Fabrication of Electrode Using 2D Materials for Electrochemical Biosensing

Contents Page:

1.0 ABSTRACT………………………………….…………………….….3

2.0 INTRODUCTION ………………………………………………… …4

2.1 AIMS AND OBJECTIVES …………………………………………..4

2.2 OVERVIEW …………………………………………………………. 4

2.3 BACKGROUND ……………………………………………………….

3.0 LITERATURE REVIEW.……………………………………………

3.1 REVIEW.…………………………………………………,……………..

3.2 DESCRIPTION OF ELECTROCHEMICAL BIOSENSING

3.3 DESCRIPTION OF 2D MATERIALS ………………….…………..

3.4 BIOSENSING IN 2D MATERIALS…………………………………

3.5 DESCRIPTION OF GRAPHENE ……………………………..…….……….

3.6 DESCRIPTION OF MOLYBDENUM DISULPHIDE (MoS₂)……..

 

1. ABSTRACT

The possibility of interacting with individual atoms in an atomically layered 2D material brings an ideal platform for highly sensitive and selective electrochemical biosensing. Graphene and its discovery has inspired researchers to try and create other 2D materials. (1) With this discovery, new avenues have been brought to light regarding biosensor development and has brought forward the possibility of other layered materials based on sensing platforms. (2) After a substantial amount of research, a distinct ‘beyond graphene’ domain has been established involving the library of non-graphene 2D materials. Most of the ongoing research is focused on material synthesis and the investigation of materials as most of 2D non-graphene materials have already been newly discovered. In addition to this, Molybdenum Disulphide (MoS2) has led to immense research regarding this materials fundamentals application and more recently, it’s potential for biosensing. (2)

 

1. INTRODUCTION

2.1 AIMS AND OBJECTIVES

This report will be based on the synthesis methods of 2D materials and getting familiarised with their property and structure. The main objectives and deliverables of this project is to carry out research into published academic/ and or technical literature relating to automatic connection between 2D materials, Graphene, MoS₂, biosensing and biosensing of Graphene. It will look at the synthesis of 2D materials including Graphene and MoS₂ and the characterisation of Graphene, Graphene Oxide and MoS₂ sheets with Raman spectroscopy and to also get their characteristic signatures for their individual identity. It will then characterise the morphology of graphene, graphene oxide and MoS₂ with scanning electron microscopy.  This report will then show the examination of preliminary electrode kinetics results of MoS₂ modified screen printed electrodes. Later, a discussion of the different methods that are used to characterise the materials will be provided in detail such as Ramen and SEM which will help understand more about each material and the various techniques that are used. Then analysis of the results and data recorded will be shown and discussion on whether any applications within biosensing use these. Finally, undertake electrochemical testing of electrodes.

2.2 OVERVIEW

Technology within the medical world has grew rapidly within the last lot of years. Devices are becoming more powerful than before due to new materials being discovered and analysed. For this report, 2D materials that are used for electrochemistry biosensing are important. Both Graphene and MoS₂ are important for this review as they are the mostly used materials. Their different properties such as their molecule arrangement, their structure, and both advantages and disadvantages will be analysed and discussed within this review. Biosensing applications and varied materials that are already being used will be discussed. Graphene is the materials that is used therefore the properties of Graphene such as its characterisation and synthesis will be shown. (2)

2.3 BACKGROUND

The developments in material science are the driving force of technological progress. It is said that the creation of new materials of different dimensionality and functionality is the one essential for any significant break throughs that may be made. Due to its high sensitivity and selectivity, the development of electrochemical sensors has also got a great deal of interest and it used more in many fields including analytical chemistry, industrial process monitoring and control, clinical diagnostics and environmental monitoring and security. (3)

For a high specificity, an enzyme can be added to the electrode surface and this was the time the biosensor research area was born. Due to the demand for disease diagnostics ad therapies, biosensors have become the known tool for the detection for o biological analytes. This includes nucleic acid, proteins and small biomolecules. (3) In addition to the development of advanced instruments and detections strategies for signal amplification, the utilization of suitable materials in biosensing applications is another important way to enhance the detection signal. (4)

Biosensing is vital for improving the quality of human life. A biosensor is an analytical device which converts a biological response into an electrical signal. The aim of this is to investigate a range of materials for their use and find out if the materials’ good to use in an electrochemical sensing application (5). Electrochemical biosensors are a special interest due to their analytical characteristics including operational simplicity, low cost, extraordinary and real-time detection. Electrochemical biosensors are also prepared for point-of-care device. Increasing research over the last few years have been put into the design of novel electrochemical biosensors as well as the improvement of their performance. (6)

Graphene is known for being the thinnest and the strongest material ever measured. It is composed of monolayers of carbon atoms arranged in a honeycombed network with six-membered rings. As a fundamental 2D carbon structure, graphene is viewed as an indefinitely extended, 2D aromatic macromolecule and can be also considered as a basic building block for carbon materials of all other dimensions. Graphene is known as an integral part of graphite and it was presumed not to exist in the free state, strictly 2D crystals were thought to be thermodynamically unstable at fixed temperatures. (7)  In previous years, two-dimensional crystal structures have become important and have attracted a lot of attention. It is known that layered crystals can be mechanically exfoliated to yield flakes of single-layer thickness just as graphene can be prepared from graphite.  (8)

Since the invention of graphene, the properties of two-dimensional (2D) materials can be different in many ways and is far superior to those of the ‘bulk counterpart’. (4) The discovery of Graphene in 2004 opened excessive research activities on 2D materials and from then, graphene has been exploited for numerous applications. (2)

Many efforts have been focusing on Graphene Oxide (GO) since the discovery of Graphene. Graphene oxides properties on its own are very useful, however, it is also promising for obtaining large quantities of this unique material hence the focusing efforts. (2)

Graphene Oxide can be visualised as individual sheets of graphene decorated with oxygen functional groups on both basal planes and edges, which has been prepared by oxidative exfoliation of graphite. The presence of oxygen makes GO amenable to chemical functionalization, yet it disrupts the extended sp2 network of the graphene hexagonal lattice. (9)  The conversion of graphene back to graphene oxide as its advantages as the chemical/ thermal reduction of GO as it is said to be the most attractive procedure because of its reliability, simplicity, high yield and low cost. (9)

Increasing further research and applications have been undertaken since the synthesis of Graphene has been found, based on their unique features. Just like graphene and other 2D materials, 2D MoS₂ offers large surface areas that enhance its electrochemical biosensing performance. MoS₂ has a direct band gap whereas all other features are similar to graphene. (10) However, due to the existence of a suitable bandgap, the overall sensitivity of devices that are based on 2D MoS₂ is much larger than that of true graphene oxide which is an insulator and has a large bandgap. Molybdenum disulphide (MoS₂) compounds have recently attracted considerable attention due to their appealing electrocatalyic properties for electrochemical reaction.  Their properties are favourable and come from the presence of catalytically active sulfur atoms on the molybdenum edges of MoS₂ planes. (10) MoS2 has a layered packed structure consisting of a single later of Mo atoms packed between two layers of sulfur atoms. (11) These stacks are piled in a graphite-like-manner to form bulk material and are held together by weak van der Waals forces. These properties lead to fabrication of MoS₂ nanosheet-based field effect biosensor for potential use in the bio-sensing applications. (10) Bulk MoS₂ is a semiconductor with an indirect bandgap of 1.3eV, which is modified to a direct band gap semiconductor of 1.8eV when it is thinned down to just a few layers. Studies have predicted, and experimental studies have confirmed that the electronic structure of MoS₂ edges is dominated by metallic 1D edge states which is in sharp contrast to the semiconducting basal plane. (11)  As a result, the electrocatalysis of atomically thin CVD grown MoS₂ films have been found to be strongly dependant on the layer number, which was correlated to the hopping of electrons in the vertical direction of MoS₂ layers. These observations have led researchers to the proposition that MoS₂ nanodots ie a few layer MoS₂ sheets with very small lateral dimensions should show enhanced catalytic activity due to a simultaneous increase an unsaturated sulfur edge sites and enhanced electron transfer accrued from the reduced number of layers. (10,11) Overall, this review will show which materials are best suited for electrochemical biosensing applications and which one is the best.

2. LITERATURE REVIEW

3.1 REVIEW

The aim of this review is to characterise the structure of graphene oxide, graphene and Mos2 whilst investigating their electrochemical performance with a view of applying them to biosensing applications. The expected contribution to knowledge for this review is to fully understand all the properties within Graphene and MoS2 molecules, their structures and to know which other 2D materials may be used for electrochemical biosensing.

3.2 DESCRIPTION OF ELECTROCHEMICAL BIOSENSING

Biosensors are attractive for both pharmaceutical and biomedical analysis because of their sensitivity and high selectivity, also sometimes their specificity, high benefit/cost and rapid data collection. The selectivity of the biosensor for the target analyte is mainly determined by the biorecognition element, whilst the sensitivity of the biosensor is greatly influenced by the transducer. (3)

Recently, the biosensor development and construction strategy includes five features.

1. The detected or measured parameter and the matrix
2. The working principle transducer
3. The chemical/biochemical model
4. The field of the application
5. The technology and materials for sensor fabrication.

Materials that are generally used for electrochemical sensors are classified as: (1) Materials for the electrode and supporting substrate. (2) Materials for improving electroanalytical performances. (3) Materials for the immobilization of biological recognition elements (5) Biological elements; the last two are for electrochemical biosensors.(3)

Materials that are used for the electrode and supporting the substrate are usually conductive materials exhibiting low currents in an electrolyte solution and free of any electroactive species generally over a relatively wide potential window. Among the most frequently used materials include the following: ‘metals (mercury, platinum, gold, silver and stainless steel), metal oxides (indium tin oxide, ITO) carbon-based materials (glassy carbon, graphite, carbon black and carbon fibre), new hybrid materials, and organic electro‐ conductive polymers or salts. Boron-doped diamond is a special case of material, because bare diamond is a non-conducting, allotropic form of carbon.’ (3)

Within the last decade, due to their toxicity, in order to avoid mercury-based electrodes and different metallic files (Au, Hg, Bi, Ag, Pb, Sn, Sb, Co, Ga, Se) that are relativity simple to achieve and easy to reproduce were developed. Reported recently was different strategies to develop two and three dimensional nanostructured electrodes with larger surfaces. (3) Below is a Figure representing the different carbon-based nanostructures that are used because of their properties for many applications. The carbon nanomaterials shown in Figure1 cover a broad range of structures beginning with zero dimensional structures (fullerene, diamond clusters) and continuing with one-dimensional (nanotubes), two-dimensional (graphene), and three-dimensional structures (nanocrystal diamond and fullerite). (3)

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Later in this review, it will be clear that out of the materials mentioned above, Graphene is the most popular for biosensing applications due to its properties and due to it being two-dimensional.

 

 

3.3 DESCRIPTION OF 2D MATERIALS

2D materials are a class of nanomaterials which are defined by their property of being merely one or two atoms thick. The study of 2D materials is one of the newest and most exciting areas of Materials Science and Engineering as they have the potential to revolutionize many electronic applications such as solar cells, transistors, digital screens and semiconductors.

For progress to be made within 2D materials, ideally materials with matching semiconductor and electronic properties combined with modern technologies for their fabrication on a commercial scale is what researchers are looking for. To date, it is said that the technology for obtaining 2D materials are characteristically related to their layered structure and the weak van der Waals bond that exist between the layers. The first scotch tape approach was transformed in to the chemical intercalation and exfoliation of 2D flakes, and has only recently turned its attention to the direct growth techniques (chemical vapor deposition). Although, metal oxides are only represented in the 2D materials family modestly which is yet again reminding for the further detailed studies needed on their growth and properties. (4) Being in fact the extreme case of surface science, the 2D materials possess the highest surface-to-volume ratio. This feature of 2D materials enhances their prospective for sensor application where the devices performance is defined by its interface occurring phenomena. So, the number of reports that are based on biosensors using 2D materials as a transducer has been constantly growing since graphene was discovered as shown in Figure 2. (4)

 

3.4 BIOSENSING IN 2D MATERIALS (NON-GRAPHENE)

A biosensor is an analytical device which converts a biological response into an electrical signal. It consists of three parts: the sensitive biological element, the transducer and the associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. (5) Biosensors and biosensing protocols can detect a wide range of compounds, sensitively and selectively, with applications in security, health care for point of care analyses of diseases and environmental safety.

It is important to note that some 2D non-graphene materials have many advantages, such as, a direct band gap which is highly promising for a number of applications. Since 2D non-graphene materials have been newly discovered, most of the research efforts are concentrated on material synthesis and the investigation of the properties of the material. These 2D non-graphene materials are still at the embryonic stages however in recent years, a number of reports have released about 2D material-based biosensors, evidencing the growing potential of 2D non-graphene materials for biosensing applications. Recent progress on the potential of using 2D non-graphene materials are similar oxide nanostructures for several types of biosensors (optical and electrochemical). (4)

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3.5 DESCRIPTION OF GRAPHENE

Since graphenes isolation in 2004, it has been described as a ‘miracle material’. Recently there have been thousands of reports based on graphenes exceptional properties and a wide range of applications it could be combined to for improvement. (12) Graphene has emerged hugely as a 2-dimensional material and has received increasing attention due to its properties such has its high surface area, excellent conductivity, high mechanical strength and its mass production. (13) Graphene has attracted strong scientific and technological interest over the years as it has shown a great promise in applications such as electronics, energy storage, fuel cells, solar cells and bioscience/biotechnologies due to its properties as mention previously. (13) It also demonstrates fascinating electrochemical properties which include wide electrochemical potential, activity and low charge transfer resistance. (4)

Graphene has served as a model for a 2D system that has captured the interest of researchers from different fields which include electrics, material science and engineering. Nevertheless, Graphene being the most well-known 2D crystal with an excess of unique properties, has its disadvantages which limits its applications. For example, the lack of an intrinsic band gap is one of the largest obstacles in its way to be fully utilized. Furthermore, the numerous studies of graphene have sparked new interest in graphene-like 2D layered nanomaterials. (12) There have been a few materials such as graphic carbon nitride, boron nitride (BN) ad transition metal oxides which have no yet been studied due to their unique physical, chemical and electronic properties. As graphene is only composed with carbon, there have been efforts to construct novel functional devices have led to the exploitation of more versatile and tunable 2D alternatives that have greater flexibility and diversity of composition structure and functionality. However, due to the attractive properties and large specific surface areas, these graphene like 2D nanomaterials have greater potential in a broad spectrum of applications such as nanoelectronics, catalysis and other specific applications. (12)

Graphene was first described as a ‘single layer in graphite intercalation compounds’ and was previously described as an isolated monolayer of carbon atoms arranged in a honeycomb like pattern. (14) Graphene is also known for being one million times thinner that paper, it is almost transparent and is believed to be the strongest material in the world. Until now, there has been no attempt made to synthesize graphene sheets, simultaneously possessing low oxygen content and small lateral dimensions with the view of examining their electroactivity. However it is said that under ambient conditions and a low concentration of oxygen (<3 at % depending on the amount of edges) will always be present, since open graphene edges can be easily terminated by oxygen groups. (15)

Biosensors employ graphene. Graphene is a zero-gap semi-conductor material which is electroactive and transparent. Graphene is an ideal material for electrochemistry due to its large 2D electrical conductivity, large surface area and low cost. Graphene can be synthesized in several ways but only some are suitable for electrochemical applications for both sensing and biosensing.

1. Graphene can be prepared by ‘peeling-off’ highly oriented pyrolytic graphite (HOPG)
2. Graphene can be epitaxially grown on silicon wafers.

These two methods are useful when studying the electronic properties of graphene.

Several graphene based nanomaterials such as graphene oxide, reduced graphene oxide, graphene quantum dots and so on have been functionalized by Biosystems integrated with nucleic acids, peptides, and enzymes and have been extensively employed for biosensor fabrication. The properties of Graphene can be altered by adopting various synthesis routes, functionalization and doping. However, it has been proven that by varying the number of layers has no significant effect on the biosensing properties of graphene, through the alignment of graphene on a substrate (vertical or horizontal to substrate) really affect the sensitivity of the device.  The choice of graphene material for biosensing solely relies on the specific sensing target and the sensing mechanism (12)

Many methods have been developed to produce graphene. In 2004 graphene sheets prepared by mechanical exfoliation (repeated peeing) of highly orientated pyrolytic graphite. This method, which was called the scotch-tape method is still widely used in laboratories to obtain pristine perfect structured graphene layers for basic scientific research and for making proof-of-concept devices However this method is not suitable for mass production. The other method that is used for producing defect-free/defect-less graphene is the mild exfoliation of graphite but the yield so far is very low. Another mass-production method is chemical or thermal reduction of GO. It is thought to be the most economical way to produce graphene. Most of graphene that is used in electrochemistry is produced with the last method is graphite oxide reduction. (13) Graphene from GO reduction, which is also called functionalized graphene sheets or chemically reduced graphene oxide, usually has abundant structural defects and functional groups which are advantageous for electrochemical applications.

With graphene, a key challenge in the synthesis and processing of bulk-quantity, graphene is to surmount the strong exfoliation energy of the

π-stacked layers in graphite, that is the high cohesive van der Waals energy adhering graphitic sheets to one another. (7)

Graphene Oxide (GO) is generally produced by graphite using strong acids and an oxidant treatment, and the GO is subsequently transformed to reduced graphene oxide (rGO). Because of the chemical synthesis, some oxygen containing functional groups and structural defects are inevitably involved on the surface of the GO and the derived rGO. Also, because the oxygen containing functional groups on the GO confine the

πelectrons within the sp² carbon nanodomains, GO can fluoresce in a wide range of wavelength from the near-infrared to ultraviolet. Generally, in most cases, these two mechanisms occur simultaneously to produce the photoluminescence emission and, the graphene ad other 2D graphene-like nanosheets have also exhibited strong photoluminescence such as g-C₃N₄ and MoS₂. (12)

For large graphene sheets, an altered electronic structure is expected at the basal plane as compared to the edge region due to its symmetrical breaking of the honeycomb lattice. Graphene has two kinds of edge terminations according to their shape named zigzag and armchair edges which have different electronic structures.  From experimental studies that have been done, a π electron called the ‘edge state’ created along the zigzag edges and there is no state present at the armchair edges.  The characteristic edge state which has a large local density of states and is spin polarized gives rise to electronic, magnetic ad chemical activities in the zigzag edges of graphene. (15) The grinding of the graphite with a small quantity of ionic liquid produces a gel due to the π- π interactions between the graphite and the ionic liquid. During this process, the ionic liquid acts as a lubricant allowing the breaking down of the graphite platelets to smaller sizes at the same time helps exfoliation of graphite layers through shear forces exerted on the graphite flake. Many applications have been found such as supercapacitors, biosensors and actuators, the production of nano-sized graphene with a low amount of oxygen has not been reported so far due to the knowledge of studies. (15) Therefore, this review will focus mainly on graphene. Getting inspired by graphene research and various other 2D materials such as MoS₂ as they have also been studied recently for their potential applications in biosensors.

3.6 DESCRIPTION OF MOLYBDENUM DISULPHIDE (MoS₂)

Molybdenum disulphide (MoS₂) is a crystal that consists of weakly coupled layers of S-Mo-S where a Mo atom is sandwiched between two layers of sulfur atoms. It offers a large direct experimental band gap of around 1.8eV and a semiconductor with an indirect bandgap of 1.3eV when it is thinned down to just a few layers. It is a naturally occurring solid that finds applications in industry in both its bulk and its dispersed forms. (11,16) Similar to graphene layers in graphite, the individual layers sandwiched together S-Mo-S are assembled together by weak van der Waals interactions in hexagonally packed structures. Because of the layers, and how they move relatively easy to move again each other, MoS2 is widely used as a dry lubricant. (16)

As shown in Figure4, the neighbouring planes in bulk MoS2 are held together by van der Waals forces making it possible to produce monolayers of MoS2 using the well-established micromechanical cleavage and liquid exfoliation techniques. (16) Recent studies have predicted and experimental studies have then confirmed that the electronic structure of MoS2’ edges is dominated by metallic one-dimensional edge states which is in sharp contrast to the semi-conducting basal plane. The result of the electrocatalysis of atomically thin CVD grown MoS2 films have been found to be strongly dependant on the layer number which is correlated to the hopping of electrons in the vertical direction of MoS2 layers. (11) These observations led researchers to the proposition that MoS2 nanodots ie a few layer of MoS2 sheets with very small lateral dimensions should show enhanced catalytic activity due to a simultaneous increase of unsaturated sulfur edge sites and enhanced electron transfer accrued from the reduced number of layers.(11)

Due to the large band-edge-excitation of the metal centred d-d transition that gives rise to the unique electronic features and therefore it is used in a several applications such as in hydrogen evolution reaction catalysis, electrochemical intercalations, hydrogen storage, elastic and coating materials. (16)

Usually, the synthesis of MoS2 nanodots are under intensive investigation and evaluation of their quality and performance for various applications including energy storage, gas sensing and optoelectronic devices which have been largely unexplored. The synthesis of MoS2 nanodots using optimized experimental conditions that involved grinding MoS2 platelets in a small quantity of room temperature ionic liquid (RTIL) which is then followed by sequential centrifugation steps. (11) As schematically shown in Figure4, the combined compressive, torsional and shear forces exerted on the MoS2 platelets can exfoliate and at the same time, break up the bulk crystals. During this grinding process, the RTIL acts as a lubricant which allows for low friction between the moving pestle and the mortar, hence facilitating the exfoliation.(11)

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The yield of the nanodots increases by prolonging the duration of grinding. It is said that the mechanical breaking point of MoS2 is 5 times small than of graphene, leading to the overall smaller lateral size. (11)  Structural defects of MoS2 including point defects, grain boundaries and edges play a huge role in sensing applications. For example, defects of MoS2 offer the possibility of surface modification and functionalization resulting in profound influence on their electrical, chemical and optical properties.  (17)

Unlike mono-layered graphene, mono layered MoS2 has three atom layer nanosheets without a wrinkle morphology and is much smoother. Before, the dislocation ad defects in MoS2 were studied and by using direct atomic resolution imaging and observed various defects of MoS2 nanosheets. Exfoliating MoS2 nanosheets into mono or few-layers not only preserves the bulk properties, but it also introduces additional characteristics ascribed to confinement effects, thus offering opportunities for various sensor applications. (17)

As MoS2-based nanocomposites have been attracting more interest in the recent years. So far in this review, there has been a summary of the preparations of methods and sensing applications of MoS2 and MoS2-based nanocomposites. Now the actual preparation of MoS2 will be discussed. (17) Similar to graphene, and other 2D materials, 2D MoS2 offers a large surface area that enhances its biosensing performance. However, the overall sensitivity of devices made that are based on 2D MoS2 is much larger than that of graphene and graphene oxides which have either no or very small bandgaps. Most stoichiometric 2D oxides in comparison, have large bandgaps that require relatively high applied energy for their electronic bad structure modulation. (18)

4. MATERIALS AND METHODS

4.1 RAMAN SPECTROSCOPY

This chapter in this review will look at the application and basic principle of Raman spectroscopy. It will look at several materials with Raman spectroscopy and then finally the characteristic properties of graphene related materials such as graphene oxide and graphite both exfoliated and synthesised.

The main spectroscopies used to detect vibrations in molecules are based on the process of infrared absorption and Raman scattering. (19) Raman spectroscopy is a scattering technique which is based on Raman Effect. For example, a frequency of a small fraction of scattered radiation is different from frequency of monochromatic incident radiation. This is based on the inelastic scattering of incident radiation through its interaction with vibrating molecules which probes the molecular vibrations. (20) They are used widely to provide information on chemical structures and physical forms to identify substances from the characteristic spectral patterns and then to determine quantitatively or semi-quantitively the amount of a substance in a sample. In Raman spectroscopy, samples can be examined in a range of physical states such as solids, liquids or vapours, in hot or cold states. It could also be in bulk, as microscopic particles, or as surface layers. This technique is very wide ranging and provide solutions to a host of interesting and challenging analytical problems. It is said that Raman scattering is less widely used than infrared absorption due to problems with sample degradation and fluorescence. (19) However, there have been improvements in recent advances in instrument technology that have simplified the equipment and reduced the problems substantially.

In Raman spectroscopy, when light interacts with matter, the photos (which make up the light) may be absorbed or scattered or may not interact with the material, but pass straight through it. (19) The scattered light that has a different frequency from the incident light is used to construct a Raman spectrum. The Raman spectra arise is due to the inelastic collision between incident monochromatic radiation and molecules sampled. (20) If the energy of an incident photon corresponds to the energy gap between the ground state of a molecule and its excited state, the photon may be absorbed and the molecule promoted to the higher energy excited state. (19) The change in this is what is measured in absorption spectroscopy by the detection of the loss of the energy of radiation from the light. However, it may also be possible for the photon to interact with the molecule ad scatter from it. If this is the case, there is no need for the photon to have an energy which matches the difference between two energy levels of the molecule. The scattered photons are observed by collecting light at an angle to the incident light beam, and as long as there is no absorption from any sort of electronic transitions, the efficiency increases as the fourth power of the frequency of the incident light. (19) When the frequency of incident radiation is high than the frequency of scattered radiation, stoke lines appear in the Raman spectrum however, when the frequency of incident radiation is lower than frequency of scattered radiation, anti-stokes lines appear in the Raman spectrum. (20)

Stokes bands are more intense than anti-Stokes as Stokes sifted Raman bands involve the transitions from lower to higher energy vibrational levels whereas the anti-Stokes bands are measured with fluorescing samples because the fluorescence causes interference with stoke bands. The magnitude of Raman shifts does not depend on the wavelength of incident radiation however, Raman scattering does depend on the wavelength of the incident radiation. A change in polarizability during molecule vibration is an essential requirement to obtain Raman spectrum of sample. As Raman scattering due to water is low, water is an ideal solvent for dissolving samples. Glass can also be used for optical components (mirror lens, sample cell) in Raman spectrophotometer. (20)

Raman scattering is a commonly used technique for example, it is widely used for measuring particle size and size distribution down to sizes less than 1um. The process of absorption is used in a rage of spectroscopic techniques. For example, it is used I acoustic spectroscopy where there is a small energy difference between the ground and excited states ad in X-ray absorption spectroscopy where there is a very large difference. (19)

A Raman spectrum is presented as an intensity-versus-wavelength shift. A Raman spectra can be recorded over a range of 4000-10cm however Raman active normal modes of vibration of organic molecules occur in the range of 4000-400cm^-1. Dependent on spectrophotometers design and optical components, typical Raman spectra cover the wavenumber region between 400-5cm^-1 and 4000-3800cm^-1. A Raman spectrum is significantly simple than their Infrared (IR) counterparts because in normal Raman overtones, combination and different bands are rare. (20)

4.2 RAMAN SPECTROSCOPY IN GRAPHENE OXIDE AND GRAPHITE

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