bond in GO, respectively. Reduction of GO resulted in a red shift of the main absorption band to 273 nm. In addition, the intensity of the absorption tail in the region at >300 nm has significantly increased. The result suggests that the GO nanosheets have been reduced and the electronic conjugation within the GO nanosheet was restored upon hydrazine reduction.

0.5 Graphene oxide 0.4 228 nm Reduced graphene oxide 0.3 273 nm

0.2 Absorbtance /a.u. Absorbtance 0.1

0 200 300 400 500 600 700 800 -1 Wavenumber / cm

Figure 2.6: UV-Vis spectra of an aqueous suspension of GO before (black) and after reduction with hydrazine at 100°C for 24 h (red).

The remarkable structural changes arising during the chemical processing from GO to reduced GO, are also reflected from their Raman spectra (Figure 2.7). As reported [14], the pristine graphite displays a prominent G peak at 1581 cm-1 as the only feature in its Raman spectrum. For GO, Figure 2.7A shows that the G band is shifted to 1592 cm-1, and the D band at 1360 cm-1 becomes prominent, indicating the reduction in size of the in-plane sp2 domains, possibly due to the extensive oxidation. The Raman spectrum of the reduced GO with hydrazine also contains both G and D bands at 1598 and 1360 cm-1, respectively (Figure

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) 2.7B). However, there is an increase of D/G ratio to 0.84 compared to that in GO (0.68). This change suggests a decrease in the average size of the sp2 domains upon reduction of the exfoliated GO [14], and can be explained if new graphitic domains were created that are smaller in size to the ones present in GO before reduction, but present in a higher density [2].

(B) Intensity (a.u.) Intensity (A)

1500 2000 2500 3000 -1 wavenumber / cm Figure 2.7: Raman spectra of GO before (A) and after (B) reduction with hydrazine at 100°C for 24 h.

2.1.2. Surface analysis of GO and rGO by hydrazine hydrate GO aqueous solution and reduced graphene/DMSO or DMF suspension can be deposited on various technologically relevant substrates by drop casting. The morphology of GO and rGO deposited by drop-casting onto silicon interfaces is displayed in Figure 2.8, which shows that the wrinkles and overlaps are the two fundamental morphologies between flexible, interacting graphene sheets which are usually convoluted in solution-processed thin films. From a cross-section SEM image the thickness of the rGO film deposited in this manner is estimated as 500±100 nm (Figure 2.8 C). Tapping mode AFM imaging [6] was also used to examine the microstructure of GO and rGO and to estimate the thicknesses of the nanosheets (Figure 2.9). From line profile measurements, a GO thickness of ~0.6-0.7 nm with a lateral dimension of about 300 nm was determined, matching well with reported apparent thicknesses for single-layered GO sheets [15]. After hydrazine reduction, the thickness decreased to 0.4 nm in accordance with the removal of most oxidized species from the surface [15].

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

(A) (B) (C)

5µm

Figure 2.8: Top view SEM images of GO (A) and rGO (B), and cross-section SEM image of rGO (C). GO and rGO are drop casted on silicon substrate.

4 nm 4 nm

0 nm 0 nm Figure 2.9: Tapping-mode AFM images of graphene oxide (GO) before (A) and after (B) reduction with hydrazine. GO and rGO are drop casted on mica.

The wettability was also investigated through contact angle measurement. The surface of a material is a very important part to determine the compatibility with its environment, especially in hybrid materials, coatings, as well as biological media, because wettability and the surface charge are essential for thrombosis formation, cell attachment, or cell proliferation [16]. As a crucial part to bridge the exceptional properties to numerous applications, the surface plays a vital role in graphene materials [17, 18]. GO and rGO were assembled into thin films by the filtration process on silcon and their wettability was evaluated through static contact angle measurements. Graphite displays a hydrophobic character with a water contact angle of 98±2°, because it consists of carbon atoms without any polarity [19]. GO film was exfoliated from oxidized graphite and exhibits a static contact angle of 23±2°, much less than 90°. Hence, GO shows a good hydrophilic property, due to the presence of a big amount of oxygen-containing functional groups on its surface, mostly in the form of hydroxyl and epoxy groups on the basal plane, with smaller amounts of hydroxyl, carboxyl, carbonyl, lactone,

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) phenol, and quinone at the sheet edges [20]. After chemical reduction of GO with hydrazine, epoxy groups have been eliminated. The resulting rGO film exhibits a good hydrophobic property with a contact angle of 110±2°. In addition, the wettability of organic solvents like DMF was also investigated. The static contact angles were <5° for GO and 18±2° for rGO, which makes rGO easily dispersible in DMF.

2.1.3. Electrical properties of GO and rGO by hydrazine hydrate Graphene is reported to have remarkable electrical properties [21-23]. One of the factors which make graphene so attractive is its low energy dynamics of electrons. This 2D crystal of sp2 hybridized carbon is a zero bandgap semiconductor which makes it a new contender for future electronic applications. Recently, it has also become popular to explore the electrochemical properties of graphene at electrode surfaces. Due to its favourable electron- transfer kinetics, and unique surface properties (such as high specific surface area), graphene can accommodate active species and facilitate their electron transfer (ET) at electrode surfaces, thus suggesting the potential applications of graphene sheets for constructing efficient biosensing, supercapatitor, energy-conversion, biomedical and other electronic systems [24-29].

2.1.3.1. Resistance of GO and rGO GO and rGO films were obtained by filtration of a dispersion through a PVDF membrane and dried under vacuum (Figure 2.10). The sheet resistances were performed by Hall effect measurement system using the four-probe method. The sheet resistance (Rs) value of graphene oxide film paper was found to be more than 1012 Ω/square, indicating that GO is a typical insulator, strongly correlated to the amount of sp3 bonding. After hydrazine reduction, the rGO film with a thickness of ~1.05 µm had a good conductivity and displayed Rs ~6768Ω/square. This resistance is much higher than the equivalent value calculated from the single or a few graphene sheets due to the non–uniformity of the graphene layers and the large interlayer resistance [30, 31]. Table 2.1 shows the sheet resistances of graphene based films fabricated by different approaches.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

(A) (C)

(B)

Figure 2.10: GO (A) and rGO (B) films on a PVDF membrane after filtration for the resistance measurements, and (C) cross-section SEM image of rGO with a thickness of 1.05 µm.

Table 2.1: Sheet resistances of graphene based thin films.

Sheet resistance Graphene film Ref. Ω/square Vacuum filtration of reduced graphene oxide suspension ~6768 our work Vacuum filtration of graphene oxide suspension, followed by 4.3 × 104 [32] reduction CVD grown graphene on Cu and roll-to-roll transfer (4 layers 30 [33] thick) Mechanical exfoliation of highly oriented pyrolytic graphite 400 [21] (thickness < 3nm) Spin coating of reduced graphene oxide 102–103 [34] Liquid-liquid assembly of graphene platelets 100 [35] LB assembly of chemically modified graphene 8 × 103 [36]

2.1.3.2. Electrochemical properties of rGO by hydrazine The electrochemical properties of glassy carbon/graphene (GC/graphene) composite electrode were determined using cyclic voltammetry. Two different redox mediators were

3-/4- used. According to McCreery, the electron transfer reaction involving [Fe(CN)6] occurs via an inner-sphere pathway with the electrode kinetics being sensitive to surface composition of the carbon-based interface, its microstructure and the density of electronic states near the

3+/2+ Fermi potential [37]. On the other hand, [Ru(NH3)6] is a nearly ideal outer-sphere redox system that is insensitive to the surface microstructure, surface oxides, and adsorbed monolayers on sp2 carbon electrodes and can thus serve as a useful benchmark in comparing

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) electron transfer rates at various carbon electrodes [38].

600 200 (A) (B) 150 rGO rGO 400 100

50 200 GC GO GC 0 GO µA i/

0 -50 i / µA i/ -100 -200 -150

-400 -200 -0.4 -0.2 0 0.2 0.4 0.6 0.8 -0.6 -0.4 -0.2 0 0.2 0.4 E / V vs. Ag/AgCl E / V vs. Ag/AgCl Figure 2.11: Cyclic voltammograms obtained on glassy carbon (grey), glassy carbon/rGO 3-/4- (red), and glassy carbon / GO (blue) electrodes in (A) 5 mM [Fe(CN)6] / KCl (0.1 M), scan -1 3+ -1 rate=100 mVs and (B) 5 mM [Ru(NH3)6] / KCl (0.1 M), scan rate= 50 mV s .

Figures 2.11 compares the electron transfer behavior of GC, GC/GO, and GO/rGO

3-/4- 3+/2+ electrodes using 5mM [Fe(CN)6] and 5mM [Ru(NH3)6] as redox couples. In both cases, GC coated with GO (in blue color) exhibited its intrinsic insulating nature and showed very weak current response. The low EP of the rGO modified GC electrode (169mV for 5mM 3-/4- 3+/2+ [Fe(CN)6] and 145mV for 5mM [Ru(NH3)6] ) indicates that the graphene film possesses the requisite surface structures and electronic properties to support rapid electron transfer for these redox systems. Enhancement in the anodic and cathode peak currents is observed in both cases with respect to the bare GC electrode, suggesting that the GC/rGO interface exhibits better electrochemical activity than unmodified GC [39]. This fast heterogeneous electron kinetics between the electrode and the solution can be ascribed to the unique electronic structure of rGO [26]. On the one hand, its zero-bandgap semiconductor character with significantly fast electron mobility at room temperature can enhance the electron transfer when its facet contacts the electrolyte directly [40, 41]. On the other hand, chemically converted graphene contains a big amount of structural defects like winkles, surface oxides, etc. These existing defects offer graphene sheet enormous specific surface area and achieve

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) high density of electronic states near the Fermi level, which results in fast electron-transfer

3-/4- reactions of the redox systems [26]. In the case of [Fe(CN)6] , the current increase is

3+ slightly lower than that recorded for [Ru(NH3)6] . The presence of remaining oxygen

3-/4- functions on the rGO might repel the negatively charged [Fe(CN)6] , thus decreasing the detected current. The stability of the graphene-modified interface was investigated by cycling the

3-/4- electrode between -0.5V and 1.3 V in 0.1 M KCl, followed by recording CV in [Fe(CN)6] . No change in the peak current height was observed which is indicative for the robustness of the interface.

In Chapter 3.1, we will investigate if the increased electron transfer kinetics compared to GCE could also have a positive effect on the detection of L-dopa, carbidopa and a mixture of both. The chemical structure of L-dopa and carbidopa is related and both molecules show a multistep oxidation process as dopamine [26]. While the superior biosensing performance of dopamine on graphene interfaces compared to other carbon-based interfaces has been reported by several authors [41-43], L-dopa and carbidopa are at present not investigated. Graphene- based electrodes have not only allowed detection limits of dopamine of 0.17 µM [41], but also permitted in parallel the discrimination between dopamine, uric acid and ascorbic acid, which coexist in biological samples but have overlapping voltammetrtic response on traditional solid electrodes. In the case of L-dopa detection, the discrimination with carbidopa is of high importance as discussed in the introduction.

2.2. Environmentally friendly reduction approaches of graphene oxide To avoid using highly poisonous hydrazine, many environmentally friendly and high- efficient reductants have been developed and used for the reduction of GO [5]. Our group has recently reported on the possibility for simultaneous reduction and functionalization of GO using tetrathiafulvalene (TTF) [6, 7] and dopamine derivatives [8, 9] (Figure 2.12).

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

Figure 2.12: Schematic illustration of the preparation of (a) rGO/TTF nanocomposites and (b) rGO/N3-dopamine.

Motivated by this finding, we investigated if alkynyl-dopamine, tyrosine and aminophenylboronic acid could be used in a similar manner. The interest of using dopamine and its derivatives in combination with GO is based on the physicochemical properties of dopamine. Dopamine has shown to be a good reducing agent for metallic salts such as

HAuCl4, or H2PtCl6 [44, 45]. At a weak alkaline pH, dopamine undergoes self-polymerization to produce an adherent polydopamine coating on a wide range of substrates, with the accompanied oxidation of catechol groups to the quinone form [46-48]. The fascinating (reduction, self-polymerization, and adhesion) properties of dopamine allow to use it simultaneously as a reducing agent of GO and also as a capping agent to decorate the resulting rGO. Alkynyl-dopamine is thus expected to be employed for simultaneous reduction and functionalization of GO, and its chemically reactive alkynyl function can be post- functionalized with thiolated precursors. Tyrosine on the other hand is one of the aromatic amino acids, whose unstable phenol structure can be easily oxidized to quinone and is thus expected to be a good reducing agent ofGO. At last, a similar case could apply to aminophenylboronic acid with the labile phenylamino group.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) 2.2.1. Reduction and functionalization of GO by alkynyl-dopamine (rGO/ Alkynyl- dopamine) and post-functionalization 2.2.1.1. Formation of rGO/Alkynyl-dopamine We [6-8] and others [49] have recently shown that the direct reaction of GO with dopamine or tetrathiafulvalene (TTF) allows not only the reduction of GO to rGO in an easy and environmentally friendly manner, but results also in simultaneous modification of the rGO nanosheets with the dopamine or TTF ligand through π-π stacking interactions. The interest in rGO functionalization with dopamine derivatives is the ease with which functional groups can be introduced via its amine groups. For example, we have shown that rGO modified with dopamine bearing azide groups allowed the post-functionalization with ethynylferrocene using Cu(I) catalyzed 1,3-dipolar cycloaddition [8]. Here, we demonstrate that rGO/alkynyl-dopamine nanocomposite material can be prepared by simple reaction of GO with alkynyl-dopamine under sonication for 4 h at 80°C (Figure 2.13).

Figure 2.13: Schematic illustration of the formation of rGO/alkynyl-dopamine nanosheets.

The interaction between alkynyl-dopamine and rGO takes advantage of strong π-π interactions between the graphene sheets and the aromatic ring of dopamine as shown previously [8, 49]. Our interest in the fabrication of rGO/alkynyl-dopamine nanohybrid materials is motivated by the fact that alkynyl functional groups can be further employed for post- functionalization by covalently linking thiolated molecules in a thiol-yne reaction mechanism. Alkynyl-terminated dopamine was thus investigated for its capablity to reduce GO while

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) simultanously incorporating into the graphene sheet. The rGO/alkynyl-dopamine nanocomposite material is readily formed through the addition of alkynyl-dopamine to GO under sonication at 80°C and neutral pH (Figure 2.13). The formed precipitate is poorly dispersible in polar solvents such as water, but forms stable suspensions in solvents such as DMF, DMSO and THF with rGO/alkynyl-dopamine concentrations of up to ≈0.5 mg/mL. The interaction between alkynyl-terminated dopamine and graphene is most likely dominated by π-π stacking between the hexagonal cells of graphene and the aromatic ring structure of dopamine. However, as the alkynyl-terminated dopamine acts as reducing agent, the GO is likely to oxidize the ortho-quinol structure of the dopamine ligand to radical or quinone intermediates which are likely to covalently bind to graphene or to each other. X-ray photoelectron spectroscopy (XPS) analysis was performed on GO before and after its reaction with alkynyl-terminated dopamine to gain further information on its chemical composition. The C1s core level XPS spectrum of GO nanosheets is displayed in Figure 2.14Aa and can be deconvoluted into four components with binding energies at about 283.8, 284.7, 286.7 and 287.9 eV assigned to sp2-hybridized carbon, C-H/C-C, C-O and C=O species, respectively. The C/O ratio of GO is 1.73 comparable to reported data in the literature [13, 50]. After reaction of GO with alkynyl-terminated dopamine, XPS analysis of the resulting product indicates significant changes in the C1s core level spectrum (Figure 2.14Ab). The band at 283.8 eV due to sp2-hybridized carbon became predominant, suggesting the reconstitution of the graphitic network. The bands at 285.2, 286.7 287.9 and 289.2 can be attributed to C-H/C-C, C-O, C=O and N-C=O/O-C=O species of the dopamine derivative and of some remaining oxygen functionalities on the rGO. The C/O ratio increased to 3.6. This value is comparable to the C/O ratio of 3.4 obtained for GO reduced with azide-functionalized dopamine [8]. The success of the incorporation of the dopamine derivative is in addition confirmed by the presence of 2.4 at. % nitrogen. Raman scattering is a useful tool to characterize the structural properties of graphene- based materials. Figure 2.14B shows the Raman spectra for GO and rGO/alkynyl-dopamine presenting the main features of graphene-based materials with a D-band at 1351 cm-1, a G- band at 1570 cm-1 and a 2D-band at ≈2700 cm-1 [51]. The ratio of the intensities of the D and

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

G bands (ID/IG) was found to be 0.71 for GO and 0.67 for rGO/alkynyl-dopamine, comparable to ID/IG=0.63 recorded for hydrazine reduced graphene oxide [52].

1 (A) rGO/alkynyl-dopamine G-band (B) 1. sp2 C 2. C-C/C-H D-band 3. C-N 4. C-O 3 2 5. C=O 4 5 2D band (b)

3 a.u. / Intensity (b) GO 2 1. sp2 C 2. C-C/C-H 3. C-O 4 4. C=O (a) (a) 1 1500 2000 2500 292 290 288 286 284 282 280 -1 Binding energy / eV Raman shift / cm

1.2 (C) 0.5

1 (D)

228nm 269nm 0.4 0.8

0.3 0.6 (b) 0.2

0.4 282.1nm Adsorbance intensity / a.u./ intensity Adsorbance 297 nm 0.1

0.2 a.u. / Intensity Adsorbance

(a) 0 0 200 300 400 500 600 700 800 240 320 400 480 560 640 720 800 Wavelength / nm Wavelength / nm Figure 2.14: (A) C1s core-level XPS spectra of GO before (a) and after reaction with alkynyl- dopamine (b); (B) Raman spectra of GO (black) and rGO/alkynyl-dopamine (red); (C) UV- Vis spectra of GO in water (a) and rGO/alkynyl-dopamine in ethanol (b); (D) UV/Vis spectra of alkynyl-dopamine in ethanol.

The formation of rGO was also validated by UV-Vis spectrosopy. The UV-Vis absorption spectra of GO in water and rGO/alkynyl-dopamine in ethanol are seen in Figure 2.14C. GO dispersed in water exhibits a maximum absorption at 228 nm, attributed to the * transition resulting from C=C bonds of the aromatic skeleton, and a broad shoulder at

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) ~297 nm due to the n-π* transition of C=O bonds from carboxylic acid functions. The spectrum of rGO/alkynyl-dopamine nanocomposite shows a broad absorption band at 269 nm. The red shift from 226 nm (GO) to 269 nm for rGO/alkynyl-dopamine is consistent with the restoration of the sp2 structure in rGO. Furthermore, the band at 269 nm is broad and can be correlated to the signal band from alkynyl-dopamine at 282.1 nm ( Figure 2.14 D). In addition, the room-temperature conductivity of rGO/alkynyl-dopamine was measured by a standard four-probe method. The nanocomposite showed a conductivity of 0.89 S m-1, which is lower than that measured on hydrazine reduced graphene (145 S m-1) using the same technique. This value is comparable to that reported recently by using azido-terminated tetrathiafulvalene [7]. The electrochemical properties of the rGO/ alkynyl-dopamine composite were evaluated

3-/4- 3+ by cyclic voltammetry using Fe(CN)6 (Figure 2.15A) and Ru(NH3)6 (Figure 2.15B) as redox couples. The rGO/alkynyl-dopamine composite material was drop casted onto a bare ITO. The drop casting process is very reproducible and leads to homogeneous rGO/alkynyl- dopamine films of ≈280 nm, comparable to rGO/azide-dopamine films (Figure 2.15C) [8]. The difference of electrochemical surface activity between ITO and ITO/rGO/alkynyl- dopamine can be easily recognized from the representative CVs of the two redox couples. The detected currents on rGO/alkynyl-dopamine greatly go beyond that of a bare ITO with a relatively strong capacitive component. However the Ep in both cases does not show a big

3-/4- change. For Fe(CN)6 , Ep of rGO/alkynyl-dopamine electrode slightly decreases to 186 3+ mV, in comparison to bare ITO with 191 mV. For Ru(NH3)6 , the redox peaks get a bit more separated on rGO/alkynyl-dopamine with Ep = 140 mV as compared to Ep = 131 mV measured on bare ITO. An estimation of the amount of alkynyl-dopamine incorporated into the rGO matrix can be obtained from cyclic voltammograms (CV) experiments. Figure 2.15D shows the electrochemical signature of the rGO/alkynyl-dopamine modified ITO interface. A redox

ox wave with E1 =0.19 V vs. Ag/AgCl is observed, characteristic for dopamine oxidation in a two-electron process. The loading of alkynyl-dopamine on the rGO surface was calculated by integrating the anodic peak area according to Г=QA/nFA , where F is the Faraday constant, n the number of electrons exchanged (n=2), and A the surface area (A= 0.12 cm2). A value of Г=

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) (2.07±0.73) ×1014 molecules cm-2 is obtained. The dopamine loading is lower than rGO modified with TTF using a similar approach (Г=8.9×1014 molecules cm-2) [6], but comparable when unmodified dopamine was used (Г =(3.05±0.73) ×1014 molecules cm-2).

Figure 2.15 Cyclic voltammograms of bare ITO (grey) and ITO/rGO/alkynyl-dopamine (red) 3-/4- -1 in (A) 5 mM Fe(CN)6 in PBS (pH=7.4) aqueous solution. Scan rate = 0.1 V s ; (B) 5 mM 3+ -1 Ru(NH3)6 in PBS (pH=7.4) aqueous solution. Scan rate = 0.05 V s ; (C) SEM image of ITO/graphene/alkynyl-dopamine formed by drop casting (thickness = 280 nm); (D) Cyclic voltammogram of an ITO electrode modified by drop casting with rGO/alkynyl-dopamine in 0.1 M PBS; scan rate = 0.05 V s-1.

2.2.1.2. Post-functionalization of rGO/alkynyl-dopamine The photochemical “click” reaction between alkynes or alkenes and thiolated molecules has attracted increasing interest during the last years because of its versatility and specificity [53-57]. It is believed that this reaction can overcome obstacles with regards to high-density deposition of ligands, processing large and bulky headgroups, and direct placement of ligands

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) possessing multiple reactive functionalities. These reactions have “click” chemistry characteristics such as a high yield, regiospecificity, mild reaction conditions and tolerance to a variety of functional groups [55, 58, 59]. Compared to the Cu(I) “click” chemistry [58], no catalyst is required as the reaction is initiated thermally or photochemically. The main difference between thiol-ene and thiol-yne reaction is the possibility to anchor two thiolated molecules in the case of thiol-yne to form a double addition product through a radical process [53, 60, 61]. Herein, our idea in the fabrication of rGO/alkynyl-dopamine nanohybrid materials is motivated by the fact that alkynyl functional groups can be further employed for the covalent linking of thiolated molecules in a thiol-yne reaction mechanism. Although less investigated than the thiol-ene reaction, thiol-yne chemistry has the advantage that the alkynyl group can react with two thiolated molecules to form a double addition product [53]. Alkynyl- terminated dopamine was thus investigated for its capablity to reduce GO while simultanously incorporating into the graphene sheet. Then we further introduce the concept of thiol-yne reaction onto rGO nanocomposite (Figure 2.16).

S-R thiolated molecules used:

O S-R CH2-(CH2)4-CH2-SH O O O Fe HS-Fc

O 2 SH-R NH  = 365 nm, O NH 30min, under nitrogen F2 F2 F2 C C C CF HS 3 HS-PF HO OH C C C C F2 F2 F2 F2 HO OH Figure 2.16: Reaction of alkynyl-dopamine with thiolated molecules through thiol-yne radical addition mechanism.

In a proof of concept experiment, 6-(ferrocenyl)-hexanethiol (HS-Fc) or 1H,1H,2H,2H- perfluorodecanethiol (HS-PF) was photochemically “clicked” onto the rGO/alkynyl- dopamine derivative (Figure 2.16). Since the thiol-yne reaction allows addition of two thiolated molecules to an alkyne, it appears to be perfectly suited to obtain high surface

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) densities of the functional group of interest on the interface. 6-(ferrocenyl)-hexanethiol (SH- Fc) and 1H,1H,2H,2H–perfluorodecanethiol have been investigated here as model systems to check the reactivity of the reduced GO modified with alkynyl-dopamine (Figure 2.16). The photochemical based thiol-yne reaction was carried out at 365 nm for 30 min under nitrogen atmosphere with a light power of 100 mW cm-2. The success of the reaction with 6-(ferrocenyl)-hexanethiol (SH-Fc) was followed by XPS. Figure 2.17A shows the high resolution XPS spectrum of the S2p core level. It can be deconvoluted into bands at 164.2 (S2p3/2) and 165.3 eV (S2p1/2) due to S-C bonds and

4+ contributions at higher energies, 166.9 (S2p3/2) and 168.1 eV (S2p1/2) due to oxidized S , in the form of S-O, with S-C/S-O ratio of 2.2. The presence of oxidized thiols was also observed in the initial 6-(ferrocenyl)-hexanethiol with a S-C/S-O ratio of 2.4. This indicates that only a small additonal fraction of 6-(ferrocenyl)-hexanethiol molecules are further oxidized during the photochemical reaction and interact non-specifically with the rGO/alkynyl-terminated dopamine nanomaterial. The atomic ratio of N/S is 1.3 (Table 2.2). Taking into consideration that about 0.65 at % of SH-Fc is oxidized rather than reacting in a thiol-yne reaction, a N/(S-C) ratio of 1.9 is determined. This value is close to the theoretically expected ratio of N/S=2.0 for a double addition rather than a monoaddition. The non specific adsorption of 6-(ferrocenyl)- hexanethiol or its oxidized counterpart onto the rGO surface was verified in a control experiment, where ITO interfaces modified with rGO/alkynyl-dopamine films were immsered in 10 mM ethanolic solution of 6-(ferrocenyl)-hexanethiol as before for 30 min but without light irradiation. As seen from Table 2.2, the presence of bands due to S2p and Fe2p indicate some nonspecific adsorption onto rGO/alkynyl-dopamine interfaces most likely due to  stacking interactions. The bonded ferrocene moiety shows a redox potential at ≈0.6 V vs Ag/AgCl and integration of the anodic peak area results in a surface coverage of =5.04 ×1014 molecules cm-2 (Figure 2.17B). This is ~2.4 times higher than the amount of dopamine ligand incorporated onto the rGO and suggests the preferential double addition together with some non-specific adsorption. Indeed it is much higher than using Cu(I) “click” chemistry on rGO/dopamine-N3 nanomatrices [8].

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

(A) 30 (B) E (ferrocene) S2p 2 S2p 3/2 20

E (dopamine) 1 10

S2p A 1/2 

0 i / i / S2p 3/2 S2p 1/2 -10

-20 172 170 168 166 164 162 160 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 Binding energy / eV E / V

Figure 2.17: (A) S2p high resolution XPS spectrum of rGO/alkynyl-dopamine after photochemical reaction (λ = 365 nm) with 10 mM 6-(ferrocenyl)-hexanethiol for 30 min at room temperature (P=100 mW cm-2). (B) CV of ITO interface modified with rGO/alkynyl- dopamine after photochemical reaction with 10 mM 6-(ferrocenyl)-hexanethiol in PBS (0.1 M), scan rate = 0.05 V s-1.

Table 2.2: Atomic percentages of rGO/alkynyl dopamine before and after reaction with 6- (ferrocenyl)-hexanethiol.

C1s O1s N1s Fe2p S2p (C-S) S2p (S-O) Interface investigated (at.%) (at.%) (at.%) (at.%) (at.%) (at.%) rGO/alkynyl-terminated 74.30 23.30 2.40 0.00 0.00 0.00 dopamine + 10 mM HS-Fc 73.18 23.60 2.20 0.50 0.37 0.15 + 10 mM HS-Fc upon 72.50 22.20 2.10 1.60 1.10 0.50 photoirradiation

Contact angle measurements further comfirmed the incorporation of thiolated ferrocene on rGO/alkynyl-dopamine through UV irradiation. Before thiol-yne reaction, the static contact angle with water on rGO/alkynyl-terminated dopamine is 54°. After reaction with 10 mM HS-Fc upon photoirradiation at 365 nm for 30 min, the static contact angle of the interface increases to 94°, confering a hydrophobic character to the surface. The surface that has undergone the reaction with 10 mM HS-Fc without photoirradiation, the static contact angle did not change much.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

(A) (B)

F1s C1s 2 C1s 1. sp C 2. C-C/C-H 1 O1s 3. C-O/C-N/C-S 4. C=O 5. CF 2 6. CF 3 3 2

N1s

5 4 S2pS2p 6

0 200 400 600 800 294 292 290 288 286 284 282 Binding energy / eV Binding energy / eV

Figure 2.18: (A) XPS survey spectrum of rGO/alkynyl-dopamine deposited onto ITO after immersion into 10 mM 1H,1H,2H,2H-perfluorodecanethiol and UV light irradiation at 365 nm for 30 min at room temperature (P=100 mW cm-2). (B) C1s core level XPS spectrum after thiol-yne reaction with HS-PF.

Given the high cross section of fluorine in XPS analysis, 1H,1H,2H,2H-perfluorodecanethiol was used as a second model compound to investigate the thiol-yne reaction. Figure 2.18A displays the XPS survey spectrum of rGO/alkynyl-terminated dopamine interface after photochemical reaction for 30 min. The success of the reaction is evidenced by the presence of F1s (688 eV) and S2p (164 eV) in addition to C1s (284.5 eV), O1s (513 eV) and N1s (400 eV) bands. The high resolution C1s XPS spectrum of the perfluorodecanethiol modified rGO matrix is shown in Figure 2.18B. The presence of –CF3 and –CF2 groups is clearly seen by the contributions at 293.1 eV and 290.7 eV, respectively. The other bands are due to sp2- hybridized carbon (283.9 eV), C-C/C-H (284.5 eV), C-S/C-N/C-O (285.8 eV) and C=O (287.8 eV). The atomic ratio of N/S is 1.3, lower than the N/S=2 expected for double addition product, indicating the preferential monoaddition product in this case. This might be due to the increase in hydrophobicity of the rGO/alkynyl-terminated dopamine with a water contact angle of 44° to 97° upon linking of perfluorodecanethiol. In conclusion, this study demonstrated that alkynyl-dopamine reacts in a similar way than dopamine, inducing simultaneous reduction of GO to rGO at room temperature under sonication and functionalization of the rGO. The motivation for using dopamine is that such reduced graphene oxide nanocomposite materials will allow for post-functionalization of the

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) alkynyl-dopamine moieties with thiolated molecules using a photochemical “click” reaction between alkynes and thiols. This approach opens up new perspectives for covalent modification of graphene composites which can be exploited for future applications.

2.2.2. Reduction of GO using tyrosine Here we describe an easy and environmentally friendly chemical method for the reduction and non-covalent functionalization of GO using tyrosine (Figure 2.19). The reaction of an aqueous solution of GO (0.5mg/mL) and tyrosine (10mM) at 100°C for 24 h produced a black precipitate that can be easily separated from the supernatant via centrifugation.

Figure 2.19: Schematic illustration of the reduction of GO using tyrosine.

(A) 223 (B) 228 270 1.5

rGO/Tyr Absorbance (a.u.) Absorbance

Absorbance (a.u.) Absorbance 0.5 274 GO Tyrosine in water 0 200 300 400 500 600 700 800 200 300 400 500 600 700 800 Wavelength (nm) Wavelength (nm) Figure 2.20: UV-Vis absorption spectra of GO before (black) and after (red) reduction with tyrosine at 100°C for 24 h (A), and the spectrum of tyrosine (50 μM) in water (blue) (B).

The success of the reaction was confirmed by UV-Vis absorption spectra (Figure 2.20A)). The UV–Vis spectra show clearly that the absorption peak red-shifted from 228 to 270 nm upon reaction with tyrosine at 100°C for 24 h. The presence of a peak at around 220

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) nm is most likely due to the incorporation of the tyrosine moieties in the reduced GO. Indeed, free tyrosine displays a strong absorption peak at 223 nm (Figure 2.20B). In addition, the intensity of the absorption tail in the region > 400 nm has significantly increased. The result suggests that the GO nanosheets have been reduced and the electronic conjugation within the GO nanosheet was restored upon reaction with tyrosine.

C1s

2 1. sp C 1 2. C-C/C-H 3. C-O/C-N N1s (C) 4. C=O 2 4 3

(B) Intensity (a.u.) Intensity 3 2 1. sp2 C 2. C-C/C-H 3. C-O (a.u.) Intensity 4. C=O 4 1 (A)

296 292 288 284 280 404 402 400 398 396 Binding energy / eV Binding energy (eV) Figure 2.21: High resolution X-ray photoelectron spectroscopy (XPS) of GO before (A) and after (B) reaction with tyrosine at 100°C for 24 h. (C) corresponds to N1s core level spectrum of rGO/Tyr composite.

X-ray photoelectron spectroscopy (XPS) analysis was performed on GO before and after its reaction with tyrosine at 100°C for 24 h to gain further information on the chemical transformations occurred on its surface. The C1s core level XPS spectrum of GO nanosheets is displayed in Figure 2.21A and can be deconvoluted into four components with binding energies at about 283.9, 285.1, 286.8 and 287.8 eV assigned to sp2-hybridized carbon, C-H/C- C, C-O and C=O species, respectively. The C/O ratio of GO is 1.73 comparable to reported data in the literature [50]. After reaction of GO with tyrosine, analysis of the resulting composite indicates significant changes in the C1s core level spectrum (Figure 2.21B). The C1s exhibits bands at 283.9 eV (sp2-hybridized carbon), 285.1 eV (C-H/C-C), 287.8 eV (C- O/C-N) and 290.7 eV (COOH). The intensity of the band at 283.9 eV increased significantly compared to GO, suggesting that the sp2 network has been restored during the process (Figure 2.21B). The ratio of carbon to oxygen increased to 6.07. Indeed, GO is a good

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) electron acceptor that can be easily reduced in the presence of electron donors. Tyrosine seems to act as a good reducing agent for GO, as previously seen for other amino acids such dopamine [8] and L-ascorbic acid, getting at the same time oxidized to a quinone-type product (Figure 2.19) [62]. In a control experiment, an aqueous solution of GO (without tyrosine) was heated at 100°C for 24 h and its oxidative degree was determined by XPS. The result was comparable to that of GO with an increased C/O ratio to 2.8 due to a slight decrease in the C-O content. However, the component due to sp2-hybridized carbon remained rather unchanged. The result suggests that water alone is not sufficient to reduce GO to rGO under these experimental conditions. Incorporation of tyrosine or its derivatives on the rGO matrix was further confirmed by the presence of nitrogen in the XPS spectrum at 400 eV (Figure 2.21C).

300 150 (A) rGO/Tyr (B) rGO/Tyr 200 100

ITO 50 ITO 100 0

0 i /i µA i /i µA -50

-100 -100

-200 -150

-300 -200 -0.4 -0.2 0 0.2 0.4 0.6 0.8 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 E / V vs. Ag/AgCl E / V vs. Ag/AgCl Figure 2.22: Cyclic voltammograms of (A) ITO (grey) and ITO/rGO/Tyr (red) in 5 mM 3-/4- Fe(CN)6 /0.1 M KCl, scan rate: 100 mV/S; (B) ITO (grey) and ITO/rGO/Tyr (red) in 5 mM 3+ Ru(NH3)6 /0.1 M KCl, scan rate: 50 mV/s.

The electrochemical properties of the rGO/Tyr composite were evaluated by cyclic

3-/4- 3+ voltammetry using Fe(CN)6 (Figure 2.22A) and Ru(NH3)6 (Figure 2.22B) as redox couples. Figure 2.22A displays the CVs of ITO before and after drop casting a film of

3-/4- rGO/Tyr using Fe(CN)6 as redox couple. The ITO electrode shows a peak separation, E of 189 mV, which increased upon coating with rGO/Tyr to 302 mV. Similarly, rGO/Tyr

3+ displayed a comparable electrochemical behavior using Ru(NH3)6 as redox couple with a E of 110 mV for bare ITO and 165 mV after coating ITO with rGO/Tyr (Figure 2.22B). For

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) both redox couples, the detected currents on rGO/Tyr exceeded that of a bare ITO with a relatively strong capacitive component.

In Section 3.2, we will show that the rGO/Tyr nanomaterial displays good electrocatalytic activity for the detection of hydrogen peroxide (H2O2) with a wide linear range from 100 µM to 2 mM, a detection limit of 80 µM and a sensitivity of 69.07 µA mM-1 cm-2.

2.2.3. Preparation of rGO-Au NPs/Tyr Recently, a new class of hybrid materials consisting of graphene or its derivates and inorganic nanostructures with controlled size and shape are developed (e.g. Au, Ag, magnetic nanoparticles, etc), displaying promising properties for applications in various areas ranging from electronics and optics to electrochemical sensing as well as energy conversion and storage [63-67]. Here we report a green, in situ approach for the synthesis of aqueous stable rGO-gold nanoparticles (rGO-Au NPs) hybrids using tyrosine as a reducing agent for both

GO and Au ions from HAuCl4 (Figure 2.23). The reaction of an aqueous solution GO (0.5mg/mL) and tyrosine (10mM) at 100°C for 24h produced a black precipitate, then 1mL

HAuCl4 (40mM) was added into the mixture while keeping the temperature at 100°C and stirring for 3h. The formed dark purple precipitate, which could be easily separated from the supernatant via centrifugation, was washed three times with Milli-Q water. The obtained rGO- Au NPs/Tyr nanocomposites can be facilely redispersed in water with the aid of ultrasonication for 20 min.

Figure 2.23: Schematic illustration of the formation of rGO-Au NPs/Tyr.

The success of the as-prepared hybrids was confirmed by UV-Vis absorption spectra (Figure 2.24). The UV–Vis spectra show clearly that the absorption peak due to π-π*

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) transition band red-shifted from 228 to 270 nm, together with the disappearance of the n-π* transition band at 300 nm. This implies the restoration of the electronic conjugation in rGO upon reaction with tyrosine at 100°C for 24 h. The first evidence of Au NPs formation was provided by the significant color changes of the solutions. The successful loading of Au NPs on the rGO surface was confirmed by the peak around 561 nm, which is the characteristic surface plasmon resonance (SPR) band of nano-gold [68, 69].

561

rGO-Au NPs/Tyr

rGO/Tyr Absorbance (a.u.) Absorbance

200 300 400 500 600 700 800 Wavelength (nm)

Figure 2.24: UV-Vis absorption spectra of GO (black), rGO/Tyr (red) and rGO-Au NPs/Tyr (green).

X-ray photoelectron spectroscopy (XPS) analysis results also confirmed the formation of rGO-Au NPs/Tyr nanocomposites (Figure 2.25) Indeed, the C1s core level XPS spectrum of the hybrid (Figure 2.25(c)) presents similar features as rGO/Tyr (Figure 2.25(b)), and can be deconvoluted into four components with binding energies at about 283.9 eV (sp2-hybridized carbon), 285.2 eV (C-H/C-C), 287.5 eV (C-O/C-N) and 290.2 eV (COOH). The ratio of carbon to oxygen was 3.73. The intensity of the band at 283.9 eV increased significantly compared to the initial GO (Figure 2.25A(a)), suggesting that the sp2 network has been restored during the reduction process with tyrosine. Incorporation of tyrosine or its derivatives on the rGO matrix was further confirmed by the presence of nitrogen in the XPS spectrum at 400 eV (Figure 2.25B) with a content of 2.04 at%. Au 4f core level spectrum (Figure 2.25C) indicated Au NPs formation. The binding energies of Au4f7/2 and Au4f5/2 electrons are 81.9 and 85.6 eV, respectively. This is consistent with the reports on gold metal, suggesting that Au NPs deposited on solid substrates exist in the metallic state [70-72]. The overall Au content

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) was 8.17 at%, indicating an important loading of Au NPs onto the rGO/Tyr interfaces.

Figure 2.25: High resolution X-ray photoelectron spectroscopy (XPS) (A) C1s core level spectra of (a) GO, (b) rGO/Tyr and (c) rGO-Au NPs/Tyr. (B) N1s core level spectrum of rGO- Au NPs/Tyr composite. (C) Au 4f core level spectrum of rGO-Au NPs/Tyr.

210 100 (A) (B) rGO-Au NPs/Tyr rGO-Au NPs/Tyr 50 105 GC GC

0 i / µA i / i µA

-50 -105

-100

-0.4 -0.2 0 0.2 0.4 0.6 0.8 -0.6 -0.4 -0.2 0 0.2 E / V vs. Ag/AgCl E / V vs. Ag/AgCl

Figure 2.26: Cyclic voltammograms of (A) GC (grey) and GC/ rGO-Au NPs/Tyr (blue) in 5 3-/4- mM Fe(CN)6 /0.1 M KCl, scan rate: 100 mV/S; (B) GC (grey) and GC/rGO/Tyr/Au NPs 3+ (blue) in 5 mM Ru(NH3)6 /0.1 M KCl, scan rate: 50 mV/s.

The electrochemical properties of the rGO-Au NPs/Tyr composite were evaluated by

3-/4- 3+ cyclic voltammetry using Fe(CN)6 (Figure 2.26A) and Ru(NH3)6 (Figure 2.26B) as

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) redox couples. Figure 2.26A displays the CVs of GC before and after drop casting a film of

3-/4- rGO-Au NPs/Tyr using Fe(CN)6 as redox couple. The GC electrode shows a peak separation, E of 121 mV, which increased upon coating with rGO-Au NPs/Tyr to 226 mV. Similarly, rGO-Au NPs/Tyr displayed a comparable electrochemical behavior using

3+ Ru(NH3)6 as redox couple with a E of 95 mV for bare GC and 160 mV after coating GC with rGO-Au NPs/Tyr (Figure 2.26B). For both redox couples, the detected currents on rGO- Au NPs/Tyr exceeded that of a bare GC with a relatively strong capacitive component. In conclusion, a green method for the synthesis of rGO-Au NPs/Tyr hydrids in aqueous solution that exploits the ability of tyrosine to operate as an effective dual reducing agent for both graphene oxide and gold salts is reported. It is well-documented that Au NPs can play a key role in electrocatalytic reactions [73-75]. So we will make full use of this advantage, and design the rGO-Au NPs/Tyr modified electrodes for the detection of some small molecules such as H2O2, hydrazine, and nitric oxide. Meanwhile, we will also utilize the good catalytic activity of rGO-Au NPs/Tyr for the reduction of nitrophenol.

2.2.4. Reduction and functionalization of graphene oxide with aminophenylboronic acid for the formation of rGO/APBA In this section, we illustrate another example of one-step functionalization/reduction of GO through π-π stacking interactions using aminophenylboronic acid. Motivated by the fact that boronic acid modified electrodes have been successfully employed for the electrochemical detection of sugars, we investigated if 4-aminophenylboronic acid (APBA) can be used in a similar manner like dopamine or TTF [6-9] for simultaneous reduction and incorporation of APBA moieties on the graphene skeleton (Figure 2.27).

Figure 2.27: Schematic illustration of the formation of rGO/APBA nanosheests.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) The direct reaction of an aqueous mixture of GO (0.5 mg/mL) and APBA (5 mM) for 12 h at 100°C resulted in the formation of a black water insoluble precipitate.

1,2 105 750 (A) rGO/APBA (B) 1 B1s 1. sp2 C 1 105 700 2. C-C/C-H 3. C-N 4. C=O 8 104 650 2 4 3 (b) 6 104 600

3 2 4 GO 4 10 550 1. sp2 C 2. C-C/C-H 2 104 3. C-O 500 4 4. C=O 1 (a) 0 450 292 290 288 286 284 282 280 195 194 193 192 191 190 189 188 Binding energy / eV Binding Energy / eV

(b)

(a)

406 404 402 400 398 396 394 Binding Energy / eV Figure 2.28: (A) C1s core-level XPS spectra of GO before (a) and after reaction with 4- aminophenylboronic acid (APBA) (b). (B) B1s high resolution XPS sepctrum of rGO/APBA. (C) N1s high resolution XPS spectrum of APBA (a) and rGO/APBA (b).

The chemical composition of GO before and after reaction with APBA was determined by X-ray photoelectron spectroscopy (XPS) analysis (Figure 2.28). The C1s core level XPS spectrum of GO nanosheets is displayed in Figure 2.28Aa and can be deconvoluted into four components with binding energies at about 283.8, 284.7, 286.7 and 287.9 eV assigned to sp2- hybridized carbon, C-H/C-C, C-O and C=O species, respectively. The C/O ratio of GO is 1.73 comparable to reported data in the literature [13, 50]. After reaction of GO with APBA, XPS analysis of the resulting product indicates significant changes in the C1s core level spectrum

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) (Figure 2.28Ab). The band at 283.9 eV due to sp2-hybridized carbon became predominant, suggesting the reconstitution of the graphitic network. The bands at 284.7 and 285.5 eV can be attributed to C-H/C-C/C-B and C-N of APBA, while the band at 287.3 eV is due to some remaining oxygen functionalities on the rGO. The C/O ratio increased to 3.8. The success of the incorporation of the 4-aminophenylboronic acid derivative is in addition confirmed by the presence of B1s and N1s contributions in the XPS spectrum (Figures 2.28B, C) with an atomic percentage of B1s of 4.51 and a B/N ratio of 1.1. The B1s high resolution XPS spectrum shows a band at 191.1 eV, corresponding to -C-B(OH)2, in accordance with the chemical compositon of APBA (Figure 2.28B). The N1s band of the nanocomposite (Figure 2.28Cb) can be deconvoluted into two bands at 398.2 eV and 399.7 eV with a ratio 2/1. This is rather different form the N1s band of the initial APBA (Figure

2.28Ca) where two bands at 399.1 eV and 400.96 eV (ratio=1/1.5) due to -NH2 and

+ protonated NH3 groups are observed. The band at 399.7 eV of the rGO/APBA matrix is due to amine (NH2) bonds, while the lower binding energy band at 398.2 eV is most likely due to imine (=N-) groups, resulting from APBA oxidation and the formation of oligomeric side products. It is indeed well known that while graphene is a good electron acceptor, aniline and its derivatives are, on the other hand, very good electron donors [76]. As a result GO nanosheets are readily reduced by aniline at elevated temperature, accompanied by simultaneous in situ polymerization of aniline monomers [77]. The successfull incorporation of APBA and the degree of GO reduction was also examined by Fourier transfom infrared (FTIR) spectroscopy. Figure 2.29A displays the FTIR transmission spectra of exfoliated GO and rGO/APBA nanocomposite. The spectrum of GO shows next to the strong OH vibration band at 3399 cm-1, the presence of C=O in carboxylic acid and carbonyl moieties (1740 cm-1), C=C (1622 cm-1), C-OH of carboxyl groups (1421 cm-1), C-O-C (1225 cm-1) and C-O of epoxy or alkoxy groups (1065 cm-1). After reaction of GO with APBA, the intensity of the OH, C-OH, C-O-C bands strongly decreased. The band of C=C at 1622 cm-1 remains, which confirms the formation of reduced GO. Some carbonyl functions seen at 1740 cm-1 remain and are most likely arising from carbonyl groups on the edge of the rGO matrix. In addition, the presence of B-OH and/or NH stretching mode (3430 cm-1), B-O stretching mode (1350 cm-1) and C-N stretching at 1281 cm-1 indicate the

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) formation of rGO/APBA. Figure 2.29B exhibits the UV-Vis absorption spectra of GO and rGO/APBA aqueous dispersions. For GO, a characteristic absorption peak is observed at 228 nm, while for rGO/APBA this peak is shifted to 272 nm with a substantial increase in absorption intensity above 300 nm. The result suggests that GO nanosheets have been reduced and the aromatic network has been restored [6, 78].

Figure 2.29: (A) Transmission FTIR spectra of GO (a, black) and rGO/APBA (b, red); (B) UV-Vis spectra of APBA (black), GO (grey) and rGO/APBA (blue) in water; (C) Cyclic voltammograms recorded on GC electrode before (black) and after modification with 3+ -1 rGO/APBA (red) in 5 mM Ru(NH3)6 /KCl (0.1M), scan rate: 50 mV s .

The electrochemical behaviour of the rGO/APBA nanocomposites deposited on a glassy carbon electrode by drop casting was examined by cyclic voltammetry. A remarkable difference of electrochemical surface activity between GC and GC/rGO/APBA recorded in

3+ Ru(NH3)6 at the same scan rate can be easily recognized from the representative CVs shown

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) in Figure 2.29C. Next to larger recorded redox currents, the capacitance of the interface is in addition highly increased for GC/rGO/APBA. The novel matrix was further investigated for its sensing capability of different sugars (fructose, mannose and glucose). The fabricated rGO/APBA sensor exhibits a wide linear range with detection limits of 100 nM for fructose, and around 800 nM for mannose and glucose. (Section 3.3).

2.3. Diamond particles (NDs) for the redcution of GO and the formation of an rGO/NDs composite material Diamond nanoparticles (also referred to as nanodiamonds, NDs) have received considerable interest for applications in tribology and nanobiotechnology owing to their chemical inertness, biocompatibility and high specific area [79]. The average diamond particle size in typical detonation diamond is ≈10 nm, but depends strongly on the surface functionalization [80, 81]. As nanodiamonds have been shown electrochemical activity, we investigated the possibility to use them as an intercalating material into GO nanosheets as well as their ability to reduce GO into rGO [82]. To inhibit agglomeration of rGO through electrostatic interactions and providing at the same time open nanochannels, incorporation of intercalating spacers is a promising strategy. We thus investigated the possibility of NDs integration into GO matrix with the concomitant reduction of GO to rGO (Figure 2.30).

100 C/ 48h

Graphene Oxide (GO) rGO/ND

Figure 2.30: Schematic illustration of the preparation of rGO/NDs nanocomposite.

Here, we report on the preparation of reduced graphene oxide/nanodiamonds (rGO/NDs) composites using a solution phase process. The direct reaction of an aqueous solution of GO

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) and NDs at different GO/NDs ratios at 100°C for 48 h gave the corresponding composites with enhanced properties. The GO matrix was partially reduced to rGO under these conditions, while the NDs particles were intercalated into the rGO sheets. Incorporation of NDs into rGO matrix resulted in a significant improvement of the dispersibility of the rGO/NDs composite in solvents such as ethanol and DMF with the suspensions being stable for several weeks. The preparation method of rGO/NDs composites is illustrated in Figure 2.30. First, GO was prepared using a modified Hummers method and a stable suspension of GO (0.5 mg/mL) in water was achieved upon exfoliation for 3h under ultrasonication. Hydroxylated- nanodiamond particles, pre-treated by a bead-assisted sonic disintegration process (BASD) were added to the exfoliated GO sheets in water and heated at 100°C for 48 h. It is likely that the OH groups of the NDs serve as reducing sites in the reduction process of GO to rGO. The molar ratio of OH active groups on NDs is believed to be sufficient to reduce GO efficiently. Reduction of GO to rGO was thus performed by keeping the amount of GO constant and varying the amount of NDs. Four different ratios of GO/NDs (1/1, 4/1, 10/1 and 20/1) were chosen. Immediately after sonication for 3h

4 h later

1 week later

Figure 2.31: Dispersibility of rGO/NDs (0.5 mg/mL) in different solvents.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) The resulting rGO/NDs composites showed a low dispersibility in water, toluene, DMSO and THF (Figure 2.31). However in other solvents such as acetone, ethanol, DMF, acetonitrile and N-methyl-2-pyrrolidone, stable rGO/NDs suspensions were achieved even after 1 week. Table 2.3 displays the size and zeta potential measurements of NDs, GO, and rGO/NDs nanocomposites at different GO/NDs ratios.

Table 2.3: Characteristics of NDs, GO and rGO/NDs nanocomposites at different GO/NDs ratios.

Product Size/ nm / mV

NDs 31±6 17±1 GO 151±1 -41±4

rGO/NDs (1/1) 225±2 -30±1 rGO/NDs (4/1) 239±7 -34±2 rGO/NDs (10/1) 289±18 -37±2

rGO/NDs (20/1) 298±9 -42±2

(A) (B)

1. sp2 C 1. sp2 C 2 2. C-C/C-H 2. C-C/C-H 3 1 3. C-O 3. C-O 4. C=O 4. C=O 2 ND (c) 3 rGO/ND (10/1) 4 (c)

1 GO heating 3 2 2 at 100°C, 48 h 4 1 (b) rGO/ND (4/1) 3 4 (b)

3 2

2 1 4 3 GO 4 1 (a) rGO/ND (1/1) (a) 294 292 290 288 286 284 282 280 294 292 290 288 286 284 282 280 Binding energy / eV Binding energy / eV Figure 2.32: C1s high resoltution XPS spectra of (A) GO, GO after heating at 100°C for 48h, ND, and of (B) different rGO/ND mixtures.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) X-ray photoelectron spectroscopy (XPS) analysis was performed on GO before and after its reaction with NDs to gain further information on its chemical composition. The C1s core level XPS spectrum of GO nanosheets is displayed in Figure 2.32Aa and can be deconvoluted into four components with binding energies at about 283.8, 284.7, 286.7 and 287.9 eV assigned to sp2-hybridized carbon, C-H/C-C, C-O and C=O species, respectively. The C/O ratio of GO is 2.53 comparable to reported data in the literature [13, 50] with an atomic percentage of oxygen of ≈ 28 at%. In the case of NDs, the C1s core level spectrum shows two contributions at 285.2 eV and 286.7 eV assigned to C-C/C-H and C-O functions, respectively (Figure 2.32Ac) with an oxygen content of 10.32 at%. After reaction of GO with NDs particles in a 1/1 ratio, analysis of the resulting composite indicates significant changes in the C1s core level spectrum (Figure 2.32Ba). The C1s exhibits the spectral signature of NDs with the bands at 285.2 eV and 286.7 eV and additional ones at 283.4 eV (sp2-hybridized carbon) and 287.9 eV (C=O). The intensity of the band at 283.4 eV increased compared to GO, suggesting that the sp2 network has been partially restored during the process. The C/O ratio increased to 3.76 with an overall oxygen content of ≈22 at%. Indeed, GO is a good electron acceptor that can be easily reduced in the presence of electron donors [7, 8]. The NDs particles seem to serve as a good reducing agent for GO, where the hydroxyl groups on the NDs might most likely be converted to C=O bonds. In a control experiment, an aqueous solution of GO (without NDs) was heated at 100°C for 48 h and its oxidative degree was determined by XPS (Figure 2.32Ab). The XPS spectrum was deconvoluted into 4 components with binding energies at about 283.8, 284.7, 286.7 and 287.9 eV assigned to sp2- hybridized carbon, C-H/C-C, C-O and C=O species, respectively. The C/O ratio had somewhat increased to 2.8 due to a decrease in the C-O content. However, the component due to sp2-hybridized carbon remained rather unchanged. Water alone seems to be not sufficient to reduce GO to rGO under these experimental conditions. Independently, we found that amine-terminated nanodiamonds (ND-NH2) were not efficient for the GO reduction under our experimental conditions. Indeed, the reaction of GO with ND-NH2 at 100°C for 48 h induced only a slight shift (226 to 242 nm) of the maximum absorption in the UV-Vis spectra (data not shown). These results suggest that the presence of OH groups on the NDs surface is a prerequisite for efficient GO reduction. The XPS spectrum of the rGO/NDs with a ratio of 4/1

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) is deconvoluted into 4 components with binding energies at 283.4 eV (sp2-hybridized carbon), 285.5 eV (C-C/C-H), 286.7 (C-O) and 287.9 eV (C=O) (Figure 2.32Bb). The intensity of the band at 283.4 eV is largely increased compared to that obtained for rGO/NDs with a ratio of 1/1 with an increased C/O ratio to 4.5 and a total O1s content of 18.2 at%. Increasing the GO/NDs ratio further to 10/1 (Figure 2.32Bc) and 20/1 did not alter the XPS spectra further but increasing slightly the GO/NDs ratio to 4.7 (10/1) and 4.9 (20/1) with oxygen contents of 17.00 at% (10/1) and 16.19 at% (20/1). The overall oxygen content is about twice that reported for other chemically reduced graphene matrices without the addition of intercalating molecules (6.98-9.07%) [29]. However, it is comparable to rGO multi-layered structures prepared by microwave-assisted hydrothermal heating at 190°C with an oxygen content of 13- 16 at% [83].

C-O R-O C-O-C OHC=O OH

rGO/ND (20/1)

rGO/ND (10/1)

rGO/ND (4/1)

rGO/ND (1/1) Transmission ND-OH

1000 1500 2000 2500 -1 3000 3500 wavenumber / cm Figure 2.33: FTIR spectra of GO (black), hydroxylated ND (grey), rGO/ND (1/1) (green), rGO/ND (4/1) (blue), rGO/ND (10/1) (red) and rGO/ND (20/1) (violet).

FTIR was in addition used to elucidate the chemical composition of the rGO/NDs nanocomposites. Figure 2.33 displays the FTIR spectrum of GO. It consists of a strong and broad absorption band at 3393 cm-1 attributed to the vibration of hydroxyl groups or/and adsorbed water molecules. The sharp band at 1622 cm-1 is ascribed to the vibrations of residual water. The strong absorption band at 1734 cm-1 is assigned to the C=O stretching of –

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) COOH groups. Vibrational bands due to carboxy (1406 cm-1), epoxy (1227 cm-1), alkoxy (1058 cm-1) groups situated at the edges of the GO nanosheets are also visible. The FTIR spectrum of the NDs particles shows strong bands at 3419 cm-1 and 1622 cm-1 due to the -OH vibrations. For rGO/NDs nanocomposites, in all cases a strong absorption band at 1734 cm-1 (C=O) is present next to the band at 1622 cm-1 due to OH vibration. The formation of rGO was also validated by UV-Vis spectrosopy. The UV-Vis absorption spectra of GO and rGO/NDs in are seen in Figure 2.34A. The spectrum of the rGO/NDs nanocomposite varies significantly with the ratio of GO/NDs employed. The absorption band at 263 nm is relatively small for GO/NDs ratio of 1/1, but is significantly increased for higher ratios. The red shift from 226 nm (GO) to 263 nm for rGO/NDs is consistent with the restoration of the sp2 structure in rGO. The high absorption intensity at wavelengths higher than 300 nm is in addition consistent with the formation of reduced graphene.

0.8 (A) (B) GO (0.5 mg/mL) D G 0.7 ND-OH (34.3 µg/mL)

226nm rGO/ND (1/1)

0.6 263nm rGO/ND (4/1) rGO-ND (20/1) rGO/ND (10/1) 0.5 rGO/ND (20/1) rGO-ND (10/1) 0.4 rGO-ND (4/1) 0.3 Intensity rGO-ND (1/1) Absorbance / a.u. / Absorbance 0.2 GO 0.1 sp3 ND 0 x5 200 300 400 500 600 700 800 1200 1600 2000 2400 2800 Wavelength/ nm -1 wavenumber / cm Figure 2.34: (A) UV-Vis and (B) Raman spectra of GO (black), ND-OH (grey), rGO/ND (1/1) (green), rGO/ND (4/1) (blue), rGO/ND (10/1) (red) and rGO/ND (20/1) (violet).

Raman scattering is a useful tool to characterize the structural properties of graphene- based materials. Figure 2.34B shows the Raman spectra for GO and the rGO/NDs nanocomposites presenting the main features of graphene-based materials with a D-band at 1351 cm-1, a G-band at 1570 cm-1 and a small contribution ≈2700 cm-1 due to the 2D-band

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO)

[19]. The ratio of the intensities of the D and G bands (ID/IG) was found to be 0.71 for GO and

0.69 for the rGO/NDs nanocomposites. This is higher than the ID/IG=0.63 for hydrazine reduced graphene oxide [84], but lower than that of GO reduced by sodium borohydride

(ID/IG >1.0) [85], suggesting that the rGO/NDs nanocomposites have relatively little defects. (A)

1:1 4:1

200 nm 200 nm

10:1 20:1

200 nm

200 nm 200 nm

Figure 2.35: (A) SEM images and (B) typical HR-TEM images of rGO/ND matrices. The HR-TEM image of ND-OH is also included.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) Scanning electron microscopy (SEM) as well as transmission electron microscopy (TEM) images of the different rGO/NDs composites are displayed in (Figure 2.35A-B). Increasing the NDs concentration results in the formation of highly porous nanocomposites. TEM images show that the NDs particles are well dispersed in the rGO matrix.

0.3 0,3 (A) (B) 0.2 0,2

0.1 0,1

0 i / mA / i i / mA / i -0.1 GC -0,1 -0.2 rGO/ND (1/1) GO rGO/ND (1/1) rGO/ND (4/1) -0,2 rGO/ND (4/1) -0.3 rGO/ND (10/1) rGO/ND (10/1) rGO/ND (20/1) -0.4 -0,3 rGO/ND (20/1) -0.4 -0.2 0 0.2 0.4 0.6 0.8 -0,6 -0,4 -0,2 0 0,2 E / V vs. Ag/AgCl E / V vs. Ag/AgCl Figure 2.36: Cyclic voltammograms of a bare GC electrode before (doted black) and after coating with rGO/NDs (1/1) (green), rGO/NDs (4/1) (blue), rGO/NDs (10/1) (red) and 3-/4- rGO/NDs (20/1) (violet) recorded in (A) Fe(CN)6 (5 mM)/PBS (0.1 M) at scan rate of 100 -1 3+ -1 mV s and in (B) Ru(NH3)6 (5 mM)/PBS (0.1 M) at scan rate of 50 mV s .

To investigate the usefulness of the rGO/NDs composites as electrode materials, their electrochemical properties were evaluated. While GO is an insulating material, partial restoration of the sp2 network (as indicated above from XPS and UV-Vis results) of the different rGO/NDs matrices together with the reported electrochemical activity of NDs [82] should provide rGO/NDs with electrochemical activity. Figure 2.36 shows cyclic voltammograms of the different rGO/NDs matrices after drop-casting 1.5 mg/mL of the

3-/4- 3+ composite material onto GC electrode using Fe(CN)6 (Figure 2.36A) and Ru(NH3)6 (Figure 2.36B) as redox couples. There was a remarkable difference of the electrochemical activity between the GC electrodes with and without coating with rGO/NDs nanocomposites.

3-/4- In the case of Fe(CN)6 , the GC electrode showed a peak separation, E of 108 mV, which increased upon coating with rGO/NDs to 168 mV (1/1), 153 mV (4/1) and 190 mV (10/1). The electrochemical behavior of the rGO/NDs (20/1) nanocomposite is comparable to that of

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) 4/1 nanocomposite. This behavior is independent of the redox mediator and did not

3+ qualitatively change using Ru(NH3)6 . The results clearly suggest that a GC electrode coated with rGO/NDs (0.5 mg cm-2 mass loading) is more conducting compared to that coated with GO, where no electrical current could be detected under similar conditions. In Chapter 4, the rGO/NDs composites have been successfully used as electrodes in supercapacitors application. The electrochemical performance including the capacitive behavior of the rGO/ND composites were investigated by cyclic voltammetry and

-1 galvanostatic charge/discharge curves at 1 and 2 A g in 1M H2SO4. The rGO/ND matrix with 10/1 ratio displayed the best performance with a specific capacitance of 186±10 F g-1 and excellent cycling stability.

2.4. Conclusion In this chapter, four different environmental friendly approaches of reduction and functionalization of GO have been demonstrated. The structure, chemical composition, morphological properties, and electrochemical behavior of these resulting rGO nanomaterials were determined using different analytical methods (XPS, Raman, FTIR, UV-Vis, TEM, SEM, CV, etc). Table 2.4 summarises and compares the various rGO nanocomposites, including the samples of our work and also others from literature. In the next chapter, based on their own feature, the rGO nanocomposites we synthetized will be investigated for sensing and energy storage applications. The nancomposites comprise reduced GO and tyrosine, phenylboronic acid or diamond nanoparticles. In the case of phenylboronic acid not only the GO matrix was reduced to rGO but the oxidant also integrated in high amount onto the rGO sheets through π-π stacking interactions, resulting in simultaneous functionalization of the rGO matrix. As will be shown in the next chapter, the matrix can be further used for the interaction with sugars. In the case of tyrosine, only a small amount integrated into the rGO matrix. Its use as nonenzymatic sensing platform of hydrogen peroxide will be demonstrated. Finally, diamond nanoparticles resulted in the reduciton of GO and intercalated into the rGO sheets. This matrix was further used as efficient electrode in electrochemical supercapacitors. For the future scope of the approaches to produce rGO nanocomposites, in consideration

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) of heterogeneity in chemically derived graphene, size or mass-selection separation methods have to be developed and employed to narrow the size distribution of the graphene sheets and thus make them commercially available. In parallel, it has to be noted that the method for achieving large-scale production of highly reproducible graphene nanosheets is still in progress.

Table 2.4: Comparison of different kinds of rGO nanocomposites and GO.

ΔEp/ mV C/O I /I Samples D G Ref. ratio (Raman) 3-/4- 3+ Fe(CN)6 (5mM) Ru(NH3)6 (5mM) (at 100mV/s) (at 50mV/s)

Improved Hummer’s 1.73 0.68 insolating insolating [11] method GO Modifed Hummer’s 2.53 insolating insolating [86] method our rGO (by hydrazine) 6.8 0.84 169 145 work rGO (by hydrazine) 10.5 1.38 - - [87] our rGO (by tyrosine) 6.1 - 302 110 work our rGO/Tyrosine/Au NPs 3.7 226 160 work rGO by L-ascorbic acid 12.5 - - - [88] our rGO/Alkynyl-dopamine 3.6 0.67 186 140 work our rGO/Aminophenylboronic acid 3.8 - - 110 work rGO/N3-dopamine 3.3 - - 120 [8] rGO/TTF 5.49 - - 220 [6] rGO/NDs 1/1 3.76 168 192 rGO rGO/NDs 4/1 4.5 0.69±0.02 153 127 /Nanodiamond our rGO/NDs 10/1 4.9 190 263 particles work rGO/NDs 20/1 4.9 192 202

GO+H2O 100°C for 48 h 2.8 - insolating insolating

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) 2.5. References [1] Park, Sungjin, Ruoff Rodney S. Chemical methods for the production of graphenes. Nature Nanotechnology. 2009;4(4):217-24. [2] Stankovich, S., Dikin D. A., Piner R. D., Kohlhaas K. A., Kleinhammes A., Jia Y., et al. Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide. Carbon. 2007;45:1558-65. [3] McAllister, Michael J., Li Je-Luen, Adamson Douglas H., Schniepp Hannes C., Abdala Ahmed A., Liu Jun, et al. Single sheet functionalized graphene by oxidation and thermal expansion of graphite. Chemistry of Materials. 2007;19(18):4396-404. [4] Gao, Xingfa, Jang Joonkyung, Nagase Shigeru. Hydrazine and thermal reduction of graphene oxide: Reaction mechanisms, product structures, and reaction design. The Journal of Physical Chemistry C. 2009;114(2):832-42. [5] T. Kuila, S. Bose, A. K. Mishra, P. Khanra, N. H. Kim, J. H. Lee. Chemical functionalization of graphene and its applications. Progress in Materials Science. 2012;57:1061-105. [6] Kaminska, Izabela, Das Manash R., Coffinier Yannick, Niedziolka-Jonsson Joanna, Woisel Patrice, Opallo Marcin, et al. Preparation of graphene/tetrathiafulvalene nanocomposite switchable surfaces. Chemical Communications. 2012;48(9):1221-3. [7] Kaminska, Izabela, Barras Alexandre, Coffinier Yannick, Lisowski Wojciech, Roy Saumya, Niedziolka-Jonsson Joanna, et al. Preparation of a responsive carbohydrate- coated biointerface based on graphene/azido-terminated tetrathiafulvalene nanohybrid material. ACS Applied Materials & Interfaces. 2012;4(10):5386-93. [8] Kaminska, Izabela, Das Manash R, Coffinier Yannick, Niedziolka-Jonsson Joanna, Sobczak Jonusz, Woisel Patrice, et al. Reduction and functionalization of graphene oxide sheets using biomimetic dopamine derivatives in one step. ACS Applied Materials & Interfaces. 2012;4(2):1016-20. [9] Kaminska, Izabela, Qi Wang, Barras Alexandre, Sobczak Janusz, Niedziolka-Jonsson Joanna, Woisel Patrice, et al. Thiol–yne click reactions on alkynyl–dopamine-modified reduced graphene oxide. Chemistry – A European Journal. 2013;19(26):8673-8. [10] Titelman, G. I., GElman V., Bron S., Khalfin R. L., Cohen Y., Bianco-Peled H. Characteristics and microstructure of aqueous colloidal dispersions of graphite oxide. Carbon. 2005;43:641. [11] Marcano, Daniela C., Kosynkin Dmitry V., Berlin Jacob M., Sinitskii Alexander, Sun Zhengzong, Slesarev Alexander, et al. Improved synthesis of graphene oxide. ACS Nano. 2010;4(8):4806-14. [12] Zalan, Zita, Lazar Laszlo, Fulop Ferenc. Chemistry of hydrazinoalcohols and their heterocyclic derivatives. Part 1. Synthesis of hydrazinoalcohols. Current Organic Chemistry. 2005;9(4):357-76. [13] Liao, Ken-Hsuan, Mittal Anudha, Bose Shameek, Leighton Christopher, Mkhoyan K. Andre, Macosko Christopher W. Aqueous only route toward graphene from graphite oxide. ACS Nano. 2011;5(2):1253-8. [14] Tuinstra, F., Koenig J. L. Raman spectrum of graphite. The Journal of Chemical Physics. 1970;53(3):1126-30. [15] Paredes, J. I. , Villar-Rodil S., Solıs-Fernandez P., Martınez-Alonso A., Tascon J. M.

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) 2008;46(13):1795-7. [46] Lee, Haeshin, Dellatore Shara M, Miller William M, Messersmith Phillip B. Mussel- inspired surface chemistry for multifunctional coatings. Science. 2007;318(5849):426- 30. [47] Lee, Haeshin, Lee Bruce P, Messersmith Phillip B. A reversible wet/dry adhesive inspired by mussels and geckos. Nature. 2007;448(7151):338-41. [48] Lee, Haeshin, Lee Yuhan, Statz Andrea R, Rho Junsung, Park Tae Gwan, Messersmith Phillip B. Substrate-independent layer-by-layer assembly by using mussel-adhesive- inspired polymers. Advanced Materials. 2008;20(9):1619-23. [49] Xu, Li Qun, Yang Wen Jing, Neoh Koon-Gee, Kang En-Tang, Fu Guo Dong. Dopamine-induced reduction and functionalization of graphene oxide nanosheets. Macromolecules. 2010;43(20):8336-9. [50] O. Fellahi, M. R. Das, Y. Coffinier, S. Szunerits, T. Hadjersi, M. Maamache, et al. Silicon nanowire arrays-induced graphene oxide reduction under UV irradiation. Nanoscale. 2011;3:4662-9. [51] Wang, Hailiang, Robinson Joshua Tucker, Li Xiaolin, Dai Hongjie. Solvothermal reduction of chemically exfoliated graphene sheets. Journal of the American Chemical Society. 2009;131(29):9910-1. [52] Fan, Zhuangjun, Yan Jun, Zhi Linjie, Zhang Qiang, Wei Tong, Feng Jing, et al. A three-dimensional carbon nanotube/graphene sandwich and its application as electrode in supercapacitors. Advanced Materials. 2010;22(33):3723-8. [53] Meziane, Dalila, Barras Alexandre, Kromka Alexander, Houdkova Jana, Boukherroub Rabah, Szunerits Sabine. Thiol-yne reaction on boron-doped diamond electrodes: Application for the electrochemical detection of DNA–DNA hybridization events. Analytical Chemistry. 2011;84(1):194-200. [54] Weinrich, D., Lin P. C., Jonkheijm P., Nuguyen U. T. T., Schroder H., Niemeyer C. M., et al. Oriented immobilization of farnesylated proteins by the thiol-ene reaction. Angewandte Chemie International Edition. 2010;49:1252. [55] Lowe, A. B., Hoyle C. E., Bowman C. N. Thiol-yne click chemistry: A powerful and versatile methodology for materials synthesis. Journal of Materials Chemistry. 2010;20:4745. [56] Caipa Campos, M. A., Paulusse J. M. J., Zuilhof H. Functional monolayers on oxide- free silicon surfaces via thiol –ene click chemistry. Chemical Communications. 2010;46:5512. [57] Jonkheijm, P., Weinrich D., Kohn M., Engelkamp H., Christianen P. C. , Kuhlmann J., et al. Photochemical surface patterning by the thiol-ene reaction. Angewandte Chemie International Edition. 2008;47:4421. [58] Kolb, HC, Finn MG, Sharpless KB. Click chemistry: diverse chemical function from a few good reactions. Angewandte Chemie-International Edition. 2001;40(11):2004-21. [59] Hoyle, C. E., Lowe A. B., Bowman C. N. Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis. Chemical Society Reviews. 2010;39:1355. [60] Hensarling, Ryan M, Doughty Vanessa A, Chan Justin W, Patton Derek L. “Clicking” polymer brushes with thiol-yne chemistry: Indoors and out. Journal of the American

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CHAPTER 2 REDUCTION AND SIMULTANEOUS FUNCTIONALIZATION OF GRAPHENE OXIDE (GO) Chemical Society. 2009;131(41):14673-5. [61] Wendeln, Christian, Rinnen Stefan, Schulz Christian, Arlinghaus Heinrich F, Ravoo Bart Jan. Photochemical microcontact printing by thiol− ene and thiol− yne click chemistry. Langmuir. 2010;26(20):15966-71. [62] Gao, Jian, Liu Fang, Liu Yiliu, Ma Ning, Wang Zhiqiang, Zhang Xi. Environment- friendly method to produce graphene that employs Vitamin C and amino acid. Chemistry of Materials. 2010;22(7):2213-8. [63] Huang, Xiao, Qi Xiaoying, Boey Freddy, Zhang Hua. Graphene-based composites. Chemical Society Reviews. 2012;41(2):666-86. [64] Singh, Virendra, Joung Daeha, Zhai Lei, Das Soumen, Khondaker Saiful I, Seal Sudipta. Graphene based materials: past, present and future. Progress in materials science. 2011;56(8):1178-271. [65] Huang, Xiao, Yin Zongyou, Wu Shixin, Qi Xiaoying, He Qiyuan, Zhang Qichun, et al. Graphene-based materials: Synthesis, characterization, properties, and applications. Small. 2011;7(14):1876-902. [66] Qi, Xiaoying, Tan Chaoliang, Wei Jun, Zhang Hua. Synthesis of graphene–conjugated polymer nanocomposites for electronic device applications. Nanoscale. 2013;5(4):1440-51. [67] Huang, Xiao, Li Shaozhou, Huang Yizhong, Wu Shixin, Zhou Xiaozhu, Li Shuzhou, et al. Synthesis of hexagonal close-packed gold nanostructures. Nature Communications. 2011;2:292. [68] Toderas, Felicia, Baia Monica, Baia Lucian, Astilean Simion. Controlling gold nanoparticle assemblies for efficient surface-enhanced Raman scattering and localized surface plasmon resonance sensors. Nanotechnology. 2007;18(25):255702. [69] Tan, Shawn J, Campolongo Michael J, Luo Dan, Cheng Wenlong. Building plasmonic nanostructures with DNA. Nature Nanotechnology. 2011;6(5):268-76. [70] Hashmi, A. Stephen K., Hutchings Graham J. Gold catalysis. Angewandte Chemie International Edition. 2006;45(47):7896-936. [71] Maldotti, Andrea, Molinari Alessandra, Juarez Raquel, Garcia Hermenegildo. Photoinduced reactivity of Au-H intermediates in alcohol oxidation by gold nanoparticles supported on ceria. Chemical Science. 2011;2(9):1831-4. [72] Kominami, Hiroshi, Tanaka Atsuhiro, Hashimoto Keiji. Gold nanoparticles supported on cerium(IV) oxide powder for mineralization of organic acids in aqueous suspensions under irradiation of visible light of λ=530nm. Applied Catalysis A: General. 2011;397(1–2):121-6. [73] Zhang, Nana, Qiu Haixia, Liu Yu, Wang Wei, Li Yi, Wang Xiaodong, et al. Fabrication of gold nanoparticle/graphene oxide nanocomposites and their excellent catalytic performance. Journal of Materials Chemistry. 2011;21(30):11080-3. [74] Bei, Fengli, Hou Xueliang, Chang Shery LY, Simon George P, Li Dan. Interfacing colloidal graphene oxide sheets with gold nanoparticles. Chemistry-A European Journal. 2011;17(21):5958-64. [75] Guo, Shaojun, Li Jing, Ren Wen, Wen Dan, Dong Shaojun, Wang Erkang. Carbon nanotube/silica coaxial nanocable as a three-dimensional support for loading diverse ultra-high-density metal nanostructures: facile preparation and use as enhanced

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS

The detection of biologically active molecules is of critical importance from a biomedical, health-care, environmental and security point of view [1]. Such detection can be carried out by a sensor. Sensors are devices, which are composed of an active sensing material (a receptor) with a signal transducer (Figure 3.1) [1-3].

Figure 3.1: Scheme of a biosensor [1].

The receptor can be any organic or inorganic material with a specific interaction with one analyte or group of analytes, while the transducer converts chemical information into a measurable signal. In the case of biosensors, the biological sensitive platform selectively recognizes a particular biomolecule through a reaction, specific adsorption, or other physical or chemical process, and the transducer converts the result of this recognition into a usable signal, which can be quantified to show some specific details of this biomolecule. Based on the transducing mechanism, sensors can be classified as electrical, thermal or optical devices. Among these, electrochemical sensors possess advantages over other platforms, because their electrodes can sense materials present within the host without damaging the system [4]. Therefore, electrochemical sensors and biosensors have received increasing attention and

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS recently been developed by the low-dimensional nanotechnology, which paves the way for new materials and devices with desirable properties and useful functions [2, 5]. Recently, graphene modified interfaces have attracted enormous interest as alternative electrode platforms for sensitive and selective sensing [6, 7]. These composite interfaces have shown numerous unexpected electrochemical properties such as favorable electron transfer kinetics and enhanced electrochemical reactivity for different biomolecules, as compared to other carbon-based electrodes [7-10]. In this chapter, a variety of electrochemical sensing and biosensing platforms based on rGO composites we have synthesized (in Chapter 2) will be presented. In Section 3.1, rGO formed from GO through hydrazine reduction will be used for electrochemical detection of two catecholamines: L-dopa and carbidopa. In Section 3.2, the nonenzymatic detection of

H2O2 will be carried out by rGO obtained through chemical reduction with tyrosine. Section 3.4 deals with a novel strategy for the development of an enzyme-free sugar sensor based on the interaction with aminophenylboronic acid modified rGO.

3.1. Voltammetric detection of L-dopa and carbidopa 3.1.1. Introduction Catecholamines are hormones released by the adrenal gland in response to stress and are thus part of the sympathetic nervous system. In the human body, the most abundant catecholamines are epinephrine (adrenaline), norepinephrine and dopamine, all of which are produced from tyrosine and phenylalanine via L-dopa formation. Their chemical structures are related, containing a catechol (3,4-dihydroxyphenyl ) group, an intermediate ethyl chain and a terminal amine group. The biosynthesis of dopamine from L-dopa can be inhibited by another catechol analogue, carbidopa [(-)-L-2-(3,4-dihydroxybenzyl)-2-hydrazinopropionic acid]. Carbidopa is one of the most widely described drugs given to people with Parkinson’s disease in order to inhibit peripheral metabolism of L-dopa. Indeed, L-dopa prescribed as an active substrate is ineffective against Parkinson’s disease, since in extra cerebral tissue, L- dopa is metabolized to dopamine by a decarboxylation process. Thus, only a small fraction of uncreated carboxylated species is transported across the cerebral tissue to the central nervous system. When carbidopa is concomitantly used with L-dopa, as inhibitor for the

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS decarboxylase activity, a high concentration of L-dopa is formed. Hence, the development of a method for the simultaneous determination of L-dopa and carbidopa is very important, since they are frequently found together in pharmaceutical formulations. Several methods have been proposed for the simultaneous detection of these and other catecholamines. The most employed methods include spectrophotometry [11], high performance liquid chromatography (HPLC) with electrochemical detection [12], and capillary zone electrophoresis with electrochemical-based detection [13]. Electrochemical detection is indeed one of the most powerful techniques for trace analysis with high selectivity, often associated with high sensitivity and low cost. In spite of this, electrochemical studies of the determination of L-dopa and carbidopa are rare. The group of Anges investigated the possibility of analyzing L-dopa and carbidopa by differential pulse voltammetry (DPV) utilizing a glassy carbon (GC) electrode coated with a Nafion film, which is selective for carbidopa [14]. Correa de Melo et al. have shown that lead dioxide modified carbon past electrodes allowed simultaneous detection of L-dopa and carbidopa [15]. While sensitive detection of dopamine at multi-walled carbon nanotubes grafted silica network/gold nanoparticles functionalized nanocomposite electrodes has been reported [16] as well as enhanced dopamine detection using GC electrodes modified with multi-walled carbon nanotubes [17], the simultaneous detection of L-dopa and carbidopa has not been investigated on such carbon based materials. We have thus investigated the possibility for a simultaneous detection of L-dopa and carbidopa at neutral pH by differential pulse voltammetry (DPV) on GC electrodes modified with hydrazine reduced graphene oxide (rGO) (Section 2.1).

3.1.2. Electrochemical behavior of L-dopa and carbidopa on rGO modified GC electrode The electrochemical behavior of a 1 mM solution of L-dopa in 0.1 M KCl (pH 7.4) on GC/rGO interface is displayed in Figures 3.2. The first oxidative scan shows a quasi-

a reversible redox process with anodic peak potential of E 1=0.34 V/Ag/AgCl. This corresponds to the oxidation of L-dopa to open-chained quinone as schematically drawn in Figure 3.3. When the potential scan is reversed, the corresponding reduction peak is observed

c E 1=0.19 V/Ag/AgCl but with a diminished intensity. A new redox couple becomes apparent

a c after continuous cycling at more cathodic potentials (E 2=0.09 V/Ag/AgCl, E 2=-0.12

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS V/Ag/AgCl). This suggests the implication of a slow chemical follow up reaction after the oxidation of L-dopa , as reported by others GC electrodes [18]. Under neutral pH, sufficient unprotonated quinones are available to favor the cyclization reaction. The second redox couple corresponds thus to the oxidation of the cyclized product, cyclodopa, to dopachrome.

400 Ea 1 300

Ea 200 2

100 i / µA / i 0

-100

-200 c E Ec 2 1 -0.5 0 0.5 E / V (vs. Ag/AgCl) Figure 3.2: Cyclic voltammograms of an aqueous solution of L-dopa (1 mM) in KCl (0.1 M, pH = 7.2) recorded on GC/graphene electrode. Scan rate = 50 mV s-1, first scan (black line), 20th scan (red line).

Figure 3.3: Mechanism of L-dopa oxidation.

In the case of carbidopa (Figure 3.4), two irreversible oxidation waves with peak

a a potentials at E 1=0.37 V/Ag/AgCl and E 1=0.81 V/Ag/AgCl are observed. Even reversing the

a potential scan after E 1 revealed no reductive current. The second redox peak might be due to the oxidation of amide groups of the carbidopa to imides (Figure 3.5) [19]. Continuous cycling did not reveal the presence of an additional redox couple as in the case of L-dopa. On the other hand, the intensity of the second redox band decreased significantly due to a probable follow up chemical reaction of the formed catechol imide.

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS

600

400 E 2 a

1 i / µA / i E 200 a

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 E / V (vs. Ag/AgCl) Figure 3.4: Cyclic voltammograms of an aqueous solution of carbidopa (1 mM) in KCl (0.1 M) recorded on GC/rGO electrode; scan rate = 50 mV s-1, first scan (black line), 20th scan (red line).

HO COO- a O COO- O COO- +2e (E 1) + +e +2H + H C NH-NH + + N NH H3C NH-NH3+ c 3 3 -2H H3C 2 HO -2e (E 1) O O Figure 3.5: Mechanism of carbidopa oxidation.

3.1.3. Electrochemical quantification of L-dopa and carbidopa Before investigation of the possibility of simultaneous detection of both catechol derivatives, the electrochemical sensitivities of L-dopa and carbidopa on graphene-based interfaces were determined by DPV. DPV is an effective, selective and sensitive technique which is suitable for the quantitative evaluation of the two catecholamines. Figure 3.6 illustrates the voltammograms recorded in positive (Figure 3.6a) and negative potential scan direction (Figure 3.6b) of 1 mM solutions of L-dopa and carbidopa in 0.1 M KCl exhibiting the same electrochemical characteristics as discussed before. L-dopa and carbidopa show redox peaks at 0.34 V/Ag/AgCl while carbidopa shows a second band at 0.81 V/Ag/AgCl. DPV was used for the quantitative evaluation of L-dopa and carbidopa, based on the linear correlation between oxidation peak current and concentration of the two analytes.

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS

70 0 (a) 60 carbidopa -10 -20 50 carbidopa L-dopa -30 40

i / µA / i -40 30 µA / i -50 20 L-dopa -60

10 -70 (b) 0 -80 -0.2 0 0.2 0.4 0.6 0.8 1 -0.2 0 0.2 0.4 0.6 0.8 1 E/V (vs. Ag/AgCl) E/V (vs. Ag/AgCl) Figure 3.6: Differential pulse voltammogramms of 1 mM aqueous solutions of L-dopa (black) and carbidopa (red) recorded on GC/rGO electrode in KCl (0.1 M); scan rate = 50 mV s-1 ; (a) anodic scan, (b) cathodic scan.

Calibration plots are shown in Figure 3.7. The linear range for L-dopa was between 3×10-6 µM and 1.4 ×10-5 µM, a slope of (2.15±0.5) ×10-6 µA µM-1 with a limit of detection of 0.8 µM were determined, independent of the scan direction.

30 i / µA / i 20

0 0 10 20 30 40 50 60 [catecholamine]/ µM

Figure 3.7: Calibration plot for the two catecholamines L-dopa (●) and carbidopa (●) when scanned in anodic direction and (closed circles, ● and ●) and when scanned in cathodic scan (open circles ○ and○): calibration equation: I(L-dopa)=0.01+2.15[L-dopa] (R= 0.9997); I(carbidopa)=0.02+0.44[carbidopa] (R=0.9998).

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS For carbidopa, a linear concentration range between 4×10-6 µM and 1.6 ×10-5 µM, a slope of (0.42±0.3)×10-6 µA µM-1 with a limit of detection of 1.8 µM were determined. This is comparable to the detection limit of 0.17 µM reported for dopamine on multilayered graphene nanoflake films [20] and to the results of Angnes et al. for L-dopa and carbidopa using GC electrodes, with an increased linear working range [14]. In the case of carbidopa, the detection limit as well as the current sensitivity are comparable to a recently published paper by Brett et al. on the use of multi-walled carbon nanotubes and poly(Nile blue A) modified GC electrodes [21]. It is somehow better than the detection limit of 3.6 µM of carbidopa with a current sensitivity of 0.041 µA µM-1 reported using ferrocene-modified carbon nanotube past electrodes [22]. A linear range between 31 µM and 470 µM with a current sensitivity of 0.028 µA µM-1 and a limit of detection of 2.16 µM were reported by Zapata-Urzua et al. [23]. The detection limit obtained for L-dopa is also comparable with the recent paper by the group of Cassia Silva Luz using Co(DMG)2ClPy/multi-walled carbon nanotubes composite immobilized on basal plane pyrolytic graphite electrode with a detection

-1 limit of 0.86 μM and a sensitivity of 4.43 μA μM [24]. The most important advantage of using graphene over GC is in the simultaneous determination of L-dopa and carbidopa. On GC, L-dopa shows some interference with carbidopa detection. Using a carbon electrode with a Nafion film, which is selective for carbidopa, helped to overcome this limitation [14]. Another report used lead dioxide modified GC, which allowed detecting clearly high positive potential processes like the oxidation of carbidopa [15]. Herein, we demonstrate that direct electrochemical analysis of both catechols can be performed with a graphene modified GC electrode. Figure 3.8A shows the reductive DPV scan in the positive scan direction of a 400 µM solution of L-dopa alone and in the presence of increasing concentrations of carbidopa with a final concentration of 400 µM. It can be clearly seen that the reductive current due to the reduction of the quinone form of L- dopa remains unchanged even when carbidopa is present in high proportions. The limit of detection remained unchanged at around 0.9 µM. On the other hand, the second redox wave of carbidopa can be used to quantitatively detect carbidopa in the presence of L-dopa. Figure 3.8B shows a CV of carbidopa in the presence and absence of L-dopa. The presence of L- dopa with concentrations 4 times higher than carbidopa has no effect on the voltammetric

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS profile.

118 -116 (A) (B) -120 116

-124 114

-128

i / µA / i i / µA / i -132 112

-136 110 -140 108 0 0,2 0,4 0,6 0,8 0,6 0,65 0,7 0,75 0,8 0,85 0,9 E/V vs. Ag/AgCl E / V vs. Ag/AgCl Figure 3.8: Differential pulse voltammogramms for (A) L-dopa (400 µM) (black) with increasing amounts of carbidopa: 32 µM (grey), 100 µM (green), 400 µM (blue). (B) Carbidopa (100 µM) with increasing amounts of L-dopa: 32 µM (grey), 400 µM (green) recorded on GC/rGO electrode in KCl (0.1 M).

The effect of interference of ascorbic acid, uric acid, glucose and dopamine on the sensor response in the presence of L-dopa and carbidopa were additionally investigated. Equal concentration of ascorbic acid and uric acid with that of L-dopa and carbidopa are not influencing the electrochemical peak currents. However, 10 times higher ascorbic acid or uric acid concentrations influence strongly the detection of both L-dopa and carbidopa. In the case of glucose, the addition of 1-100 mM glucose resulted in a strong decrease of the peak currents due to L-dopa and carbidopa oxidation. This effect is most likely due to adsorption of glucose onto the graphene matrix [25], blocking partly the charge transfer through the matrix. Due to the almost equal redox potential of dopamine and L-dopa, the detection of L-dopa is highly influenced by the presence of dopamine, formed by biosynthesis from L-dopa.

3.1.4. Conclusion Glassy carbon electrodes, modified by drop casting with rGO, were successfully used to investigate the electrochemical behavior of L-dopa and carbidopa by cyclic voltammetric measurements. Voltammetric peak currents showed a linear response for both catecholamines in the range of 1-16 µM. The detection limit was about two times lower for L-dopa than

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS carbidopa being 0.8 µM and 1.8 µM, respectively with a current sensitivity of (2.15±0.5) and (0.48±0.3) µA µM-1. Simultaneous detection of both catecholamines can be achieved on these electrodes. Equivalent amounts of L-dopa and carbidopa have no effect on the detection limit of L-dopa. The results of this study indicate that the sensitivity of L-dopa on rGO electrodes exceeds 4 times that of carbidopa. The selectivity was achieved without the use of any additional surface modification step or membrane. The distinction of both catecholamines is simple based on the difference in the oxidative redox potential and the oxidation mechanism. While L-dopa showed a reversible oxidation in neutral solutions, carbidopa exhibits an irreversible oxidation followed by a slow chemical reaction of the electro-oxidative products. The technique reported in this work represents thus an alternative analytical approach to control the content of these catecholamines in pharmaceuticals.

3.2. Electrochemical detection of hydrogen peroxide (H2O2) using tyrosine reduced GO (rGO/Tyr) 3.2.1. Introduction . Reactive oxygen species (ROS), including hydroxyl radicals (HO ), superoxide anion

.- (O2 ) and hydrogen peroxide (H2O2) are produced in various physiological processes and can be used as an early indicator for cytotoxic events and cellular disorders [26, 27]. Among all

ROS, H2O2 is the most stable species and can be particularly harmful since it can diffuse across membranes through water channels and cause biological modifications such as peroxidation of cell membrane lipids, DNA bases and backbone hydroxylation at distal areas

[26]. H2O2 is also a by-product of many oxidative biological reactions, including those of glucose oxidase, cholesterol oxidase, alcohol oxidase, galactose oxidase, etc, and an essential mediator in food, pharmaceutical, clinical, industrial and environmental analyses [28]. Thus it is of prime importance to design biosensors for detection of H2O2 with high sensitivity, good stability, large detection range and good selectivity. By taking advantage of the sensitivity and selectivity of enzymatic reactions, highly sensitive electrochemical detection schemes of

H2O2 were usually achieved by modifying the surface of electrodes with enzymes. While enzymatic biosensors for H2O2 detection have shown good performances with low detection

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS limits, there are several drawbacks associated with enzyme-modified electrodes such as the high cost of enzymes, long-term stability and complexity of immobilization [28]. Furthermore, the enzyme activity is highly affected by the temperature and the pH of the sensing medium. To alleviate these hurdles, there has been recently a huge focus on the development of nonenzymatic sensors for the detection of H2O2 and other bio-relevant species [3]. In this area, graphene has shown promise for non-enzymatic H2O2 detection. Li et al. have prepared a nonenzymatic H2O2 sensor based on manganese dioxide (MnO2)/graphene oxide (GO) nanocomposite with a good activity in alkaline medium [29]. The sensor displayed a detection limit of 0.8 µM and a long-term stability. The good performance of the sensor was attributed to the high surface area of GO combined to the good catalytic activity of MnO2 nanoparticles. Zhang et al. developed a novel electrocatalytic sensor based on functionalized reduced graphene oxide (rGO) with conducting polypyrrole graft copolymer, poly(styrenesulfonic acid-g-pyrrole) (PSSA-g-PPY/rGO) via π-π non covalent interaction [30]. The nanocomposite showed high electrocatalytic activity toward H2O2 oxidation in neutral media with a detection limit of 10 nM. Graphene/metal nanoparticles have also been investigated for enzyme-free detection of H2O2 [31-48]. The sensors exhibited detection limits ranging from 50 nM to 35 µM, depending on the metal nature, the deposition mode and the supporting electrode material. Graphene decorated with CdS nanocrystals has been successfully applied for electrochemiluminescence (ECL) detection of H2O2. The sensor exhibited a linear range from 5 µM to 1 mM with a detection limit of 1.7 µM with excellent reproducibility and long-term stability [23]. Similarly, sensors based on Prussian blue/GO [49], Nafion/GO/Co3O4 [50] and 3D carbon micropillars/rGO [51] have achieved good performances for enzyme-free detection of H2O2. Zhang et al. reported on the use of graphene quantum dots (GQDs), 30 nm in diameter, assembled on Au electrode for the detection of H2O2 in human breast adenocarcinoma cell line MCF-7. The H2O2 release was triggered by injecting phorbol myristate acetate (PMA), a compound that can induce H2O2 generation in cells [52]. In this section, we discuss our results on nonenzymatic hydrogen peroxide detection using tyrosine-reduced GO (rGO/Tyr) (refer to Section 2.22 for the composite preparation and characterization).

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS

3.2.2. rGO/Tyrosine for nonenzymatic amperometric H2O2 detection

To assess the electrocatalytic activity of rGO/Tyr for hydrogen peroxide (H2O2) reduction, linear sweep voltammetry was performed on the rGO/Tyr/GC electrode in PBS solution (pH 7.4) by addition of H2O2 (Figure 3.9A). Addition of increasing amounts of H2O2 resulted in an increase of the reduction peak at -0.5 V with a linear range up to 2mM (Figure 3.9B).

0 0 mM -100 0.2 mM -20 0.8 mM -80 -40 1 mM -60

-60 i / µA / i

i / µA / i -80 -40 -100 -20 -120 (A) (B) 5 mM -140 0 -1 -0,8 -0,6 -0,4 -0,2 0 0 1 2 3 4 5 6 [H O ] / mM E / V (vs. Ag/AgCl) 2 2 Figure 3.9: (A) Linear sweep voltammograms of rGO/Tyr/GCE in PBS solution (pH 7.4) with different concentrations of H2O2: 0 (a), 200 µM (b), 800 µM (c), 1 mM (d), 5 mM (e). Scan rate: 50 mV/s; (B): Calibration curve (linear up to 2 mM).

Chronoamperometic detection of H2O2 was performed in addition. Figure 3.10 exhibits the typical current-time plot of the rGO/Tyr/GCE in PBS solution (pH 7.4) on successive step change of H2O2 concentrations under optimized conditions.

When an aliquot of H2O2 was added into the stirring PBS solution, the electrode responded rapidly to the substrate and the current rose steeply to reach a stable value. At the applied potential of -0.55 V, the cathode current of the sensor increased significantly and achieved 95 % of the steady-state current within 25 s, indicating a fast amperometric response behavior. The calibration curve of the sensor is shown in the inset. A linear detection range from 0.1 to 2 mM (r = 0.999) and a sensitivity of 69.07 µA mM-1 cm-2 can be estimated. A detection limit of 80 µM at a signal-to-noise ratio of 3 was achieved using the rGO/Tyr/GCE sensor. This value is lower than those reported for metal nanoparticles/rGO sensors (Table 3.1). However given the ease of preparation of the electrode material and the good sensitivity

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS of detection, there is a room for improvement of the detection limit by incorporating metal or metal oxide nanoparticles in the rGO/Tyr matrix.

-60

-50

-40

-60

-30 -50

-40

-30 -20 µA / i

-20

-10 -10 0 0 0,5 1 1,5 2 2,5 3 [H O ] / mM 0 2 2 0 5 10 15 20 25 30 time / min. Figure 3.10: Amperometric response curve at rGO/Tyr/GCE polarized at -0.55 V vs. Ag/AgCl with subsequent addition of H2O2 (200 µM), inset: calibration curve ([H2O2] =-1.34-20.72x, R=0.999).

It is well known that some co-existing electroactive species such as uric acid (UA), ascorbic acid (AA), dopamine (DA) and glucose in real samples will affect the sensor response. These species generally show serious interference for electrochemical H2O2 detection, which limits the practical application of the sensor. Herein, the influence of common interference species such as UA, AA, DA and glucose was thus investigated using their relevant physiological levels by chronoamperometry [34]. Figure 3.11 exhibits the amperometric response to the consecutive injection of 2 mM H2O2 and 0.1 mM of interfering species: UA, AA and DA, and 5 mM glucose. The working potential was hold at -0.5 V. An obvious increase of the current was observed when 2 mM H2O2 was added to the PBS solution. However, when the interfering substances were injected in the PBS solution containing 2 mM H2O2, no evident increase of the current was noticed. The results suggest that the interfering effect caused by these electroactive species is quite negligible, indicating a high selectivity of rGO/Tyr/GCE electrode for H2O2 detection.

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS

Table 3.1: Comparison of the performance of various nonenzymatic H2O2 sensors

Detection Electrode Sensitivity Linear range Reference limit -1 -2 GO/MnO2 0.8 µM 38.2 µM mM cm 5-600 µM [29] PSSA-g-PPY/rGO 10 nM 673 µM mM-1 cm-2 3x10-8-2.8x10-5 M [30] AgPd/rGO 1.4 µM - 0.01-1.4 mM [53] Pt/rGO 0.8 µM - 2.5-6650 μM [31] PtNPs/PANI/rGO 50 nM 857 µA mM-1 cm-2 - [45] AuEPG 0.1 µM 75.9 µA mM-1 0.5 μM - 4.9 mM [40] AuNPs/rGO paper 2 µM 236.8 μA cm-2 mM-1. 0.005-8.6 mM [44] AuNPs/rGO 6 µM 3 µA µM-1 cm-1 20 -280 µM [36] AgNPs/PQ11/rGO 28 μM - 100 μM - 40 mM [43] AuNPs/POM/rGO 1.5 μM 58.87 μA cm-2 mM-1 5μM – 18mM [48] AgNCs/rGO 3 μM 183.5 µA cm-2 mM-1 20μM – 10mM [33] AgNPs/rGO 1.80μM - 0.1 - 60 mM [34] AgNPs/rGO 0.5 μM - 0.1 - 100 mM [43] AgNPs/rGO 31.3 μM - 100 μM - 100 mM [54] AgNPs/Aniline/rGO 7.1 μM - 100 μM - 80 mM [37] AgNPs/rGO/AuE 1.9 μM - 0.1mM - 20mM [38] AgNPs/rGO 3.6 μM - 0.1 – 100mM [55] AgNPs/PDDA/rGO 35 μM - 100μM - 41mM [56] PtNPs/PMAA/rGO 80 nM - 1μM - 500μM [57] PtAuNPs/rGO/CNTs 0.6 μM - 2.0 - 8561 μM [47] PdNPs/rGO 0.05 μM - 0.1 μM to 1.0 mM [40] CdS/rGO 1.7 μM - 5 μM – 1 mM [23] PB/GO 0.122μM 408.7 µA mM-1 cm-2 5.0μM - 1.2mM [49] -1 -2 Nafion/EGO/Co3O4 0.3 µM 560 µA mmol L cm - [58] 3D carbon - 0.07 µA µM-1 cm-2 250 µM - 5.5 mM [51] micropillars/rGO GQDs/Au 0.7 μM - 0.002 - 8 mM [52]

Finally, the stability of the rGO/Tyr/GCE electrode was examined after 3 days storage in a refrigerator at 4ºC. The sensor retained about 95 % of its initial sensitivity for 1mM H2O2 detection, indicating that the electrode is of good stability. The result is comparable to those reported for other graphene-based electrode materials for H2O2 detection [34, 36]. It is to be noticed that the stability of the sensor is most likely linked to the preparation method.

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-150

H O 2 2 H O -100 2 2 UA AA DA Glucose

-50 i / µA i/

50 0 5 10 15 20 Time/min

Figure 3.11: Chronoamperometry curves of the rGO/Tyr/GCE exposed to H2O2 (2 mM), UA (0.1 mM), AA (0.1 mM), DA (0.1 mM), glucose (5 mM), and H2O2 (2 mM). Applied potential: - 0.5 V; PBS: 0.1 M, pH 7.4.

3.2.3. Conclusion The interest of using a tyrosine-reduced graphene oxide matrix as hydrogen peroxide sensing platform was demonstrated. Glassy carbon electrodes modified with rGO/Tyr exhibited good electrocatalytic activity toward H2O2 reduction with sensitivity of 69.07 µA mM-1 cm-2, a linear range between 0.1 to 2 mM and a limit of detection of 80 µM.

3.3. Electrochemical detection of sugars using 4-aminophenylboronic acid modified reduced graphene oxide (rGO/APBA) interfaces 3.3.1. Introduction Diabetes has been assigned next to cancer, cardiovascular and chronic respiratory diseases as the most leading causes of death and disability. A close monitoring of blood glucose concentrations plays a significant role in the diagnosis and prevention of diabetes. Tremendous efforts have been put into the development of efficient, reliable and sensitive methods to determine blood glucose and electrochemical-based sensors have been considered as excellent analytical tools for rapid and inexpensive glucose-recognition [28, 59]. The most serious problem connected with enzyme-based glucose sensors is the insufficient stability of the enzyme. The electrochemical determination of glucose concentrations without using

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS glucose oxidase (GOx) is one goal that many research teams have been trying to make come true. Initial research focused on the use of noble metals, such as mesoporous platinum [60]. The desire for better and cheaper electrocatalysts has resulted in the development of bimetallic systems [61] or in the fabrication of diamond nanowires [62] and diamond nanowires coated with nickel nanoparticles [63] for direct glucose oxidation in alkaline solutions. A different approach in exploiting enzyme-less electrochemical sensor designs is based on the use of boronic acid compounds [64-69]. Boronic acid derivatives are important ligands for specific recognition of cis-diol containing molecules such as saccharides by forming five or six-membered cyclic esters in alkaline solutions, while the cyclic esters dissociate when the medium is changed to acidic pH. Multi-walled carbon nanotubes (MWCNT) have been reported as an ideal supporting matrix for the electrocatalytic oxidation of glucose [70]. Unfortunately, the electrocatalytic effect of MWCNT was observed only under basic conditions. Another carbon-based material that has received much attention in recent years is reduced graphene oxide (rGO) [71-74]. The large surface area of rGO together with its good electrical conductivity, possibility of production in bulk quantities and ease of processing have made it particularly attractive for electrochemical-based enzymatic glucose sensing [75-83]. There are however only some reports on nonenzymatic graphene based glucose sensors using rGO [55, 84, 85] operated in alkaline media. Herein, we report on the fabrication of reduced graphene oxide-aminophenyl boronic acid (rGO/APBA) hybrid material, which can directly detect sugars such as fructose, mannose or glucose with high sensitivity without the need for any enzyme using differential pulse voltammetry (DPV). The preparation and characterization of rGO/APBA is discussed in Section 2.2.4.

3.3.2. Electrochemical sugar sensing It is well known that phenylboronic acid derivatives can preferentially bind with vicinal diols via cyclic ester bond formation (Figure 3.12A).

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS

(A)

(B)

Figure 3.12: (A) Mechanism by which -glucose can be linked to rGO/APBA nanocomposite interfaces and (B) detection principle employed.

4- The voltammetric behaviour of a negative charged redox couple such as Fe(CN)3 will be modulated in the presence of rGO/APBA at different pH. Figure 3.13A shows CVs of the

4- Fe(CN)3 redox species on rGO/APBA modified GCE at pH 7.4 and pH 12. A clear visible CV was observed under acidic conditions in both cases, indicating that the redox reaction of

4- Fe(CN)3 occurred unhindered across the rGO/APBA matrix. In contrast, the CV was significantly attenuated at pH 12 for rGO/APBA due to the electrostatic repulsion between the negatively charged redox species and the negatively charged rGO/APBA matrix originating from the addition of OH- ions to the boron atom in alkaline medium (Figure 3.12A). To ensure that the change is not linked to the rGO matrix itself, hydrazine-reduced GO modified electrode was tested and no pH dependent variation of the redox current was observed

3+ (Figure 3.13C). The use of ruthenium hexamine ([Ru(NH3)6] ), a positively charged redox

3+ couple, was also tested. No reproducible results could be achieved as [Ru(NH3)6] was unstable at basic pH and showed significant changes with rGO even in the absence of APBA.

4- The change in current of Fe(CN)3 can however be used to determine the surface pKa value of the rGO/APBA matrix [69] (Figure 3.14A), which was estimated as pKa = 10.6±0.2 for rGO/APBA.

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS

(A) (B) 300 300 in the absence of fructose in the presence of 50 mM fructose pH=7.4 pH=12 200 200

pH=7.4 pH=12 100 100

i / µA i / 0

i / µA / i 0

-100 -100

-200 -200

-0,4 -0,2 0 0,2 0,4 0,6 0,8 -0,4 -0,2 0 0,2 0,4 0,6 0,8 E / V vs. Ag/AgCl E / V vs. Ag/AgCl

300 (C) pH = 12 pH = 7.4 200

100

i / µA / i 0

-100

-200

-300 -0,4 -0,2 0 0,2 0,4 0,6 0,8 E / V vs. Ag/AgCl 4- Figure 3.13: Cyclic voltammograms recorded in 5 mM Fe(CN)6 /PBS on GC/rGO/APBA in the absence (A) and presence of 50 mM fructose (B), and on rGO prepared through GO reduction using hydrazine (C) at pH 7.4 (black) and pH 12 (grey); scan rate: 100 mV s-1.

Similar measurements were performed in the presence of fructose, mannose or glucose (Figure 3.13B). In the case of fructose, the CV recorded at pH 7.4 showed a decreased peak current when compared to the same measurements without the formation of the boronic acid- fructose complex (Figure 3.13A). This indicates a priori that already at this pH the effect of sugar binding is already non-negligible. From Figure 3.14A, a pKa of 7.9±0.2 was deduced for rGO/APBA after fructose binding, being more acidic than rGO/APBA. In the case of mannose and glucose interactions the pKa values were, as expected, less acidic with a pKa=8.5±0.2 for mannose and pKa=9.3±0.2 for glucose in line with the selectivity observed for phenylboronic acid based sensors for sugar detection being most sensitive to fructose.

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(A) (B) 140 -10 rGO/APBA rGO/APBA/fructose 120 rGO/APBA/mannose rGO/APBA/glucose -15 100

80 -20 i / µA / i 19 µM

60 i / µA / i

40 -25

20 0.5 µM rGO/APBA/fructose -30 0 µM 0 4 6 8 10 12 14 pH 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

-25

-20

i / µA / i -15

-10

-5 glucose mannose fructose 0 0 10 20 30 40 50 60 70 80 [sugar] / µM 4- Figure 3.14: (A) Change in anodic peak currents for 5 mM Fe(CN)6 /PBS as a function of solution pH on GC/rGO/ABPA (black) and GC/rGO/APBA-fructose (green), GC/rGO/APBA- mannose (red), GC/rGO/APBA-glucose (blue); (B) Differential pulse voltammograms of rGO/APBA modified GCE at different fructose concentrations (0, 0.5 µM, 19 µM); (C) Calibration curves of rGO/APBA modified GC electrodes to D-glucose (red, pH 8.2) , D- mannose (blue, pH 7.7) and D-fructose (green, pH 7.4).

Differential pulse voltammetry (DPV) was used to investigate the behavior of rGO/APBA for their potential as sugar sensors (Figure 3.14B). A linear relation between current and fructose concentration in the range of 0.2-60 µM was obtained with a correlation coefficient of r=0.999 according to i(µA)=-29.89+0.38[fructose] (Figure 3.14C). The detection limit of fructose was determined to be 100 nM from five blank noise signals (95 % confidence level). The detection of nanomolar levels of fructose has until now only been possible with the use of optical sensors such as surface plasmon resonance [86]. The

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS reproducibility of the electrodes is expressed in terms of the relative standard deviation which is found to be 4.3 % at a fructose concentration of 20 µM. For mannose and glucose, a detection limit of 0.8 µM with a linear range between 1-60 µM (i(µA)=-29.99+0.33[glucose]; i(µA)=-29.96+0.36[mannose]) were observed. The high sensitivity for fructose is in line with the selectivity observed for phenylboronic acid based sensors for sugar detection [59, 87]. Electrochemical detection schemes using phenylboronic acid based sugar sensors are reported to be suitable for the detection of micro and millimolar levels of sugar [67-69]. The detection limit of glucose is comparable to other non-enzymatic glucose sensors based on Ni nanowires arrays (LOD=0.1 µM) [88] or Cu nanoparticles modified graphene (LOD=0.5 µM) [89]. These sensors work however only under strongly alkaline conditions (0.1 M NaOH). In order to examine the suitability of the sensor within more demanding matrices, the rGO/BA matrix was used to detect the saccharide concentration in apple juice. The sample was diluted 10000 times and the overall saccharide concentration was determined by using the fructose calibration curve in Figure 3.14C. The saccharide content was determined as 40 µM (7.2 µg/mL) which results in a saccharide concentration of 0.4 M (72 mg/mL) in the apple juice. This sugar concentration is comparable to the concentration of 74 mg/mL determined using the well-established phenol-sulfuric acid colorimetric method for the analysis of carbohydrates [90], underlining the interest of the analytical approach described in this work (Figure 3.12).

3.3.3. Conclusion The interest of a reduced graphene oxide matrix modified with 4-aminophenyl boronic acid as sugar sensing platform has been demonstrated. The phenylboronic acid modified rGO

4- showed large redox currents at neutral pH using Fe(CN)3 as redox species, which were strongly modulated by the presence of glycans. In the case of fructose, a remarkable detection limit of 100 nM was obtained. For mannose and glucose, the detection limits were about eight times higher (800 nM). The sensor proved to be suitable for the investigation of sugar content of more complex matrices such as apple juice and thus represents an interesting alternative to other sensors so far reported. An additional advantage is the long term stability of the sensing matrix (rGO/APBA) and thus the sensor when stored at 4°C as a powder in the case of the

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS rGO/APBA matrix and in water once deposited on the electrical interface. The electrochemically detected signal of a 1 µM fructose solution after 2 months storage is comparable (±2 %) to the initial current reading.

3.4. Summary In this chapter, the utility of reduced graphene oxide and functional reduced graphene oxide matrices for different sensing applications has been shown. Table 3.2 overviews their notable performances and compares them with other materials reported in the literature.

Table 3.2: Reduced graphene oxide composites as electrochemical sensor and biosensors.

Performance Applications Samples Ref. Detection limit Linear range 0.8 μM for L-dopa 1-14 μM for L-dopa rGO by hydrazine Our work 1.8 μM for carbidopa 3-16 μM for carbidopa Sensing L- A carbon paste electrode modified 25 μM for L-dopa 0.2-1.2 mM for L-dopa dopa and with lead dioxide immobilized in a [15] 3.7 μM for carbidopa 32-150 μM for carbidopa carbidopa resin 5.1 2μM for L-dopa Glassy carbon - [91] 2.16μM for carbidopa rGO by tyrosine 80 µM 0.1 to 2 mM Our work Detection of AuNPs/rGO 6 µM 20 -280 µM [36]

H2O2 GO/MnO2 0.8 µM 5-600 µM [29] PSSA-g-PPY/rGO 10 nM 3x10-8-2.8x10-5 M [30] 10 0nM for fructose 0.2-60 µM for fructose rGO/aminophenylboronic acid 0.8µM for mannose 1-60 µM for mannose Our work 0.8µM for glucose 1-60 µM for glucose 0.2 -2 μmol/L (DPV) Sugar sensing GO- thionine–Au nanocompostites 0.05 µM for glucose [55] 2 - 22 µM (LSV)

Graphene wrapped Cu2O nanocubes 3.3 μM for glucose 0.3 to 3.3 mM for glucose [56] GOD/rGO 10 ± 2 μM for glucose 0.1–10 mM for glucose [79] GOD/rGO/Pt NPs/chitosan 0.6 μM for glucose 0.15 - 4.2 mM for glucose [92]

More work remains to be done in facilitating the practical applications of rGO composites and broadening the scope of their application fields in the future. Specific attention should be paid on a range of current challenges, such as more facile and controlled

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CHAPTER 3 REDUCED GRAPHENE OXIDE BASED ELECTROCHEMICAL SENSORS AND BIOSENSORS processing of rGO composites with high quality and longevity, the large-scale production and the microstructure management so that they can be used in lab-on-top nanodevices. The improvement of these properties will afford materials suitable for sensing, being more sensitive, stable, and so on. Undoubtedly, the future of rGO composites remains very bright and exciting.

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES

With the ever increasing demand for energy coupled with power shortages and high prices in today’s globalized world, there has been a tremendous drive to study advanced energy storage and management devices. A supercapacitor, also called ultracapacitor or electrochemical capacitor, is a device based on rapid and reversible storage and release of energy within electrochemical double-layer capacitor [1]. Supercapacitors have attracted particular attention in light of their high energy density, high power density, fast charge propagation and charge-discharge processes, long cycling life, as well as low maintenance [2, 3]. These devices can thus be used for a multitude of purposes such as portable electronics, memory back-up systems and environmentally friendly hybrid electrical vehicles [2-5]. A typical supercapacitor comprises dual electrodes separated via a porous separator in an electrolyte medium (Figure 4.1). When a voltage is supplied, accumulation of opposite charges at two electrodes would happen and these charges would generate an electric field that allows the supercapacitor to store energy [6].

Figure 4.1: Schematic of 2 cell electrode configurations with both electric double layer capacitance (EDLC) and pseudocapacitance.

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES What appeals to the researchers are two main parameters: the energy and power density. Figure 4.2 shows the Ragone plot of energy density vs. power density for various energy- storing devices, and reveals the promise and advantages of supercapacitors, in comparison with other energy storage devices.

Figure 4.2: Comparison of power and energy density obtained with different energy storage devices [6].

Energy density is the ability to store energy and it determines how long the supercapacitor can act as a power source. The energy storage capability E of a supercapacitor is represented by equation 4.1. 1 E= CV2 (Eq. 4.1) 2 where C represents the capacitance and V the cell voltage. Since the energy density is just simply the energy stored per unit volume or per unit mass and shows a positive linear dependence on the capacitance, therefore much research has been conducted in order to achieve the highest possible capacitance in supercapacitors. Capacitance is the ratio of charge stored to voltage applied and the specific capacitance C (F g-1) can be described by equation 4.2.

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES

A C= ε ε (Eq. 4.2) 0 r d

-1 where ε0 (Fm ) and εr (a dimensionless constant) represent the permittivity of vacuum and the relative permittivity, respectively, A (m2 g-1) is the specific surface area of the electrode accessible to the electrolyte ions, and d (m) is the effective distance between the electrodes. From the equation 4.2, it can be observed that one of the main ways to improve the capacitance is to increase the surface area of the electrodes. As graphene has an extensive surface area, it makes it a good candidate as an electrode material. Power density is another important parameter of supercapacitors as it determines how fast the energy could be discharged or charged. The maximum power achievable by the supercapacitor is given in equation 4.3.

V2 P= (Eq. 4.3) 4 ESR where V is the cell voltage and ESR in ohms is the equivalent series resistance. Therefore, one incentive to improving the power density would be to reduce the resistance of the electrode. Based on their capacitance generating mechanism, supercapacitors can be categorized into two forms [6]. The electric double layer capacitance (EDLC) type generates capacitance from the separation of charges at the interface between the electrode and the electrolyte and this attribute can be improved by optimizing the pore volume and size distribution, hierarchical structure and interconnection between macropores and mesopores as well as by expanding the specific surface area of the material. Due to the nature of EDLC, charges are not transferred between the electrode and the electrolyte thus making charge storage highly reversible and this increases the cycling stability of the supercapacitor [7]. The pseudocapacitance, on the other hand, generates capacitance from rapid Faradaic reactions within the electrode material [8, 9]. These fast faradaic reactions may include redox reactions, intercalation and electrosorption processes [7]. EDL capacitors have good electrochemical cyclic stability, but their capacitance value is often low. Pseudo-capacitors exhibit high

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES capacitance but relatively poor cyclic stability. Comprehensive utilization of both electrode materials has been confirmed to be an effective method by which high performance may be achieved. The superior supercapacitor is based on predominantly electrostatic storage of electrical energy and is determined by the combination of a high surface area activated material and a nanoscopic charge separation at the electrode-electrolyte interface [10]. Carbon materials such as activated carbon, mesoporous carbon and carbon nanotubes usually display good stability, but the capacitance values are limited by the microstructures in the materials [11, 12]. Graphene is an obvious material of choice for supercapacitors, offering high intrinsic electrical conductivity, an accessible and defined pore structure, good resistance to oxidative processes and high temperature stability [13]. Currently the prototype graphene-based electrochemical supercapacitors (Figure 4.3) lead the field in capacitance as well as energy and power densities [14]. To exploit the potential of graphene-based materials for supercapacitor applications, different approaches have been considered [14-16].

Figure 4.3: Schematic illustration of a graphene based supercapacitor device. Two high surface area graphene-based electrodes (blue and purple hexagonal planes) are separated by a membrane (yellow). Upon charging, anions (white and blue merged spheres) and cations (red spheres) of the electrolyte accumulate at the vicinity of the graphene surface. The ions are electrically isolated from the carbon material by the electrochemical double layer that is serving as a molecular dielectric [10].

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES In this chapter, the promising supercapacitance behavior of reduced graphene oxide/nanodiamond particles (rGO/NDs) composites has been explored, which expands the potential applications in the field of supercapacitors.

4.1. Introduction of rGO based supercapacitors Chemically reduced graphene oxide, synthesized through hydrazine reduction of graphene oxide (GO), has been the first explored graphene-based electrochemical double- layer capacitor with specific capacitance values of 135 F g-1 in aqueous electrolytes [14]. Chen et al. investigated the capacitive properties of partially reduced graphene oxide, prepared by the reaction of GO with hydrobromic acid. The presence of oxygen functional groups on the rGO facilitated the penetration of the electrolyte, introducing additional pseudocapacitive effects. As a result, specific capacitance values of 348 and 158 F g-1 have been measured in 1M H2SO4 and 1-butylimidazolium hexafluorophosphate (BMIPF6), respectively at a current density of 0.2 A g-1 [17]. Graphene-based electrodes prepared simply through chemical and/or thermal reduction still do not have sufficient large pores due to agglomeration of the rGO sheets and do not facilitate the access of the electrolyte. Consequently, high specific capacitance values were in most cases only achievable by charge/discharging at current densities below 1 A g-1 [18, 19]. Less-agglomerated graphene- based electrodes with suitable pore sizes are still highly demanded. Self-assembled graphene hydrogels formed by chemical reduction of GO with sodium ascorbate has shown to possess well-defined and cross-linked 3D porous structure with specific capacitance of 240 F g-1 at a

-1 discharge current density of 1.2 A g in 1M H2SO4 [19]. To inhibit agglomeration of reduced graphene oxide through electrostatic interactions and providing at the same time open nanochannels, incorporation of intercalating spacers is a promising strategy. Organic molecules such as 1-pyrenecarboxylic acid [20] or tetrabutylammonium hydroxide [21] as well as metallic nanoparticles [22] were used as spacers and resulted in specific capacitances of 120 F g-1 (in 6 M KOH) [20], 194 F g-1 (at 1 A

-1 -1 g in 2M H2SO4) [21] or 269 F g in 0.5 M H2SO4 [22]. The use of carbon nanotubes for supercapacitors and as spacer in graphene-based hybrid films has shown to increase the specific capacitance to 385 F g-1 at a scan rate of 10 mV s-1 in 6 M KOH [23].

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES In this chapter, we report on the preparation of reduced graphene oxide/nanodiamonds (rGO/NDs) composites using a solution phase process. The direct reaction of an aqueous solution of GO and NDs at different GO/NDs ratios at 100°C for 48 h gave the corresponding composites with enhanced properties. The GO matrix was partially reduced to rGO under these conditions, while the NDs particles were intercalated into the rGO sheets. This results in a significant improvement of the dispersibility of the rGO/NDs matrix in polar solvents such as ethanol and nonpolar ones like DMF with the suspensions being stable for several weeks.

4.2. Evaluation method of the supercapacitor behavior of rGO/NDs The supercapacitor behavior of rGO/NDs was investigated using a three-electrode system comprised of rGO/NDs as a working electrode, Pt foil as a counter electrode and Ag/AgCl (3M KCl) as a reference electrode. The CV and galvanostatic charge/discharge tests were performed using a VMP3 Bio Logic Science Instruments (France). To prepare rGO/NDs electrodes, rGO/NDs particles and polyvinylidene fluoride (PVDF) at the weight ratio of 90:10 were dispersed in N-methyl-2-pyrrolidone at a concentration of 20 mg/mL and 20 µL of the dispersion were deposited onto a platinum foil (A=0.5 cm2). The active mass is around

400 µg. For all electrochemical tests 1M H2SO4 was used as electrolyte, which was purged with N2 for 10 min to remove dissolved oxygen. CV curves were recorded from 0 to 0.8 V with a scan rate of 10 mV s-1. The galvanotastic charge/discharge tests were performed from 0 to 0.8 V at the current density of 1 and 2 A g-1. The specific capacitance C (F g-1) was calculated by integrating the area under the CV curve according to Equation 4.4:

1 V C = ∫ U I(V)(dV) (Eq. 4.4) m∙v∙(VU-VL) VL

-1 where m is the mass of active material (g), v is the scan rate (V s ), VU and VL are the upper and lower voltage limits (V), and I the current (A). The specific capacitance C (F g-1) was in addition calculated from the slope of the galvanostatic discharge curve after the ohmic drop according to Equation 4.5:

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES

i C= dV (Eq. 4.5) m ( ⁄dt) where i is the applied galvanostatic current (A), dV/dt is the slope of the discharge curve, and m is the active mass.

4.3. Supercapacitor behavior of rGO/NDs To check if the observed electrochemical conductivity is elevated enough to enable current collection; we investigated the supercapacitor behavior of the rGO/NDs composite electrodes. In contrast to conventional high surface area materials such as conducting polymers and nonporous electrode materials like carbon nanotubes, the effective surface area of graphene and its derivatives is not solely dependent on the distribution of pores in the solid state [14]. The chemical state is an additional important parameter to take into consideration. The presence of oxygen functional groups enhances generally the wettability and the capacitance values of graphene-based supercapacitors [24]. More specifically, quinone type functions provide pseudo-capacitance through Faradic redox reactions, which are pH dependent and more pronounced when the pH of the electrolyte is below 3 [25]. In the case of rGO/NDs, quinone type functions are present on the NDs surface due to oxidation of C-OH to C=O during the reduction of GO to rGO. The rectangular shape indicates that the main contribution to the capacitance is the charge and discharge of the double layer, which is enhanced by the presence of NDs working probably as spacers. The rGO/NDs (10/1) matrix shows in addition a pseudocapacitance contribution, mostly likely due to the presence of quinone-functions in the matrix. Similar results have been reported for rGO prepared by electrochemical reduction of GO in N2-purged PBS [24] or through reaction with hydrobromic acid [17]. They are also present on rGO and both might be responsible for a pseudocapacitance contribution. Figure 4.4A shows the cyclic voltammograms of the different interfaces in 1 M H2SO4. Beside the rGO/NDs (1/1) composite, rectangle-like shaped curves characteristic of capacitive behavior are observed with specific capacitance varying between 9-186 F g-1 depending on the rGO/NDs nanocomposite (Table 4.1). The best

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES performance is observed for rGO/NDs (10/1) with a specific capacitance of 186 F g-1. Matrices with rGO/NDs ratio of 20/1 resulted in a lower capacitance of 120 F g-1, indicating that maximal values are achieved with a 10/1 ratio. Highly NDs loaded rGO matrices (1/1) show low specific capacitance values of 9 F g-1, increasing to 114 F g-1 for the 4/1 matrices (Table 4.1).

Figure 4.4: (A) Cyclic voltammograms of rGO/ND nanocomposites in 1 M H2SO4; scan rate = 10 mVs-1; (B) Galvanostatic charge/discharge profile of the different rGO/NDs nanocomposites at current density of 1 A g-1 (dotted line) and 2 A g-1 (full line) in the potential window ranging between 0–1 V; (C) Cyclic voltammograms of rGO/NDs (10/1) -1 nanocomposites (blue: reduced with hydrazine) in 1M H2SO4; scan rate=10 mVs . (D) Specific capacitance of rGO/ND 10/1 versus cycle number at a current density of 2 A g-1.

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES The supercapacitor behavior of the different nanocomposites was further studied by galvanostatic tests at different current densities. Due to the poor capacitance of rGO/NDs (1/1), this sample was not investigated further. Figure 4.4B shows galvanostatic charge/discharge profiles at current densities of 1 or 2 A g-1. The discharge curves somewhat deviate from linearity due to the ohmic drop. The specific capacitance of each sample was calculated from the linear part of the discharge curves (0 – 0.3 V). As shown in Table 4.1, the values of the specific capacitance obtained by the galvanostatic discharge curves agree with the values obtained by the CV curves. The decrease in specific capacitance at higher current densities is due to fast kinetics.

Table 4.1: Characteristics of NDs, GO and rGO/NDs nanocomposites

Specific capacitance (F/g) in 1M H2SO4 electrolyte

Product Size/nm Zeta/mV Galvanostatic Galvanostatic CV discharge discharge (10 mV/s) 1 A g-1 2 A g-1

NDs 31±6 17±1 - - -

GO 151±1 -41±4 - - -

rGO/NDs (1/1) 225±2 -30±1 9±2 - -

rGO/NDs (4/1) 239±7 -34±2 114±8 137±15 125±11

rGO/NDs (10/1) 289±18 -37±2 186±10 169±13 143±11

rGO/NDs (20/1) 298±9 -42±2 120±5 122±10 111±10

Furthermore, we investigated the capacitive properties of rGO/NDs reduced through hydrazine. In fact, it is well established that GO reduction with hazardous hydrazine gives the best performance in terms of electrical conductivity. The reduction of GO/NDs (ratio=10/1) in the presence of hydrazine under otherwise similar conditions afforded rGO/NDs composite with a specific capacitance of 241 F g-1 (Figure 4.4C). This is higher than the capacitance value (186 F g-1) recorded for GO/NDs (ratio=10/1) although the shape of the voltammogram is very similar with the presence of a pseudocapacitive component. This result suggests that

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES even a strong reducing agent such as hydrazine is not capable of removing all electrochemically active oxygen-containing groups even though the electrical properties of the composite have been improved (Figure 4.4C). Due to the most promising performance of rGO/NDs 10/1, the stability of this sample in a long term galvanostatic cycling was tested. Figure 4.4D shows the specific capacitance of rGO/NDs 10/1 versus cycle number. The sample was cycled at 2 A g-1 for 1000 cycles showing a stable performance upon cycling. The performance of rGO/NDs 10/1 is comparable to the graphene-based supercapacitors reported elsewhere [14, 26-28].

4.4. Conclusion Conducting reduced graphene oxide matrices with intercalated nanodiamond particles of different densities were prepared by simple mixing GO with nanodiamonds and heating at 100°C for 48 h. Electrochemical investigation of the different rGO/NDs nanocomposites showed that the electrochemical behavior depends on the initial ratio of rGO/NDs used for the formation of the nanocomposites. The rGO/NDs composites have been successfully used as electrodes in supercapacitors with a maximum specific capacitance of 186±10 F g-1 in 1M

-1 H2SO4 at a current density of 1 A g . These findings may have important consequences for technological applications in the field of supercapacitors. Table 4.2 compares the supercapacitor behavior of rGO/NDs (10/1) composites investigated in this work with some other graphene based nanocomposites reported in the literature. Although the characteristics of graphene supercapacitors are very encouraging, there are still issues which must be addressed before the commercial use of such systems [10]. In particular, the irreversible capacitance of graphene-based supercapacitors is still too high, which could probably be improved by controlling the number of defects or choosing a better electrolyte.

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES

Table 4.2: Comparison of supercapacitor behavior of a range of graphene based materials.

Specific capacitance Graphene nanocomposites Cyclic ability Ref. (F/g)

186±10 (at 10 mV/s) rGO/NDs (10/1) Stable after 1000 cycles our work 169±13 (at 1 A/g)

Graphene/nickel foam 164 (at 10 mV/s) 61% remained after 700 cycles [29]

rGO, thermal reduction (dry) 150 (at 0.1 A/g) 100% remained after 500 cycles [30]

135 (in an aqueous dispersion)

rGO, chemical reduction 99 (in organic dispersion) - [14]

at constant current of 10 mA

rGO from graphite oxide 205 (at 0.1 A/g) 90% remained after 1200 cycles [16]

Ultrathin graphene film 135 (at 0.75 A/g) - [31] (25-100 nm thick)

Graphene/CNT/PANI 1035 (at 1 mV/s) 94% remained after 1000 cycles [32]

Graphene/CNT sandwich 385 (at 10 mV/s) 80% remained after 2000 cycles [23]

Nickel (II) hydroxide

nanocrystals deposited on 1335 (at 2.8 A/g) - [33]

graphene nanosheets

Graphphene/cobalt(II) hydroxide 972.5 (at 0. 5 A/g) - [34] nanocomposite

97.9% remained after 1000 RuO2/Graphene nanosheets 570 (at 0.1 A/g) [35] cycles

Graphene/PANI 1046 (at 1 mV/s) - [36] (in situ polymerization)

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES 4.5. References [1] Winter, Martin, Brodd Ralph J. What are batteries, fuel cells, and supercapacitors? Chemical Reviews. 2004;104(10):4245-70. [2] Sun, Yiqing, Wu Qiong, Shi Gaoquan. Graphene based new energy materials. Energy & Environmental Science. 2011;4(4):1113-32. [3] Brownson, Dale AC, Kampouris Dimitrios K, Banks Craig E. An overview of graphene in energy production and storage applications. Journal of Power Sources. 2011;196(11):4873-85. [4] Brownson, Dale AC, Banks Craig E. Graphene electrochemistry: an overview of potential applications. Analyst. 2010;135(11):2768-78. [5] Brownson, Dale AC, Kampouris Dimitrios K, Banks Craig E. Graphene electrochemistry: fundamental concepts through to prominent applications. Chemical Society Reviews. 2012;41(21):6944-76. [6] Tan, Yu Bin, Lee Jong-Min. Graphene for supercapacitor applications. Journal of Materials Chemistry A. 2013. [7] Halper, Marin S, Ellenbogen James C. Supercapacitors: A brief overview. The MITRE Corporation, McLean, Virginia. 2006. [8] Perera, Sanjaya D, Mariano Ruperto G, Nijem Nour, Chabal Yves, Ferraris John P, Balkus Jr Kenneth J. Alkaline deoxygenated graphene oxide for supercapacitor applications: An effective green alternative for chemically reduced graphene. Journal of Power Sources. 2012;215:1-10. [9] Ramaprabhu, S. Poly (p-phenylenediamine)/graphene nanocomposites for supercapacitor applications. Journal of Materials Chemistry. 2012;22(36):18775-83. [10] Novoselov, KS, Fal VI, Colombo L, Gellert PR, Schwab MG, Kim K. A roadmap for graphene. Nature. 2012;490(7419):192-200. [11] Zhang, Li Li, Zhao X. S. Carbon-based materials as supercapacitor electrodes. Chemical Society Reviews. 2009;38(9):2520-31. [12] Futaba, Don N., Hata Kenji, Yamada Takeo, Hiraoka Tatsuki, Hayamizu Yuhei, Kakudate Yozo, et al. Shape-engineerable and highly densely packed single-walled carbon nanotubes and their application as super-capacitor electrodes. Nature Materials. 2006;5(12):987-94. [13] Geim, Andre K, Novoselov Konstantin S. The rise of graphene. Nature Materials. 2007;6(3):183-91. [14] Stoller, Meryl D., Park Sungjin, Zhu Yanwu, An Jinho, Ruoff Rodney S. Graphene- based ultracapacitors. Nano Letters. 2008;8(10):3498-502. [15] Wang, Da-Wei, Li Feng, Wu Zhong-Shuai, Ren Wencai, Cheng Hui-Ming. Electrochemical interfacial capacitance in multilayer graphene sheets: Dependence on number of stacking layers. Electrochemistry Communications. 2009;11(9):1729-32. [16] Wang, Yan, Shi Zhiqiang, Huang Yi, Ma Yanfeng, Wang Chengyang, Chen Mingming, et al. Supercapacitor devices based on graphene materials. The Journal of Physical Chemistry C. 2009;113(30):13103-7. [17] Chen, Yao, Zhang Xiong, Zhang Dacheng, Yu Peng, Ma Yanwei. High performance supercapacitors based on reduced graphene oxide in aqueous and ionic liquid electrolytes. Carbon. 2011;49(2):573-80.

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES [18] Balandin, Alexander A., Ghosh Suchismita, Bao Wenzhong, Calizo Irene, Teweldebrhan Desalegne, Miao Feng, et al. Superior thermal conductivity of single- layer graphene. Nano Letters. 2008;8(3):902-7. [19] Xu, Yuxi, Sheng Kaixuan, Li Chun, Shi Gaoquan. Self-assembled graphene hydrogel via a one-step hydrothermal process. ACS Nano. 2010;4(7):4324-30. [20] Wang, Kun, Liu Qian, Wu Xiang-Yang, Guan Qing-Meng, Li He-Nan. Graphene

enhanced electrochemiluminescence of CdS nanocrystal for H2O2 sensing. Talanta. 2010;82(1):372-6. [21] Zhang, Yao, Sun Xiumei, Zhu Longzhang, Shen Hebai, Jia Nengqin. Electrochemical sensing based on graphene oxide/Prussian blue hybrid film modified electrode. Electrochimica Acta. 2011;56(3):1239-45. [22] Si, Yongchao, Samulski Edward T. Exfoliated graphene separated by platinum nanoparticles. Chemistry of Materials. 2008;20(21):6792-7. [23] Fan, Zhuangjun, Yan Jun, Zhi Linjie, Zhang Qiang, Wei Tong, Feng Jing, et al. A three-dimensional carbon nanotube/graphene sandwich and its application as electrode in supercapacitors. Advanced Materials. 2010;22(33):3723-8. [24] Yang, Jiang, Gunasekaran Sundaram. Electrochemically reduced graphene oxide sheets for use in high performance supercapacitors. Carbon. 2013;51(0):36-44. [25] Bleda-Martínez, María Jesús, Maciá-Agulló Juan Antonio, Lozano-Castelló Dolores, Morallon E, Cazorla-Amorós Diego, Linares-Solano A. Role of surface chemistry on electric double layer capacitance of carbon materials. Carbon. 2005;43(13):2677-84. [26] Li, Yueming, van Zijll Marshall, Chiang Shirley, Pan Ning. KOH modified graphene nanosheets for supercapacitor electrodes. Journal of Power Sources. 2011;196(14):6003-6. [27] Lee, Jeong Woo, Ko Jang Myoun, Kim Jong-Duk. Hydrothermal preparation of nitrogen-doped graphene sheets via hexamethylenetetramine for application as supercapacitor electrodes. Electrochimica Acta. 2012;85(0):459-66. [28] Sun, Dongfei, Yan Xingbin, Lang Junwei, Xue Qunji. High performance supercapacitor electrode based on graphene paper via flame-induced reduction of graphene oxide paper. Journal of Power Sources. 2013;222(0):52-8. [29] Chen, Yao, Zhang Xiong, Yu Peng, Ma Yanwei. Electrophoretic deposition of graphene nanosheets on nickel foams for electrochemical capacitors. Journal of Power Sources. 2010;195(9):3031-5. [30] Du, Xian, Guo Peng, Song Huaihe, Chen Xiaohong. Graphene nanosheets as electrode material for electric double-layer capacitors. Electrochimica Acta. 2010;55(16):4812-9. [31] Yu, Aiping, Roes Isaac, Davies Aaron, Chen Zhongwei. Ultrathin, transparent, and flexible graphene films for supercapacitor application. Applied Physics Letters. 2010;96(25):253105--3. [32] Shao, Yuyan, Wang Jun, Engelhard Mark, Wang Chongmin, Lin Yuehe. Facile and controllable electrochemical reduction of graphene oxide and its applications. Journal of Materials Chemistry. 2010;20(4):743-8.

[33] Wang, Hailiang, Casalongue Hernan Sanchez, Liang Yongye, Dai Hongjie. Ni(OH)2 nanoplates grown on graphene as advanced electrochemical pseudocapacitor materials. Journal of the American Chemical Society. 2010;132(21):7472-7.

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CHAPTER 4 NANODIAMOND PARTICLES/REDUCED GRAPHENE OXIDE COMPOSITES AS EFFICIENT SUPERCAPACITOR ELECTRODES [34] Chen, Sheng, Zhu Junwu, Wang Xin. One-step synthesis of graphene−cobalt hydroxide nanocomposites and their electrochemical properties. The Journal of Physical Chemistry C. 2010;114(27):11829-34. [35] Wu, Zhong-Shuai, Wang Da-Wei, Ren Wencai, Zhao Jinping, Zhou Guangmin, Li

Feng, et al. Anchoring hydrous RuO2 on graphene sheets for high-performance electrochemical capacitors. Advanced Functional Materials. 2010;20(20):3595-602. [36] Yan, Jun, Wei Tong, Shao Bo, Fan Zhuangjun, Qian Weizhong, Zhang Milin, et al. Preparation of a graphene nanosheet/polyaniline composite with high specific capacitance. Carbon. 2010;48(2):487-93.

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CHAPTER 5 CONCLUSION AND PERSPECTIVES

The discovery of graphene by Geim and Novoselov in 2004 sent ripples of excitement through the whole scientific community. In the past years, the initial interest in the so-called “wonder” materials has not faded, with a huge number of potential applications already proposed ranging from sensors to transparent conductive films, liquid crystal devices, photocatalysts, and supercapacitors. There is obviously still much work to be done to understand the properties of graphene and fully develop its potential. One specific branch of graphene research dealing with GO (graphene oxide) and rGO (reduced graphene oxide) composites has been popular and a matter of a huge focus from the scientific community. This is specifically motivated by the potential applications expected from these composite materials. Compared to pristine graphene, the presence of functional groups and structural defects (edge plane like-sites/defects) confers to GO and rGO some unique properties (such as the possession of electrochemical active sites, favorable electron transfer, and electrocatalytic activity), and offers enormous possibilities in many fields including sensors and energy storage and conversion devices. Specific attention is currently paid to the development of environmentally friendly chemical approaches for the synthesis of rGO on a large scale. In this thesis, we have developed four new approaches to achieve this goal based on the use of different organic reducing agents. Alkynyl-modified dopamine, tyrosine, 4-(aminophenyl) boronic acid and diamond nanoparticles were investigated in this work for their reducing power and the possibility to simultaneous functionalize the formed rGO. In the case of alkynyl-dopamine modified rGO, the robustness of the developed matrix, based on non-covalent π-π stacking of dopamine derivative on the graphene skeleton, was evaluated by the post-functionalization with thiolated molecules via a photochemical “click” reaction. Using tyrosine as reducing agent allowed simultaneous reduction of gold ions and GO and thus formation of rGO/gold nanoparticle composites. rGO nanocomposites showed

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CHAPTER 5 CONCLUSION AND PERSPECTIVES remarkable performance in electrochemical sensing and biosensing, and also in energy storage applications. The favorable electron transfer kinetics on rGO allowed for the simultaneous detection of L-dopa and carbidopa, two catecholamines, in an easy manner. Non-enzymatic electrochemical detection of glucose was achieved on 4-aminophenylboronic acid modified rGO. The specific interaction between the boronic acid functions on the rGO and the cis-diol groups of the glycan was used as sensing strategy. The fabricated sensor exhibited a wide linear range with detection limits of 100 nM for fructose, and around 800 nM for mannose and glucose. Similarly, tyrosine reduced rGO was successfully used for nonenzymatic hydrogen peroxide detection with a wide linear range from 100 µM to 2.5 mM, a detection limit of 80 µM and a sensitivity of 69.07 µA mM-1 cm-2. On the other hand, the promising supercapacitance behavior of rGO/nanodiamond particles composites has been explored. The intercalation of nanodiamonds particles and its oxidative strength towards the reduction of GO, produced conductive, less-agglomerated, and porous rGO material with a good specific capacitance of 186 F g-1 and excellent long term stability. These findings may have important consequences for technological applications in the field of supercapacitors. More work remains to be done in improving the properties and facilitating the practical applications of rGO composites and broadening the scope of their application fields in the future. Specific thoughts should be paid on a range of current challenges, such as more facile and controlled processing of rGO composites with high quality and longevity, the configuration of exquisite rGO electrodes with faster and more stable electron transfer, the large-scale production and the microstructure management so that they can be used in lab-on- top nanodevices. The improvement of these properties will afford materials suitable for sensing, being more sensitive, stable, and so on. Undoubtedly, the future of rGO composites remains very bright and exciting. However, the potential of other carbon based materials for sensing should not be underestimated. While not discussed in this manuscript, diamond interfaces have shown to be of great interest for biosensing applications mainly due to their physicochemical stability and biocompatibility. We have thus devoted some time to this material during my PhD and have

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CHAPTER 5 CONCLUSION AND PERSPECTIVES shown that boron-doped diamond (BDD) electrodes and BDD nanowires can be used efficiently for the development of electrochemical sensors [1, 2]. The diamond nanowires were obtained by a maskless reactive ion etching process with oxygen plasma using polycrystalline boron-doped diamond (BDD) films as starting material. The resulting nanowire interfaces showed enhanced electrochemical activity towards the direct electrochemical detection of glucose under strong basic (pH 12) condition with a detection limit of 60 μM and a linear range up to 8 mM [2]. The BDD NWs allowed also the simultaneous detection of two amino acids: tryptophan and tyrosine, when the ratio of tryptophan/tyrosine is ≤ 0.5. These electrodes work moreover well under physiological conditions and allowed for an accurate analysis of both amino acids in human serum spiked with tyrosine and tryptophan, making them appealing materials for electroanalysis [1] . Furthermore, amine-terminated undoped diamond films when modified with horseradish peroxidase proved to be efficient sensors for the detection of hydrogen peroxide in the nanomolar concentration regime [3].

Reference [1] Wang, Qi, Vasilescu Alina, Subramanian Palaniappan, Vezeanu Alis, Andrei Veronica, Coffinier Yannick, et al. Simultaneous electrochemical detection of tryptophan and tyrosine using boron-doped diamond and diamond nanowire electrodes. Electrochemistry Communications. 2013;35(0):84-7. [2] Wang, Qi, Subramanian Palaniappan, Li Musen, Yeap Weng Siang, Haenen Ken, Coffinier Yannick, et al. Non-enzymatic glucose sensing on long and short diamond nanowire electrodes. Electrochemistry Communications. 2013;34(0):286-90. [3] Wang, Qi, Kromka Alexander, Houdkova Jana, Babchenko Oleg, Rezek Bohuslav, Li Musen, et al. Nanomolar Hydrogen Peroxide Detection Using Horseradish Peroxidase Covalently Linked to Undoped Nanocrystalline Diamond Surfaces. Langmuir. 2011;28(1):587-92.

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CHAPTER 5 CONCLUSION AND PERSPECTIVES

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APPENDIX EXPERIMENTAL PART

6.1. Materials 6.1.1. Chemicals All chemicals were reagent grade or higher and were used as received unless otherwise specified. Graphite powder (< 20 micron), potassium permanganate (KMnO4), sulphuric acid

(H2SO4), phosphoric acid (H3PO4), hydrogen peroxide (H2O2), hydrazine hydrate (N2H4·H2O), ammonia (NH3), dimethylformamide (DMF), dimethylsulfoxide (DMSO), potassium chloride

(KCl), potassium ferrohexacyanide [K4Fe(CN)6], potassium ferricyanide [K3Fe(CN)6],

3+ ruthenium hexamine [Ru(NH3)6] , dopamine·HCl, L-dopa, S-(−)-Carbidopa, L-tyrosine, potassium bromide (KBr), gold(III) chloride trihydrate (HAuCl4), 6-(ferrocenyl)-hexanethiol (HS-Fc), phosphate buffered saline (PBS), 1H,1H,2H,2H-perfluorodecanethiol (HS-PF), phenol, ethanol (EtOH), acetone, tetrahydrofuran (THF), 4-aminophenylboronic acid hydrochloride, D-(+)-Glucose, D-(+)-Mannose, D-(−)-Fructose, acetonitrile (CH3CN), toluene, N-methyl-2-pyrrolidone (NMP), polyvinylidene fluoride (PVDF), sodium hydroxide (NaOH), hydrochloride (HCl), platinum wire and indium tin oxide (ITO, sheet resistivity: 15-25 Ω/sq) were purchased from Sigma-Aldrich. Silicon wafers were purchased from Siltronics. Glassy carbon (3 mm in diameter) and Ag/AgCl reference electrodes were obtained from Cambria Scientific. Hydroxylated diamond particles (ND-OH particles) were obtained from the International Technology Centre, Raleigh, NC, USA. Zirconia beads were obtained from Tosoh Co. (YTZ Grinding Media, 0.05 mm). The water used throughout the experiments in this thesis was purified with a Milli-Q system from Millipore Co. (resistivity = 18MΩ.cm). All glassware or PTFE (polytetrafluoroethene) containers were cleaned with a piranha

(H2SO4: H2O2=3:1 mixture) solution for 30 min, followed by copious rinsing with Milli-Q water until the water pH becomes neutral, then dried under a nitrogen stream. Before using, all containers were dried in an oven at 80°C more than 1 h.

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Safety considerations

The mixture H2SO4/H2O2 (piranha) solution is a strong oxidant. It reacts violently with organic materials. It can cause severe skin burns. It must be handled with extreme care in a well-ventilated fume hood while wearing appropriate chemical safety protection. HF is a hazardous acid which can result in serious tissue damage if burns were not appropriately treated. Etching of silicon should be performed in a well-ventilated fumehood with appropriate safety considerations: face shield and double layered nitrile gloves.

6.1.2. Synthesis of pro-2-yn-1-yl-5-((3,4-dihydroxyphenetyl)amino)-5-oxopentanoate (alkynyl-terminated dopamine)

Dopamine hydrochloride (1.453 g, 9.49 mmol) was dissolved in MeOH (50 mL). Triehylamine (0.959 g, 9.49 mmol) and 2, 5-dioxopyrrolidon-1-yl-prop-2-yne-1-yl glutarate (2.31 g, 8.63 mmol, formed from 5-oxo-5-(prop-2-yn-1-yloxy)pentanoic acid by reaction with N-hydroxysuccinimide)) dissolved in MeOH (20 mL) were added slowly. The solution was stirred overnight at room temperature under N2. The solvent was evaporated and the product dissolved in CH2Cl2 (100 mL). The organic layer was washed with HCl (0.5 M, 50 mL), water

(2×50 mL) and dried over Na2SO4. After filtration, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography (SiO2/ CH2Cl2: MeOH 10:1) affording the alkynyl-terminated dopamine in 80% yield as a white solid. 1H NMR (300 MHz, DMSO (d6)), δ (ppm from TMS): δ 1.73 (quin, 2H), 2.08 (t, 4H), 2.32 (t, 2H), 3.15 (q, 2H), 3.55 (t, 1H), 4.68 (d, 2H), 6.42 (dd, 1H), 6.56 (d, 1H), 6.62 (d, 1H), 7.86 (t, 1H), 8.66 (bs, 1H), 8.76 (bs, 1H).

6.2. Preparation of reduced graphene oxide (rGO) 6.2.1. Preparation of graphene oxide

A 9:1 mixture of concentrated H2SO4/H3PO4 (90:10 mL) was added to a mixture of graphite flakes (0.75 g) and KMnO4 (4.85 g). The reaction was then heated to 50°C and stirred for 12 h. The reaction was cooled to room temperature and poured into ice (100 mL) with slowly adding 30% H2O2 (0.75 mL). The solid product was separated by centrifugation.

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It was first washed with 30% HCl 2 times, secondly washed with 5% HCl solution 3-5 times until the sulphate ions are removed. Then washed with distilled water repeatedly until it becomes free of chloride ions and the pH of the solution is neutral, and finally washed 3–4 times with ethanol. The material remaining after this extended multiple-wash process was coagulated with 100 mL of ether, and the resulting suspension was filtered over a PVDF membrane with a 0.45 μm pore size. The solid graphite oxide obtained on the filter was vacuum-dried overnight at room temperature. A homogeneous yellow brown suspension (0.5 mg/mL) of GO sheets in water was achieved by ultrasonication for 3 h.

6.2.2. Reduction of graphene oxide with hydrazine hydrate In a typical procedure, hydrazine hydrate (0.50 mL, 32.1 mM) was added to 5 mL of the yellow-brown GO aqueous suspension (0.5 mg/mL) in a round bottom flask and heated in an oil bath at 100°C for 24 h over which the reduced GO gradually precipitated out the solution. The product was isolated by filtration over a PVDF membrane with a 0.45 μm pore size, washed copiously with water (5×20 mL) and methanol (5×20 mL), and dried [2].

6.2.3. Reduction of graphene oxide using tyrosine 10 mM L-tyrosine was added to 0.5 mg/mL homogeneous water suspension of GO nanosheets. The mixture was kept under stirring for 24 h at 100°C. The resulting black precipitate (rGO /Tyr) was separated from the aqueous supernatant by centrifugation at 14 000 rpm for 20 min. After washing with ethanol (3 times) and Milli-Q water (3 times), the precipitate was dried in an oven (80°C) and then dispersed in DMF with the aid of ultrasonication for 30 min.

6.2.4. Preparation of rGO/Tyr/Au NPs The reaction of an 20 mL aqueous solution GO (0.5 mg/mL) and tyrosine (10 mM) at

100°C for 24 h produced a black precipitate, then 1 mL HAuCl4 (40 mM) was added into the mixture while keeping the temperature at 100°C and stirring for 3 h. The formed dark purple precipitate, which could be easily separated from the supernatant via centrifugation, was

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APPENDIX EXPERIMENTAL PART washed three times with Milli-Q water. The obtained rGO/Tyr/Au NPs nanocomposite can be facilely re-dispersed in water with the aid of ultrasonication for 20 min.

6.2.5. Preparation of reduced graphene oxide (rGO) modified with 4- aminophenylboronic acid (rGO/APBA) 1 mL of a homogeneous GO suspension (0.5 mg/mL) in distilled water was added 1 mL aminophenylboronic acid (10 mM in ethanol) and stirred for 12 h at 100°C. The resulting black precipitate was separated from the supernatant by centrifugation (20 min at 14000 rpm), washed with ethanol (three times) and water (three times), and then dried in an oven at 80°C for 6 h.

6.2.6. Preparation of graphene/alkynyl-terminated dopamine (rGO/alkynyl-dopamine) The stock GO (0.5 mg/mL) suspension was diluted in water to obtain 0.05 mg/mL solution (1:10). To 1 mL of GO was added 1 mL of alkynyl-terminated dopamine (10 mM) and the mixture was kept in an ultrasonic bath for 4 h at 80°C. The resulting black precipitate was separated from the aqueous supernatant by centrifugation at 14 000 rpm for 20 min. After washing with water (3 times), the resulting precipitate was dried in an oven (80°C) and then dispersed in water with the aid of ultrasonication for 30 min.

6.2.7. Preparation of rGO/NDs nanocomposites The as-received hydroxylated nanodiamond (ND–OH) particles were treated by the bead-assisted sonic disintegration process (BASD) to break down the persistent particle agglomerates. An ultrasonicator equipped with a horn-type sonotrode (Branson, Ultrasonic- Homogenizer Sonifier II W-450 with a 4.8 mm microtip) was used. The ultrasonication vial was charged with 40 g of zirconia beads, 200 mg of ND–OH particles and 20 mL of DMSO and treated two times for 60 min (amplitude: 70%; pulse on/off, 0.3 s/0.2 s). During the BASD process the slurry in the vial was cooled with ice. Zirconia beads were removed by centrifugation at 10 000 rpm and the ND–OH particles were purified by consecutive washing/centrifugation cycles in water. The BASD treated ND-OH particles were mixed with GO (0.5 mg/mL) at different

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GO/NDs ratios: (1/1, 2/1, 4/1, 10/1, 20/1) and ultrasonicated in a Fisher Transonic TI-H-10 ultrasonication bath for 6 h. Then the mixture was heated at 100°C for 48 h. The formed black precipitate was separated from the aqueous supernatant by centrifugation at 14000 rpm for 20 min. After washing with water (3 times), the resulting precipitate was dried at 80°C before being re-dispersed in ethanol with the aid of ultrasonication for 30 min. In a control experiment, the BASD treated ND-OH particles were mixed with GO (0.5 mg/mL) at GO/NDs ratio=10/1 and ultrasonicated in a Fisher Transonic TI-H-10 ultrasonication bath for 6 h. Then hydrazine (10 mM) was added to the mixture and heated at 100°C for 48 h. The resulting black precipitate was separated from the aqueous supernatant by centrifugation at 14000 rpm for 20 min. After washing with water (3 times) and methanol (3 times), the precipitate was dried at 80°C.

6.3. Post-functionalization: Thiol-yne reaction on rGO/alkynyl-dopamine rGO/alkynyl-dopamine modified ITO electrodes were prepared by casting 50 µL of the rGO/alkynyl-dopamine solution in THF onto previously cleaned ITO substrate and heated at 70°C to ensure solvent evaporation. rGO/alkynyl-dopamine modified ITO surfaces were immersed into a solution of 6-(ferrocenyl)-hexanethiol (10 mM) or 1H,1H,2H,2H- perfluorodecanethiol (10 mM) or a mixture of both (5 mM each) in absolute ethanol under nitrogen atmosphere. The interface was exposed to UV light irradiation at λ = 365 nm, P = 100 or 500 mW cm-2 for 30 min at room temperature. The resulting surface was thoroughly washed with ethanol then with water and dried under nitrogen stream.

6.4. Preparation of graphene coated GC electrodes Glassy carbon electrodes (GCEs) were polished with alumina and diamond paste and then sonicated in a mixture of ethanol/acetone for 15 min before modification. Graphene modified glassy carbon electrodes were prepared by casting 20-60 µL of a DMSO solution of the reduced graphene oxide matrix (0.3 mg/mL) 5 times onto GCE followed by subsequent drying at 70°C in an oven for 24 h. rGO/alkynyl-dopamine modified ITO electrodes were prepared by casting 50 µL of rGO/ alkynyl-dopamine in THF onto the ITO substrate followed by heating at 70°C until full THF

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APPENDIX EXPERIMENTAL PART evaporation. The rGO/Tyr modified GC electrodes were prepared by casting 30 µL of rGO/Tyr-DMF suspension (0.5 mg/mL) onto the GCE (A= 0.28 cm2) followed by heating at 80°C until full DMF evaporation.

6.5. Determination of sugar content in apple juice (colorimetric approach) A standard calibration curve for fructose was generated by mixing aliquots of aqueous phenolic solution (5 wt%, 60 µL) and concentrated H2SO4 (900 µL) to a series of 60 mL aliquots of aqueous fructose solutions (0-200 µg/mL). After reaction for 10 min the absorption spectrum was recorded using as a blank a phenol-H2SO4 mixture containing only 60 µL of water. The absorbance of the solution was measured at two wavelengths: λmax=495 nm

(absorption band of fructose complex) and λmax=570 nm (background) and the absorbance difference (A495–A570) were plotted against the concentration of fructose. For the determination of the sugar content in apple juice, the sample was diluted 1050 times and 60µl apple juice dilution was mixed with aliquots of aqueous phenolic solution (5 wt%, 60 µL) and concentrated H2SO4 (900 µL). After reaction for 10 min the absorption spectrum was recorded using as a blank a phenol-H2SO4 mixture containing only 60 µL of water. The overall saccharide concentration determined by comparing the relative absorbance obtained to the fructose calibration curve.

6.6. Instrumentation 6.6.1. X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS) experiments were performed on a PHl 5000 VersaProbe-Scanning ESCA Microprobe (ULVAC-PHI, Japan/USA) instrument at a base

-9 pressure below 5×10 mbar. Monochromatic AlKα radiation was used and the X-ray beam, focused to a diameter of 100 µm, was scanned on a 250×250 µm surface, at an operating power of 25 W (15 kV). Photoelectron survey spectra were acquired using a hemispherical analyzer at pass energy of 117.4 eV with a 0.4 eV energy step. Core-level spectra were acquired at pass energy of 23.5 eV with a 0.1 eV energy step. All spectra were acquired with 90° between X-ray source and analyzer and with the use of low energy electrons and low

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APPENDIX EXPERIMENTAL PART energy argon ions for charge neutralization. After subtraction of the Shirley-type background, the core-level spectra were decomposed into their components with mixed Gaussian- Lorentzian (30:70) shape lines using the CasaXPS software. Quantification calculations were performed using sensitivity factors supplied by PHI. The samples were prepared by casting 50 μL aqueous or ethanol suspension of GO or rGO composites on a clean silicon wafer or ITO surface followed by drying in an oven at 80°C to make sure the solvent was removed.

6.6.2. FTIR spectroscopy Fourier transform infrared (FTIR) spectra were recorded using a ThermoScientific FTIR instrument (Nicolet 8700) at 4 cm-1. Dried GO or rGO composites (1 mg) were mixed with KBr powder (100 mg) in an agate mortar. The mixture was pressed into a pellet under 10 tons load for 2–4 min, and the spectrum was recorded immediately. Sixteen accumulative scans were collected. The signal from a pure KBr pellet was subtracted as the background.

6.6.3. Raman spectroscopy Micro-Raman spectroscopy measurements were performed on a Horiba Jobin Yvon LabRam HR Micro-Raman system combined with a 473-nm laser diode as excitation source. Visible light is focused by a 100x objective. The scattered light is collected by the same objective in backscattering configuration, dispersed by a 1800-mm focal length monochromator and detected by a CCD The samples were prepared by casting 50 μL aqueous or ethanol suspension of GO or rGO composites on a clean silicon wafer or ITO surface followed by drying in an oven at 80°C to make sure the solvent was removed.

6.6.4. UV-Vis measurements Absorption spectra were recorded using a Perkin Elmer Lambda UV-Vis 950 spectrophotometer in quartz cuvettes with an optical path of 10 mm. The wavelength range was 200–800 nm. 20 μg/mL of GO or rGO composites were nicely dispersed in water or ethanol with the

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APPENDIX EXPERIMENTAL PART aid of 3 h ultrasonication and then filled into the quartz cuvettes for measurements.

6.6.5. Scanning electron microscopy (SEM) SEM images were obtained using an electron microscope ULTRA 55 (Zeiss) equipped with a thermal field emission emitter and three different detectors (EsB detector with filter grid, high-efficiency In-lens SE detector, Everhart-Thornley secondary electron detector). The samples were prepared by casting 50 μL aqueous or ethanol suspension of GO or rGO composites on a clean silicon wafer or ITO surface followed by drying in an oven at 80°C to make sure the solvent was removed.

6.6.6. Transmission electron microscopy (TEM) Transmission electron microscope (TEM) images were taken by using Icon analytical 200 KeV microscopes. Samples were drop-casted from aqueous or ethanolic dispersions of GO or rGO onto carbon coated TEM grids and the solvent was evaporated under gentle heating by a UV lamp.

6.6.7. Atomic force spectroscopy (AFM) GO or rGO dispersions (5 µL) were deposited onto freshly cleaved mica (Sigma-Aldrich) and let to dry under nitrogen flow before imaging. The samples were imaged with a Dimension 3100 Model AFM (Veeco, Santa Barbara, CA) equipped with a Nanoscope IV controller (Digital Instruments) under ambient conditions (relative humidity ~30%, temperature ~22-24 °C). Rectangular single beam silicon cantilevers (AFM-TM Arrow, Nanoworld) with spring constants of ~42 N m-1 and typical resonant frequencies between 250 and 300 kHz were used. All AFM images were acquired in tapping mode at a constant force of 5-50 pN.

6.6.8. Zeta potential and size The average diameter and polydispersity index (PI) were determined by dynamic light scattering using a Zetasizer® Nano ZS (Malvern Instruments S.A., Worcestershire, UK). The zeta potential was measured using the electrophoretic mode with the Zetasizer®. All the

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APPENDIX EXPERIMENTAL PART batches were diluted at 1/100 (v/v) in distilled water (filtered over 0.22 μm) prior to the analysis and analyzed in triplicate.

6.6.9. Electrochemical measurements Electrochemical measurements were performed using an Autolab PGSTAT 101 potentiostat (Eco Chimie, Utrecht, the Netherlands). The electrochemical cell consisted of graphene samples as the working electrode, Ag/AgCl as the reference electrode, and platinum wire as the counter electrode. All electrochemical experiments were performed at room temperature.

3-/4- Cyclic voltammetric measurements were performed in PBS (0.1 M), Fe(CN)6 (5

3+ mM)/PBS (0.1 M) or Ru(NH3)6 (5 mM)/PBS (0.1 M) solutions at scan rates of 50 or 100 mV s-1. Differential pulse voltammetric (DPV) measurements were performed using a pulse

-1 height of 25 mV, a pulse width of 250 ms and an effective scan rate of 2 mV s .

Chronoamperometric detection of H2O2 on rGO/Tyr modified GC electrode was performed under N2-saturated steady-state condition in stirring phosphate buffered saline (pH 7.4) by applying a constant potential of -0.55 V to the working electrode. When the background current became stable (after 60 s), a subsequent addition of H2O2 was realized and the current was measured. The supercapacitor behavior of rGO/NDs was investigated using a three-electrode system comprised of rGO/NDs as the working electrode, Pt foil as the counter electrode and Ag/AgCl (3M KCl) as the reference electrode. The CV and galvanostatic charge/discharge tests were performed using a VMP3 Bio Logic Science Instruments (France). To prepare rGO/NDs electrodes, rGO/NDs particles and polyvinylidene fluoride (PVDF) at the weight ratio of 90:10 were dispersed in N-methyl-2-pyrrolidone at a concentration of 20 mg/mL and 20 µL of the dispersion were deposited onto a platinum foil (A=0.5 cm2). The active mass is around 400 µg. For all electrochemical tests 1M H2SO4 was used as electrolyte, which was purged with N2 for 10 min to remove dissolved oxygen. CV curves were recorded from 0 to 0.8 V with a scan rate of 10 mV s-1. The galvanotastic charge/discharge tests were performed from 0 to 0.8 V at the current density of 1 and 2 A g-1.

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PUBLICATIONS

1. Qi Wang, Nareerat Plylahan, Manjusha V. Shelke, R. R. Devarapalli, Musen Li, Palaniappan Subramanian, Thierry Djenizian, Rabah Boukherroub and Sabine Szunerits. “Nanodiamond particles/reduced graphene oxide composites as efficient supercapacitor electrodes.” Carbon. (Accepted) 2. Qi Wang, Musen Li, Sabine Szunerits and Rabah Boukherroub. “Environmentally

friendly reduction of graphene oxide using tyrosine for nonenzymatic amperometric H2O2 detection.” Electroanalysis. (Accepted) 3. Qi Wang, Alina Vasilescu, Palaniappan Subramanian, Alis Vezeanu, Veronica Andrei, Yannick Coffinier, Musen Li, Rabah Boukherroub, Sabine Szunerits. “Simultaneous electrochemical detection of tryptophan and tyrosine using boron-doped diamond and diamond nanowire electrodes.” Electrochemistry Communications. 35, (2013): 84-87. 4. Qi Wang, Izabela Kaminska, Joanna Niedziolka-Jonsson, Marcin Opallo, Musen Li, Rabah Boukherroub and Sabine Szunerits. "Sensitive sugar detection using 4- Aminophenylboronic acid modified graphene." Biosensors and Bioelectronics 50, (2013): 331-337. 5. Qi Wang, Palaniappan Subramanian, Musen Li, Weng Siang Yeap, Ken Haenen, Yannick Coffinier, Rabah Boukherroub and Sabine Szunerits. "Non-enzymatic glucose sensing on long and short diamond nanowire electrodes." Electrochemistry Communications 34, (2013): 286-290. 6. Qi Wang, Manash R. Das, Musen Li, Rabah Boukherroub and Sabine Szunerits. "Voltammetric detection of L-Dopa and carbidopa on graphene modified glassy carbon interfaces." Bioelectrochemistry. 93, 2013, 15-22. 7. Qi Wang, Alexander Kromka, Jana Houdkova, Oleg Babchenko, Bohuslav Rezek, Musen Li, Rabah Boukherroub and Sabine Szunerits. "nanomolar hydrogen peroxide detection using horseradish peroxidase covalently linked to undoped nanocrystalline diamond surfaces." Langmuir 28, (2011): 587-592. 8. Kaminska, Izabela, Qi Wang, Alexandre Barras, Janusz Sobczak, Joanna Niedziolka- Jonsson, Patrice Woisel, Joel Lyskawa, William Laure, Marcin Opallo, Musen Li, Rabah Boukherroub and Sabine Szunerits. "Thiol–yne click reactions on alkynyl–dopamine-

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PUBLICATIONS

modified reduced graphene oxide." Chemistry – A European Journal 19, (2013): 8673- 8678. 9. Oprea, Raluca, Serban F. Peteu, Palaniappan Subramanian, Qi Wang, Emmanuelle Pichonat, Henri Happy, Mekki Bayachou, Rabah Boukherroub and Sabine Szunerits. "Peroxynitrite activity of Hemin-functionalized reduced graphene oxide." Analyst 138, (2013): 4345-4352. 10. Huang, Yujie, Qi Wang, Mei Wang, Zhenyi Fei and Musen Li. "Characterization and analysis of DLC films with different thickness deposited by Rf magnetron PECVD." Rare Metals 31, (2012): 198-203.

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