\r\n\tThe most phenomena of colloids in the surrounding ecosystem appear in the blue color of the sky, which due to the scattering of light by colloidal particles in the air, and this phenomenon is named Tyndall effect. Also, seawater seems blue due to scattering of light by the colloidal particle present in seawater. In our body, blood is a colloidal solution. Some examples in our daily life include milk, cream, gelatin, colored glass, butter, jelly, and river mud. Colloids are useful for human health, commercial, and industrial use. Colloids and colloidal systems are essential and play a significant role in our life. Important applications of colloids are in medicines, sewage disposal, purification of water, cleansing action of soap, the formation of the delta, industry, mining, smoke precipitation, photography, electroplating, artificial rain, agriculture, the rubber industry, and others.
\r\n\r\n\tThis book aims to gather recent researches by outstanding experts in the field of colloids (chemistry, physics, biology, medicine, nanotechnology, pharmacology, and others), which include colloid synthesis, modification, and application.
",isbn:"978-1-83962-979-2",printIsbn:"978-1-83962-969-3",pdfIsbn:"978-1-83962-980-8",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"55025219ea1a8b915ec8aa4b9f497a8d",bookSignature:"Prof. Mohamed Nageeb Rashed",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10505.jpg",keywords:"Colloids, Biotechnology, Industry, Colloidal Systems, Human Health, Nutrition, Intravenous Therapy, Pharmacology, Drugs, Wastewater Treatment, Nanocolloids, Green Nanocolloids",numberOfDownloads:439,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"June 30th 2020",dateEndSecondStepPublish:"July 21st 2020",dateEndThirdStepPublish:"September 19th 2020",dateEndFourthStepPublish:"December 8th 2020",dateEndFifthStepPublish:"February 6th 2021",remainingDaysToSecondStep:"6 months",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:"Professor of analytical and environmental chemistry, member of several national and international societies, and winner of the Egyptian State Award for Environmental Researches.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"63465",title:"Prof.",name:"Mohamed Nageeb",middleName:null,surname:"Rashed",slug:"mohamed-nageeb-rashed",fullName:"Mohamed Nageeb Rashed",profilePictureURL:"https://mts.intechopen.com/storage/users/63465/images/system/63465.gif",biography:"Prof. Mohamed Nageeb Rashed is a professor of analytical and environmental chemistry, a previous vice-dean for environmental affairs, Faculty of Science, Aswan University, Egypt. In 1989 he received his Ph.D. in analytical environmental chemistry from Assiut University, Egypt. His research interest has been analytical and environmental chemistry with special emphasis on soil and water pollution monitoring, control and treatment; bioindicators for water pollution monitoring; wastewater impact; advanced oxidation treatment; photocatalysis, nanocatalyst, nanocomposite and adsorption techniques in water and wastewater treatment. Prof. Rashed supervised several M.Sc. and Ph.D. thesis in the field of pollution, analytical and environmental chemistry. He participated as an invited speaker in 30 international conferences worldwide. Prof.Rashed acts as editor-in-chief and an editorial board member in several international journals (30) in the fields of chemistry and environment. His society membership includes several national and international societies. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"50978",title:"Electrospun Graphene Oxide-Based Nanofibres",doi:"10.5772/64055",slug:"electrospun-graphene-oxide-based-nanofibres",body:'\nThe discovery of graphene around this decade changed the focus direction of material science into its unique and magnificent characteristics. Researchers throughout the world are competing in finding ways to synthesis and explore the possible applications of graphene-based materials. Graphene oxide (GO) is an intermediate material that has better hydrophilicity and easier modification due to the presence of oxygen functionalities, compared to graphene. A broad range of applications have been reported over the years, including tissue engineering, sensor, power storage, coating and photonics. Electrospinning is one of the techniques that make use of GO materials in nanofibrous composites. This state-of-the art technique has been very popular in facile 1-D nano-scaled fibres production and gives materials their excellent properties in relation to high specific surface area.
\nThe novel nanostructure of graphene has been showing promising characteristics not only in fundamental science but also in variety of applications. Its remarkable chemical and physical properties urged into many attempts of synthesizing graphene in mass production. However, it fails to give adequate productivity for large-scale use [1]. Graphene is not easy to be handled when it comes to incorporating it with other materials and to transform it into other form. Current development in this field recognizes graphene oxide (GO) as the best alternative to alleviate this problem, by having a more solvent friendly structure [2]. The solubility of GO in water and other solvents is likely in favour to the large commercialization of this novel material.
\nGO is basically a graphene sheet with oxygen functional groups, either epoxy, carboxyl or hydroxyl. It can be easily exfoliated from graphite through oxidation and sonication processes. Since graphene is a one-atomic layer of graphite, the structure of GO also consists in exfoliated layers of graphite oxide. Simplistically, the structural process is shown in Figure 1. The carbon compounds of Figure 1 are organized in a honey-comb structure. One sheet of graphene contains six-carbon-atom hexagons, bonded by strong π and σ bonds.
\nStructural process of graphene oxide.
The synthesis of GO involves oxidation of graphite into graphite oxide and exfoliation of graphite oxide into graphene oxide (Figure 1). There are several routes that can be utilized to oxidize graphite. The Brodie method, Brodie–Staudenmaier method and Hummers method are the earliest routes developed, but they lead to the release of toxic gases like NOx and ClOx [3]. Later, a typical modified Hummers method was introduced, and it has been used in many reported works in literature [4–6]. In general, graphite–graphene oxide conversion process can be summarized down into three steps [7]. First, the conversion of graphite into graphite intercalation compound (H2SO4–GIC), then, as a second step, H2SO4–GIC is converted into pristine graphite oxide (PGO). Finally, the conversion of PGO into GO takes place by the reaction of PGO with water.
\nThe structure of GO on its own has become a research area interest among material scientists. This is due to the complexity of its non-stoichiometric composition [8]. The most recent and acceptable structure is constructed by Lerf and Klinowski. The Lerf–Klinowski model ignores the lattice-based model and focuses on a nonstoichiometric-amorphous alternative. It contains unoxidized benzene rings and aliphatic six-membered rings with epoxy, hydroxyl, carbonyl and/or carboxyl groups on the graphene sheet or at the graphene edges. Each oxygen atom is covalently bonded to two carbon atoms, forming an epoxy ring. Reduced graphene oxide (rGO) structure contains both sp2 and sp3 hybridized carbons and a high carbon/oxygen atoms ratio (C:O). sp2 carbons bind to three other atoms, sharing a double bond with one of them, while a sp3 carbon binds to a four other atoms.
\nA study on atomic and electronic structure of GO using annular dark field (ADF) imaging and electron energy loss spectroscopy (EELS) was conducted by Mkhoyan et al. in 2009 [9]. They found out that oxygen atoms randomly attach to graphene sites and convert sp2 carbon bonds into sp3 bonds. By ab initio calculated structure, two carbon atoms bonded to an oxygen atom are pulled above graphene plane. This configuration is fairly stable with −3.12 eV absorption energy. The bond length between these two carbon atoms expands to 1.514 Å from graphene 1.407 Å [9]. This is close to the diamond sp3 bond length of 1.54 Å. The sp3 hybridization and π and σ bonds of graphene oxide contribute the most to its unique physical and chemical properties. The extent of polar groups on the surface and edges of the graphene oxide depends on the amount of the oxidizing agent and the time and temperature of the oxidation reaction.
\nSpotted and closely studied only since 2004, graphene has already caught wide attention in research history after one by one of its interesting properties were reported. Being a single atomic layer of graphite, graphene is very thin and light. Yet, it is the strongest material ever discovered with intrinsic strength of 42 Nm−1 [10], so robust that it is 200 times stronger than steel and can outperform the hardness of diamond. It is transparent, but it can absorb light [11]. Having Young’s modulus to approximately 1 TPa [10], it is elastic and pliable but, at the same time, is impermeable to all gases and liquids, except water [12]. It was also reported that graphene possesses superior thermal conductivity in the range of ~(4.84 ± 0.44) × 103 to (5.30 ± 0.48) × 103W/mK [13]. These values suggest that it can far exceed carbon nanotubes in conducting heat. Another important character of graphene is its exceptional electrical properties for having high intrinsic mobility of 2 × 105 cm2 v−1s−1, compared to the highest known inorganic semiconductor (carbon nanotubes, with ~1 × 105 cm2 v−1s−1) [14, 15].
\nDespite of being the building block of graphite layers, the synthesis of graphene is not a straight-forward exfoliation process. The method that discovered graphene sheets in 2004, using cellophane tape, does not appear to be a readily scalable process. Graphite exists in large quantities and is not expensive, yet producing sufficient graphene sheets is still a huge challenge [3]. Even the use of chemical vapour deposition (CVD) to deposit graphene on arbitrary substrates is not possible at low temperatures and is incompatible for mass production processes. Another concern is that graphene is a difficult material to be incorporated into various polymer matrices. Although graphene has unique properties, due to the strong π and σ bonds, these also contribute to its weak reactivity and low solubility in common organic solvents.
\nIn order to prepare graphene–polymer composites or graphene-based hybrid materials, stable dispersion of graphene with other matrices must be obtained. This is when GO becomes useful. The sp3 hybridization of GO contributes to a larger amount of free bonds, therefore increasing reactivity [16]. When graphite is oxidized, the functional groups make its hydrophilic and improve its exfoliation in aqueous media or organic solutions. The surface functionalization and solvents are the critical factors in determining interaction with functional groups of polymer or other matrixes. Several studies were done to determine the behaviour of GO towards a wide variety of solvents. Paredes et al. [17] studied the dispersibility of GO in acetone, methanol, ethanol, 1-propanol, ethylene glycol, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), pyridine, tetrahydrofuran (THF), dichloromethane (DCM), o-xylene and n-hexane. They have successfully identified some type of solvents (DMF, NMP, THF and ethylene glycol) that can maintain a stable GO solubility. Later, another similar study compared GO and rGO solubility in eighteen different solvents. That investigation suggests that polarity of solvents is not the only factor for good dispersibility [2]. NMP and ethylene glycol, besides water, were double-proved to be the best solvents for long-term stability.
\nThe covalent character of C–O bonds in GO disrupts the sp2 conjugation of pure graphene and gives GO its insulating properties. Electrical conductivity can be recovered by restoring the π–network. GO, due to the presence of abundant oxygen, can be easily modified. Control of oxidation can provide tunable electronic properties, including the possibility of accessing zero-band gap graphene via bonds. GO, which contains a C/O content ratio of 2/1, is non-conductive (less than 1 S/m). GO can be partially reduced to conductive graphene-like sheets by removing the oxygen-containing groups. Restoration of the π–network and conjugated structure can be obtained by reducing GO to 6/1 of C/O atomic ratio. GO reduction is a process that converts sp3 to sp2 carbon, and thus recovers the important electrical properties. Some researchers make use of the oxygenated functionalities to bind with functional groups of conducting polymers, such as polyaniline, to improve the conductivity.
\nAt nanoscale, materials exist in 0, 1, 2, or 3 dimensions. One dimensional nanofibres can be synthesized by several techniques such as phase separation, self-assembly, template synthesis and electrospinning. Among those techniques, electrospinning has been recognized as a facile process in fabricating polymeric fibres in nano-size [18]. It promptly gained popularity since its establishment, due to its potential implementation in various fields such as tissue engineering, chemical and biological sensing, energy generation and textiles.
\nThe first observation on electrospinning dates back from the study of the electrospraying method used by Rayleigh (1897) and Zeleny (1914) [19]. Formhals, however, is the one who patented the electrostatic force experimental setup of polymer filaments production. In 1934, Formhals published a patent in which describing the production of textile yarn from cellulose acetate [20]. Since then, further research was carried out in order to assemble fibre processing using electrostatic force. Later, the study of electrically driven jets, by Taylor in 1969, became a strong foundation in understanding the electrospinning process. Only around 1994, the electrospinning term was used. Along with its popularity and rapid establishment in many fields, electrospinning has undergone many modifications and upgrading, in order to maximize its production and effectiveness in fabricating versatile nanofibres. There are several advanced techniques and setups of electrospinning instruments used favourably according to the required characterizations. Coaxial electrospinning, multilayer and mixed electrospinning, forced air-assisted electrospinning and air-gap electrospinning are a number of innovative processing techniques to enhance the function of electrospun fibres [21].
\nBasically, the typical basic components of any electrospinning setup are the high-voltage power supply, the spinneret (normally syringe with blunt-tip needle) and the collector (normally grounded). This basic experimental setup is shown in Figure 2. The polymer melt or solution placed into the syringe is held by retort stand (for vertical setups) or with a syringe pump (for vertical and horizontal setups). The tip of the syringe needle is connected to the positive electrode of the voltage supply. From here, the polymer liquid is pumped out of the syringe through the needle and brought to the grounded collector beneath the syringe or placed horizontally.
\nElectrospinning basic experimental setup.
This unique approach for producing continuous fibres from polymer melt or solution, works by the principle of surface tension acting on the polymer drops. By the time, electricity is supplied onto the polymer, the fluid droplet will gain electrostatic charges, being positive charged. Due to the interaction between two forces, a Taylor cone is formed. The increase in high-voltage supply then eventually achieves a certain critical point, at which the electrostatic repulsion can overcome the surface tension holding the polymer droplet. It results in a liquid jet ejection targeting the collector. Figure 3 shows a clearer depiction of the principle behind electrospinning technique.
\nDescription of electrospinning principle.
The polymer jets onto grounded collector from the spinneret can be divided into two segments. There are streaming jet and whipping jet (Figure 4). The streaming jet can be seen with appropriate lighting, while the whipping jet is invisible to the naked eye. The flow of the streaming jet may give a wise initial prediction on the morphology of collected nanofibres. Leach et al., in 2011, indicated that streams behaving inconsistently, non-uniformly, in short and oscillating, might produce fibres with poor alignment, beading, wavy pattern and splattering [22]. Therefore, researchers engaging in electrospinning studies need to carefully study various parameters (which will be further explained in the next section) before the optimized nanofibre formation procedure can be achieved.
\nStreaming and whipping jets.
Electrospinning deals with many working parameters in order to fabricate the required morphology and properties in the as-spun nanofibres. Other than the electrospinning experimental setup, like having different kind of collectors or spinnerets, electrospun nanofibres are also mainly affected by working parameters. The factors to be adjusted are the properties of polymer solution or melt, operational properties and ambient parameters [18, 22]. The electrospinning solution and operational properties are among the major parameters that influence electrospun nanofibres traits. Solution viscosity, surface tension and conductivity, along with electric field strength, distance between spinneret tip with collector and also the flow rate, are thoroughly investigated by researchers to maintain optimized fibre formation. Meanwhile, most researchers prefer to keep ambient parameters such as solution temperature, humidity and surrounding air velocity as constants throughout their studies. Table 1 displays some factors taken into account for tuning the electrospun fibre morphology. Note that whenever finer or smaller diameter fibres are spun, they tend to get beading. Therefore, it is important for researchers to recognize their requirements and determine the optimized conditions for electrospun fibre formation. There are also some parameters that need minimum requirements to be executed in order to induce electrospinnability. The applied voltage and tip-to-collector distance, for example, are some of those parameters, having certain minimum values which differ for every electrospinning material. Other parameters that rule electrospinning process are the molecular weight of the material used, types of collector and of solvent used, and ambient parameters [18, 23].
\nParameter | \nEffect on fibre morphology | \n
---|---|
Viscosity | \nFibre diameters increase with solution viscosity. Also, higher viscosity results in more beading | \n
Concentration | \nHigh solution concentration results in larger fibre diameter | \n
Applied voltage | \nHigher-voltage supply jets more fluid and cause larger fibre diameter Increase in electrical potential also results in rougher fibres | \n
Surface tension | \nReduction in surface tension minimizes fibre beads | \n
Feed-rate | \nLow feeding rate: decrease in fibre diameter High feeding rate: increase in bead generation and Taylor cone instable | \n
Tip-to-collector distance | \nMinimum distance required for dry fibres. Excessively far distance will lessen the deposited amount of fibres on the collector | \n
Conductivity | \nDecrease in fibre diameter with increase in conductivity, bending instability and diameter distribution | \n
The establishment of GO incorporation into polymeric electrospun nanofibres was only initiated around this decade. Since 2010, at least 34 literature works were published involving about 14 types of synthetic and natural polymers. There are some distinct reasons to add GO and its derivatives into electrospun nanofibres. The major purpose is to improve the mechanical and electrical properties of the electrospun material. Others include the morphological enhancement to meet certain diameters or porosity of the spun fibres. Table 2 summarizes the reported polymer/GO combinations.
\nGraphene oxide | \nPolymer | \nReferences | \n
---|---|---|
Graphene oxide | \nPoly (vinyl acetate) (PVAc) | \n[56] | \n
Graphene oxide | \nPolycaprolactone (PCL) | \n[42, 56] | \n
Functionalized graphene oxide | \nPoly (lactic acid) (PLA) | \n[26, 41] | \n
Graphene oxide | \npoly(lactic-co-glycolic acid) (PLGA) and collagen (Col) | \n[40] | \n
Graphene oxide | \npoly(lactic-co-glycolic acid) (PLGA) | \n[32, 40] | \n
Graphene oxide | \nPLA and polyurethane (PU) | \n[38] | \n
Graphene oxide | \nPolyurethane (PU) | \n[38, 53, 55] | \n
Graphene oxide | \nPolyethylene oxide (PEO)/chitosan (CS) | \n[44] | \n
Graphene oxide | \nPoly (vinyl alcohol) (PVA) | \n[31, 35, 44, 48] | \n
Reduced graphene oxide | \nPoly (vinyl alcohol) (PVA) | \n[24, 27] | \n
Graphene oxide | \nPVA and chitosan CS | \n[33, 47] | \n
Graphene oxide | \nNylon 6-6 | \n[52] | \n
Reduced graphene oxide | \nNylon-6 | \n[57] | \n
Graphene oxide | \nNylon-6 | \n[34] | \n
Reduced graphene oxide | \nPoly(carbonate urethane) (PCU) | \n[30] | \n
Reduced graphene oxide | \npolyvinyl pyrrolidone (PVP) | \n[25] | \n
Reduced graphene oxide | \npolyacrylonitrile (PAN) | \n[25, 29, 36] | \n
Functionalized graphene oxide | \npolyacrylonitrile (PAN) | \n[28] | \n
Graphene oxide | \nPolyacrylonitrile (PAN) | \n[28, 50] | \n
Reduced graphene oxide | \nPolyvinyl Butyral (PVB) | \n[46] | \n
Graphene oxide | \nPolyacrylic acid (PAA) | \n[54] | \n
Reported literatures on electrospun GO.
A fundamental study by Park et al. in 2013 described the effect of GO distribution and its location in 1D polymer composites. By controlling the distribution and location of the embedded nanomaterials in 1D nanoscale composites, the macroscopic properties can be enhanced and thus enable many practical applications. In their study, three types of polymers and two different solvents were used. The type of polymers and solvents are two processing conditions to manipulate the distribution and location of the GOs. Homogeneous colloidal dispersions of GO in the polymer solutions were successfully electrospun into polymer nanofibres, without the formation of beads or aggregates [24]. The location of GO was influenced by phase separation during the electrospinning process, which was in turn dependent on the compatibility between the two components and the vapour pressure of the solvents. The thermal and mechanical properties of polymeric composite nanofibres were effectively improved by adding GO as nanofiller.
\nGOs have a curved morphological structure and form powder materials after exfoliation from graphite oxides. This powder structure can be used in paints, adhesives, coatings and lubricants, where GOs act as multifunctional additives. However, for applications where structured thickness is a limitation, the levelling of the graphene sheets in a plane is required. Thus, an effort to study the levelling of graphene sheets through electrospinning was recently carried out [25]. Those authors used PAN and PVP as examples of polar polymers for nanofibre matrices. GO was reduced using different agents, but later it was confirmed that the curved features of GO sheets cannot be prevented using arGO coating strategy. Nonetheless, the strategy of using electrospinning to extend single GO sheets seems effective. Levelled graphene sheets were successfully developed and showed good conductivity in the order of 103 Sm-1.
\nThe effect of GO loading into PCL was investigated in presence of different oxidation levels [26]. GO dispersibility, wettability, polarity and charge relaxation time, which assess the fibre diameter, increase with oxidation level. Surface tension of PCL solution is invariable in the presence of GO, resulted in diameter variations of the spun fibres. The diameter of PCL nanofibres decreases with GO loading, due to the reduction of solution viscosity and increase in the conductivity. Another morphological study attempted to blend rGO into PVA [27]. The addition of a nanofiller seems to increase the viscosity and conductivity, but the nanofibre exhibited smaller diameters than those without nanofillers. Some GO particles were also observed to be protruded from the composite nanofibres under transmission electron microscope (TEM).
\nThe manufacturing of commercial high-performance carbon nanofibre (CNF) has been carried out using PAN as precursor, due to its high carbon yield and superior fibre strength. It includes a typical process of oxidative stabilization and carbonization steps. Nevertheless, most continuous CNFs produced to date have relatively poor graphitic structure, which is important for higher elastic moduli and thermal and electrical conductivities. This can be solved by increasing the carbonization temperature, because high temperature graphitization significantly improves the size and alignment of the graphitic crystallites in PAN-based CNFs. This high-temperature condition is, however, unlikely since fibre processing at such conditions requires a specialized expensive equipment. Therefore, Papkov and co-workers, in 2013, carefully made an effort to maximize the orientation of the polymer chains inside the polymer precursor fibre by incorporating oriented inclusions that had high surface area and could strongly interact with polymer chains [28]. GO was used since it can both organize the polymer chains and simultaneously serve as a ‘templating’ agent for the formation of graphitic crystallites during carbonization. Their close examination revealed the presence of crumpled GO particles inside the PAN matrix. The irregular shape of the radially crumpled sheets closely matches the popular model of the high-strength carbon fibre structure, which consists of irregular, longitudinally wrinkled turbostratic graphitic stacks with preferred axial orientation. It is very useful to represent a natural template to achieve similar high-strength CNFs structure in economic manufacturing protocol. In addition, the crumpled graphene oxide particles may provide multiple nucleation sites for graphitic crystal growth during carbonization, especially because the carbon atoms along the crumpled folds are more reactive.
\nThe paramount role of graphene-based embedded in polymer matrix via electrospinning is to enhance the mechanical properties of the polymeric nanofibres. Wang et al., in 2015, examined PAN/GO composite nanofibres with lateral force microscopy and force-distance curves, in order to assess the surface properties like friction force and elasticity. The experiment results indicated that, with increasing GO concentrations, both the surface friction force and adhesive force increased [29]. A mechanical characterization of high-performance GO incorporated into an aligned fibro-porous PCU membrane was made, under static and dynamic conditions [30]. The electrospun membrane indicated that 55% of the tensile strength increased, a 127% rise in toughness, and the achievement of maximum strength reinforcement efficiency was reported at 1.5 wt.% GO loading. An even smaller loading of GO (0.02%) in PVA diluted aqueous solution was able to produce elevation of tensile strength of electrospun nanofibres by 42 times [31].
\nAnother study on significant improvements in thermomechanical and surface chemical properties of poly(
There were also some experiments carried out with produce electrospun GO/polymer nanofibres for morphological modification, in order to meet certain requirements for applications. A spider-wave-like nano-net was fabricated from GO blend with nylon-6 which holds polyelectrolytic and polymorphic characteristics [34]. The amide (CO-NH) group of nylon-6 molecule can easily interact with GO by means of hydrogen bond formation or donor-acceptor complexes. The strategy was to incorporate foreign materials into nylon-6 in order to change the morphology and crystallization of nanofibres. The pristine nylon-6 electrospun mat appears well defined with no spider-wave-like nano-net. As GO was added into the polymeric solution, a double-layered morphology (bimodal fibre diameter distributed mat) was noticeable. Further increase of GO in the polymeric solution not only decreased the spider-wave-like structure but also decreased the spinnability and formed a film-like structure.
\nThe widespread use of electrospinning technique in tissue engineering application is no longer new. Many researchers have been exploring this subject. GO/polymer composites also found their way through this area, since they showed potential biocompatibility and noncytotoxicity characteristics. Scaffolds play an important role in tissue engineering as they not only provide support for cell attachment, but they also allow cell growth into tissue until the tissue is able to sustain itself. There are several types of GO/polymer composites been developed for tissue engineering scaffolds, such as PVA/GO [35], PAN/rGO [36], PVA/CS/GO [37], PLA/PU/GO [38], PLA/hydroxyapatite (HA)/GO [39], GO/PLGA/Col [40], GO-g-PEG/PLA [41] and PCL/GO [42]. The polymeric scaffolds attain better thermal, mechanical and electrical properties, due to the intrinsic properties of GO. Cells adhesion and proliferation onto these nanofibrous scaffolds have been tested morphologically and quantitatively. Cells like mouse osteoblastic cells (MC3T3-E1), adipose-derived stem cells (ADSCs), skeletal myoblasts (C2C12), mouse marrow mesenchymal stem cells (MSCs) and low-differentiated rat pheochromocytoma cells (PC12-L) were cultured onto the samples. They confirmed that a small amount of GO did not restrain the proliferation and viability of cells, which demonstrated the appreciable cell affinity of GO. Cells are likely to spread on the composite scaffolds and some even enhance the cell growth by having similar rate of cell proliferation to that of tissue culture plates.
\nTissue engineering is very important for regeneration and replacement of damaged tissues in the human body. Scaffolds for musculoskeletal and skin tissues have caught the attention of researchers in the last years, aiming to help many patients with injuries. On the other hand, tissue engineering for organs or tissues without regenerative abilities is an extensive area that needs serious exploration. Vascular tissue engineering is one of its branches, and it aims to fabricate functional vascular grafts to be used in vivo, in order to replace blood vessels and help their regeneration. Current commercial synthetic grafts used by surgeons are woven poly (ethylene terephthalate) (Dacron) and extended polytetrafluoroethylene (ePTFE). Although the aim was to substitute large diameter blood vessels, they are not suitable for small diameter grafts due to thrombosis and intimal hyperplasia risks. A thermoplastic polyurethane (TPU)/GO scaffold was fabricated using grounded rotation mandrel as electrospinning collector to obtain fibrous small diameter tubular scaffolds [43]. The tensile strength (Young’s modulus) and scaffolds hydrophilicity increased as GO amount increased. The suture retention strength and burst pressure of tubular TPU/GO scaffolds containing 0.5 wt.%. GO exceeded the mechanical requirements of human blood vessels. In addition, results of platelet adhesion tests indicated that a low loading of GO is unlikely to cause thrombosis on the scaffolds. Endothelial cells could form a layer on the inner surface of the tubular scaffolds which resembles the native blood vessel structure, indicating that the TPU/GO electrospun scaffolds, with low GO loading level, have the potential to be used in small diameter vascular graft applications.
\nAnother impressive function of scaffolds that needs to be expanded is the ability to control drug release onto specific tissues or organs. A targeted drug delivery system that can efficiently transport drugs is a way for a scaffold to improve the efficacy of the drug and to reduce the systemic toxicity. Electrospun nanofibres are very important materials due to the high surface area and high porosity of the nanofibres, which are able to provide higher drug encapsulation efficiency and better stability, compared to other systems. An electrospun scaffold for anti-cancer drug delivery was tested using a polyethylene oxide (PEO)/chitosan (CS)/GO nanocomposite [44]. In the nanofibre carrier system, GOs act as nanocarriers which control doxorubicin (DOX) release, an anthracycline antibiotic used in cancer chemotherapy intravenous administration. DOX and GO surface can make a strong bond through π–π stacking interactions as a non-covalent type of functionalization that may provide controlled release of drug. GO also contains epoxy and carboxylic functional groups that can react with the amine groups of CS. The π–π stacking interaction between DOX and GO with the fine pores of nanofibrous scaffolds allowed high drug loadings (98%).A strong pH dependence was shown by the DOX release result at pH 5.3 and 7.4. At pH 5.3, faster drug release can be seen which may resulted from the unstability of hydrogen-bonding interaction between GO and DOX.
\nAlong with the development of tissue engineering field, the fabrication of scaffolds is also advancing and succeeding in providing a mimic environment to the real body system. Currently, combining biomaterial with living cells for tissue scaffolds is a critical approach. Luo et al., in 2015, have reported a fabrication of GO-doped ply (lactic-co-glycolic acid) (PLGA) nanofibrous scaffold mixed with human MSCs for osteogenic differentiation [45]. The porous structure and the diameter of produced fibres are similar to the topological structure of natural extracellular matrices (ECM), and therefore facilitating cell attachment and proliferation. GO plays two roles in this scaffold. First, it enhances the hydrophilic performance and protein- and inducer-adsorption ability of the nanofibres. Second, it accelerates the human MSCs adhesion, proliferation and differentiation towards osteoblasts.
\nGraphene-based materials have extraordinary electronic transport properties, strong mechanical strength, excellent electrocatalytic activities, high surface area, ease of functionalization, and thus have contributed to ion detector and biosensing applications. A sensor for Cu (II) detector based on rGO/polyvinyl butyral (PVB) nanofibres was fabricated by Ding and co-workers in 2015. Electrospun GO/PVB on glassy carbon electrode (GCE) was reduced through an electrochemical method, to form reduced graphene oxide (RGO)/PVB nanofibres [46]. Cu (II) is one of the heavy metals that contaminate water sources through casual discharge from mining, machinery manufacturing and metal smelting. Although copper is an essential element in human body, the excessive intake of copper might results in Wilson’s disease and Menke’s syndrome [46]. The rGO/PVB nanofibres modified glassy carbon electrode (GCE) were used for Cu (II) detection by differential pulse anodic stripping voltammetry (DPASV) [46]. The as-fabricated sensor based on rGO/PVB nanofibres showed good analytical performance with the linear range of 0.06−22 μM, a low detection limit of 4.1 nM (S/N=3), high sensitivity of 103.51 μA ∙μM−1 cm−2, good selectivity and excellent reproducibility (RSD = 0.49%) [46].
\nA novel platform for enhanced biosensing of electrospun PVA/CS/GO was also engineered [47]. Authors used glucose oxidase (GOD) as a model enzyme. Nanofibres produced from electrospinning are an ideal biosensing platform because of the large surface area they provide, leading to high enzyme loading capacity and low hindrance for mass transfer. Electrospinning is also a simple, durable and adjustable prove to provide stability and reproducibility of biosensors. GOs can bind enzyme molecules well, through electrostatic interactions, and thus avoiding the leaching of enzymes from modified electrodes. Nanomaterials are needed in nanofibre platform to bind the enzyme, since accessibility of the substrate to enzyme will be inhibited when the enzyme is confined inside the nanofibres. The fabricated biosensing platform displayed optimal oxidation and reduction capabilities towards electro-active species, even when compared to the bare Pt electrode, due to the synergy effects of PVA/chitosan nanofibres and GO nanosheets [47].
\nPower generation is one of the essential needs of human daily life. The increasing demand for green and rechargeable energy sources to power up many electronic devices urged into a variety of power storage system alternatives. Knowing the superior properties of graphene and high specific surface area of CNFs, a number of attempts have been made to incorporate these materials into the fabrication of electrodes for Li-batteries and supercapacitors. Current freestanding anode for Li-ion batteries of graphite has limited theoretical capacity (372 mA h g−1). Xu et al., in 2014, proposed the use of Si, a nontoxic and abundant element, as the anode, due to its highest ever-known theoretical capacity up to 4200 mA h g−1 [48]. However, Si has its limitations, as it can undergo up to 280% volumetric expansion upon lithiation, which leads to electrode pulverization and loss of electrical contacts, resulting in severe capacity fading. The solution to this problem is to convert bulk Si into particles of low dimensions and to uniformly disperse Si in an inactive and conductive matrix, like GO. Co-axial nozzle was used to electrospun Si/CNF/GO anode, in order to avoid agglomeration during the spinning process. The resulting electrode achieved a high rate capacity of 567 mA h g−1 at 1 mA h g−1 current density. It obtained excellent cyclic performance with a discharge capacity of 872 mA h g−1after 50 cycles and 91% capacity retention. Another approach is the synthesis of core-shell structured nanofibres that consist of rGO/PAN (shell) and ZnO/carrier polymer (core) [49]. The current use of ZnO has actually gained a good theoretical capacity of 978 mA h g−1. Unfortunately, it also carries a huge volume expansion for approximately 228%, which causes damage of mechanical integrity. Considering the same solutions, Shilpa et al., in 2015, encapsulated ZnO nanoparticles in the hollow core of glassy carbon–reduced graphene oxide (C–rGO) where the void spaces should provide a buffer zone, accommodating the volume changes associated with lithiation/delithiation of the metal oxide and prevent attrition of nanoparticles by detachment and fragmentation. The outcome of this study gained capacitance of 815 mA h g−1 at a current density of 50 mA g−1 with a capacity retention of almost 80% after 100 cycles [49]. A freestanding electrode eliminates the use of current collector, which is an inactive material that only adds to the battery mass and volume.
\nAnother CNF composite in form of fibre mats was prepared by electrospinning blend of GO and PAN in N,N-dimethylformamide (DMF) for supercapacitor electrodes [50]. Porous carbon material of CNFs was further improved by graphene-based electrical conductivity, through carbonization, to obtain CNF/graphene composites. GO concentration was the major factor responsible for the morphology and pore structures, where the optimized-specific surface area and conductivity were achieved at 5 wt.%. The electrode material (5 wt.% GO) exhibited enhanced electrochemical performance with a highest specific capacitance of 146.62 F g−1, and highest energy density of 14.83–19.44 W h kg−1 in the power density range of 400–20,000 W kg−1. Over the last decades, there was another upcoming power storage system that has been successfully applied in numerous fields. Latent heat storage using phase change material (PCM) makes use of the solid–liquid phase change of a material in order to store thermal energy. Fatty acid esters showed potential significant energy storage ability, as organic PCMs, with satisfactory thermal properties, thermal conductivity and thermal reliability. Nevertheless, the use of organic PCMs may undergo energy leakage. The synthesis of PCM with a supporting material is crucial to overcome the leakage problem and improve its thermal conductivity. Ke et al., in 2014, report on the preparation of methyl stearate (MES) fatty acid ester with PAN/GO by electrospinning [51]. GO was selected as the heat transfer/diffusion promoter and also as supporting material in the phase change composite. The results suggest that by adding GO, beneficial effects on mechanical properties, thermal stability and thermal energy storage/release performance were gained.
\nA work has been published dealing with the use of nylon 6-6 excellent mechanical resistance, with functionalized GO as a corrosion protection composite coating [52]. 2% GO loading in the electrospun nylon 6-6 possessed coating capacitance in the order of 10−7 F/cm2, compared to 10−4 F/cm2 for coating prepared by deposit. This value suggests charge storage capacity for the coating condition. Thus, the efficiency of deposited coating is better than electrospun coating. The well-known physicochemical properties of PU have been widely used in coating technology. However, its low hydrophilicity and mechanical properties are the main setbacks for its application. Meanwhile, GO-polymer composite fibres emerged as the subject of enormous interest because polymer nanocomposites, with graphene materials as fillers, have shown dramatic improvements in such needed properties. Electropinning of in situ synthesized PU/GO has been conducted for surface coating of metallic stents [53]. This study showed that the incorporation of small amounts of GO sheets through PU nanofibres could enhance the stability of coated layer on the surface of nonvascular stent. The grafted PU chains on the surface of GO can facilitate the beneficial stress transfer from the PU matrix to the GO sheets. Moreover, proper incorporation of GO sheets into the polymer matrix can improve the hydrophilicity of the materials, without any adverse biological effect. These improved properties of PU mat, induced by GO, make it a potential candidate for surface coating of medical devices.
\nIn order to fabricate water-swellable rubber (WSR), a multi-scale hybrid nanofibre with enhanced elasticity, resilience, toughness and water swelling ability was electrospun [54]. WSR can be produced by chemical and physical methods. The chemical method is more complicated and more environmentally unfriendly than the physical method. In the physical method, common hydrophobic rubbers are mixed with various water-absorbent materials, known as superabsorbent polymers (SAPs) (hydrogel form). Hydrophilic super water-absorbent resin does not disperse well in hydrophobic rubber, expecting easy break off of physical-synthesized WSR. Eventually, swelling ability will lose, strength is remarkably decreased, and water is polluted. The addition of hydrophilic fibres can fix then as they can act as internal water channels in hydrophobic materials for superabsorbent nanocomposites. These fibres can help to transfer water from the surface of the rubber matrix to the hydrophilic SAP particles, as well as between encapsulated SAP particles in the rubber matrix, enhancing the water swelling ability of WSR composites. Fabrication of PAA/ethylene glycol (EG)/hyperbranched bis-MPA polyester-64-hydoxyl (HB)/GO showed synergistic effects on water swelling ability compared to without HB and GO. The electrospun fibre mats were added into conventional rubber by simple physical mixing, and the rubber composites showed considerable enhancement of water swelling ability, mainly due to the water channels built.
\nFunctional shape memory composite nanofibres with GO have also been fabricated with PU shape memory [55]. Shape memory PU/GO (SMPU/GO) has better shape memory effect (SE) and lower thermal shrinkage, compared to SMPU nanofibre mats. When the loading amount of GO increased to 4 wt.%, the thermal shrinkage ratio of the composite nanofibrous mats reached as low as 4.7 ± 0.3%, while the average fixation ratio and recovery ratio were as high as 92.1 and 96.5%, respectively. The study indicates that GO is a desirable reinforcing filler for preparing shape memory nanofibres with improved properties. Electrospun polymer with GO and modified GO fillers have been extensively investigated for a wide range of applications. Apart from all applications mentioned, electrospinning of polymer/GO also stands out as an optical element in fibre lasers [56] and photocatalysts [57], showing their important role in the area of photonics.
\nGraphene oxide and its derivatives show comparable properties as alternatives to graphene. Their outstanding performance can easily be manipulated into a wide variety of applications. An important highlight is that several researchers have shown that the biocompatibility of produced composites is still preserved, with no significant cytotoxicity appearing at low GO loading. Utilizing electrospinning to fabricate polymer/GO composites is a wise choice to obtain high specific surface area fibres at the nanoscale. It displays a promising advantage in achieving optimum properties out of the electrospun composites. Nonetheless, further research towards commercializing polymer/GO electrospun fibres in applications and improvement on electrospinning machines needs to be done, so that one can make use of their outstanding advantages to a larger extent.
\nChlorophyll and carotenoid are important pigments that have been used as intrinsic optical molecular probes to observe plant performance during different phases of development. Chlorophyll and carotenoid are biosynthesized in chloroplast and their metabolism is closely related with the chloroplast development. Chlorophyll biosynthesis begins with the formation of 5-aminolevulinic acid (ALA) from glutamate (Glu) via Glu-tRNA synthetase, Glu-tRNA reductase (GluTR) and Glu-1-semialdehyde aminotransferase (GSA-AT) [1]. Eight molecules of ALA are condensed, eventually forming the symmetric metal-free porphyrin, protoporphyrin IX (Proto IX), which is a common precursor of haem and chlorophyll. The biosynthesis of chlorophyll continues by insertion of Mg2+ into Proto IX and followed by several steps in the chlorophyll cycle to create protochlorophyllide.
\nFurther, reaction is one of the most interesting steps because this is the first step in chlorophyll biosynthesis that requires light: the NADPH:protochlorophyllide oxidoreductase converts protochlorophyllide into chlorophyllide. This reaction is then continued to produce chlorophyll (chl) a and b. So, when dark-grown etiolated seedlings are exposed to light, protochlorophyllide is immediately converted to chlorophyllide and then further to synthesis of chl. Once chl a and b are formed and properly incorporated into the thylakoid membranes and associated photosystems, chloroplast is fully functional to do photosynthesis [2].
\nPlant carotenoids are synthesized and accumulated exclusively in plastids, most importantly, chloroplast and chromoplast [3]. There are two types of plant carotenoid: carotene, which is cyclized and uncyclized hydrocarbons, and xanthophylls, which are oxygenated derivatives of carotenes. Carotenoid synthesis is initiated by the formation of C40 compound phytoene by the head-to-head condensation of two molecules of geranylgeranyl diphosphate (GGDP) by phytoene synthase and then to a series of 4 sequential desaturation reactions, by two separate enzymes to produce lycopene, which has 11 conjugated double bonds [4]. Lycopene is then cyclized to α-carotene or β-carotene, which is then further hydroxylated to produce colorful xanthophylls such as lutein, β-cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin and neoxanthin. The biosynthesis and accumulation of carotenoids in the dark-grown etiolated seedling are essential for the assembly of membrane structure and benefits the development of chloroplast when seedlings emerge into the light [5]. Understanding the relationship between structure and photophysical properties of these pigments can provide insights into a better study of how photosynthesis works at the molecular level in chloroplast.
\nThe photophysical properties and functions of chlorophyll and carotenoid reside in their chemical structure. Chlorophylls are defined as cyclic tetrapyrroles carrying a characteristic isocyclic five-membered ring that are functional in light-harvesting or in charge separation in photosynthesis [6]. The chemical structure with IUPAC numbering scheme of chl a is shown in Figure 1. It is a squarish planar molecule, about 10 Å on a side. An Mg atom in the center of the planar portion is coordinated to four nitrogen atoms. The five rings in chlorophylls are lettered A through E, and the substituent positions on the macrocycle are numbered clockwise, beginning in ring A. Chlorophyll has two molecular axes: y-axis is defined as passing through the N atoms of rings A and C and x-axis passing through the N atoms of rings B and D. The delocalized π electron system extends over most of the molecule, except for ring D, in which the C-17—C-18 double bond is reduced to a single bond. The tail is formed by condensation of four isoprene units and is then esterified to ring D. It is often called phytol tail, after the polyisoprenoid alcohol precursor that is attached during biosynthesis. Because of the reduced ring D, plant chlorophylls such as chl a and b are classified as chlorins rather than porphyrins. These types of pigments have (in organic solvents) absorption bands around the blue and red spectral regions (Figure 2a), which are called B (or Soret) and Q bands, respectively, and arise from π→π* transition of the four frontier orbitals [7, 8]. One band each pair is polarized along the x-axis (Bx, Qx) and other along y-axis (By, Qy). The strong absorption band at the maximum absorption wavelength (λmax) 660 nm is called Qy transition band, which corresponds to the electronic transition polarized along the y-axis. The Qx-band of chl a shows a weak band near 550 nm, while the two overlapping Soret (B) bands show at about 430 nm. The chemical structure of Chl b is identical to chl a except at the C-7 position, where a formyl group replaces the methyl group. This structural change results in a shift of the Qy maximum absorption to shorter wavelength. The fluorescence spectrum of chlorophylls peaks at slightly longer wavelengths than the absorption maximum. The fluorescence emission (Figure 2b) is polarized along the y molecular axis, as it is emitted from the Qy transition. Shift of the emission to the longer wavelength side of the main transition is known as Stokes shift. In light reaction, chlorophyll plays as key pigment in the collection of light energy in the light-harvesting complexes and to carry out reversible photochemical redox reaction (Krasnovsky reaction) in the reaction centers.
\nChemical structure of Chl a (a), Chl b (b), lycopene (c), β-carotene (d), zeaxanthin (e) and lutein (f) with IUPAC numbering system.
(a) UV–Vis absorption of Chl a (black) and Chl b (red) in MeOH, (b) fluorescence emission spectra of Chl a (red) and Chl b (black) in MeOH, (c) β-carotene (red), zeaxanthin (black) and lutein (blue) in EtOH, (d) lutein in several organic solvents; MeOH (black), acetone (pink), diethyl ether (purple), hexane (light blue), EtOH (blue).
Structure of carotenoid is characterized by a linear chain of conjugated π-electron double bonds (Figure 1). In oxygenic organisms, carotenoid usually contains ring structures at each end, and most carotenoids contain oxygen atoms, usually as part of hydroxyl or epoxide groups. The primary molecular factor that gives rise to their strong absorption bands in the visible spectral region is the number of π-electron conjugated double bonds, N. The position of the absorption maxima is affected by the length of the chromophore, the position of the end double bond in the chain or ring and the taking out of conjugation of one double bond in the ring or eliminating it through epoxidation. Progressive movement to longer wavelengths (bathochromic shift) is illustrated by the absorption spectra of the acyclic carotenoid of increasing chromophore length. Carotenoids show different optical characteristics in various solvents, depending on the polarizability of the solvent [9, 10]; however, generally they have a typical three-peaked absorption spectrum with well-defined maxima and minima (fine structure) (Figure 2a). A ring closure as in β-carotene produces a less-defined fine structure. The introduction of a carbonyl group in conjugation with the polyene system produces a bathochromic shift and the loss of fine structure [4]. The influence of other substituents such as OH is negligible, for example, β-carotene, cryptoxanthin and zeaxanthin all have very identical absorption spectrums. Owing to the double bonds in the molecule, all carotenoids exhibit cis-trans isomerization (stereomutation). A cis double bond implies a configuration with the highest-priority group on the same side, whereas in the trans configuration they are on opposite sides. The absorption spectrum of a cis isomer presents a subsidiary peak in the near-ultraviolet, the cis peak; generally, it is located 143 nm from the longest wavelength maximum. For example, cis peak will appear at 330 nm if the longest wavelength maximum is 473 nm. In photosynthetic systems, carotenoid has essential functions. First, carotenoid is an accessory pigment in the collection of light energy in the spectral region which chl does not absorb and in transferring energy to a chl pigment [11, 12]. Second, carotenoid functions in a process called photoprotection by quenching triplet state of chl before it reacts with oxygen to form singlet oxygen species (ROS) [13, 14]. Third, carotenoid regulates energy transfer in the light-harvesting antenna through a process called xanthophyll cycle, to avoid over-excitation of the photosynthetic system by safely dissipating excess energy [15, 16].
\nIn the chloroplast interior, there are four main constituents in plant thylakoids, that is, photosystem II (PSII), cytochrome b6f, photosystem I (PSI) and the ATP synthesis. Chlorophylls and carotenoids are embedded in PS II and PSI, large pigment-protein clusters, the structures of which are perfectly adopted to ensure that almost every absorbed photon can be utilized to drive photochemistry. Both PSII and PSI consist of two moieties, that is, core complex or the reaction center that is responsible for charge separation and light-harvesting antenna complexes that surround the core complex and have functions to increase the capture of light energy and energy transfer to the reaction center in the core complex.
\nOne can detect chlorophyll and carotenoid bound in PSII and PSI in chloroplast by measuring their absorption and fluorescence spectra. Figure 3a (solid red line) shows the absorption spectrum of diluted chloroplast that is indicated by red shift of Chl a, Chl b and carotenoid’s bands because these molecules are bound as pigment-protein complexes in chloroplast. The Soret band of chl a in the complexes was detected at 438 nm while in the MeOH it was found at 432 nm (Figure 2a black line). The fluorescence emission spectra (Figure 3b) indicate a strong emission band of PSII complexes with maximum wavelength at (λmax) about 682 nm and weak emission band of PSI complexes with λmax at about 730 nm. It is shown here that Chl a acts as the main contributor to the excitation band at 434 nm and it shows that excitation at 434 nm (Soret band) produces stronger emission intensity, while the excitation at 475 and 512 nm, correspond to Chl b Soret band and carotenoid, respectively, produces weaker emission intensity. If we monitor the emission at 682 nm and measure the excitation spectrum, it shows that the PSII emission at 682 nm is the result of contribution from Chl a, Chl b and carotenoids (Figure 3a solid black lines) with bands at λmax about 414, 434, 475 nm, respectively.
\n(a) Overlaid of UV–Vis absorption (red) and fluorescence excitation (black) (λem = 682 nm) spectra of chloroplast and (b) emission spectra of chloroplast with excitation at 434 (black), 475 (red) and 512 (blue) nm. Measurements were conducted at ambient temperature. The isolation of chloroplast was carried out as follows: 20 g of suji leaves (Pleomele angustifolia) were washed with running water and cut. The leaves were then homogenized in 200 mL ice-cold isolation buffer (300 mM sorbitol, 50 mM HEPES-KOH pH 7.5, 2 mM EDTA, 80% acetone, 0.1% BSA) for 10 min in a cold environment, followed by filtration using cloth. Centrifugation was conducted in 2 steps, to discard cell debris at 200 g, 4°C, 20 min and to harvest chloroplast pellet at 3000 g, 4°C, 20 min. Final chloroplast pellet was collected and subjected to spectrum UV–VIS (Shimadzu UV-1700)and fluorescence measurement (Jasco FP-8500).
The current high-resolution structural models of antenna complexes have been obtained only for LHCII (2.72 Å) and recently for CP29 (2.8 Å) from PSII of spinach [17, 18]. Here we focus more on the LHCII structure. LHCII shows trimeric structure. Each monomeric contains three transmembrane α-helices, a, b and c (Figure 4a). One monomeric subunit contains eight chlorophyll (Chl) a pigments, six Chl b, two luteins (Lut), neoxanthin and one additional xanthophyll [17, 19]. The 14 chlorophylls are non-covalently attached in the protein cavity. Four carotenoid binding sites per monomer have also been characterized, but in this case the type of carotenoid bound can vary. Typically, two lutein molecules are in groves on both sides of helices a and b and have been likened to a cross-brace. A third carotenoid, 9-cis neoxanthin, is located in the Chl b-rich region near helix c. The fourth carotenoid is located at monomer-monomer interfaces in the trimer. It has been suggested that this site accommodates carotenoids that can participate in the xanthophyll cycle. It depends on the external stress level of the plant; the fourth carotenoid is either violaxanthin (no or low stress) or zeaxanthin (high stress) [20]. In this structure, the carotenoids are in van der Waals contact with the chlorophylls [9]. This is essential as carotenoids in LHCII act as accessory light-harvesting pigments and photoprotectors. The accessory light-harvesting function represents singlet-singlet energy transfer from the carotenoid to the chlorophylls. Since the singlet excited state lifetime of the carotenoid is quite short, approximately 200 fs, the carotenoid must be in close distance to a chlorophyll molecule if the energy transfer is to be efficient. Photoprotection function represents the quenching of triplet excited state of chlorophylls and so preventing the formation of singlet oxygen. This triplet-triplet exchange reaction also requires the carotenoid to be in close contact with the chlorophylls. Regarding CP29, it binds 3 carotenoids and 13 chlorophyll molecules [18]. The position of some chlorophyll binding sites in CP29 differs from LHCII.
\n(a) A view looking down on the top of trimeric complex of LHCII structure from spinach. Each monomer is colored magenta, yellow and pale green. The three-transmembrane helices (a, b and c) present in a monomer are labeled and are easily visible. Chl a molecules are in red, Chl b colored green and carotenoids colored orange. (b) Side-view of LHCII structure shows chlorophyll and carotenoid molecules are packed densely and close to each other (within van der Waals contact), enabling the crucial photo-protective role of these molecules to function by quenching triplet chlorophyll excited states. (c) Structure of PSII from Thermosynechococcus elongatus [28], a side-view representation of the overall dimer perpendicular normal with the pseudo-twofold symmetry axis. (e) PSII core reaction center is shown; component co-factors of the electron transport chain viewed along the membrane plane. The two branches are related by the pseudo-twofold symmetry axis. The respective pairs of pigments on the branches are labeled to indicate whether the Mg2_ is coordinated by D1, D2. (d) Structure of PSI from Synechococcus elongatus [29]; overview of the complete trimer looking along the membrane normal from the stromal side with each polypeptide of the trimer colored differently and chlorophyll molecules given in green. The two main proteins that comprise a monomer are PsaA (yellow) and PsaB (magenta). The electron transport chains are in the center of each monomer. (f) PSI core reaction center component co-factors of the electron transport chain are viewed along the membrane plane. The two branches are related by the pseudo-twofold symmetry axis. The representative pair of chlorophyll molecules on the branches are labeled A or B indicating whether the Mg2+ is coordinated by PsaA or PsaB. The iron–sulfur center of Fx involves residues from both PsaA and PsaB, while FA and FB are located in an extrinsic subunit called PsaC.
The current high-resolution crystal structure of PS II and PSI core complexes is limited to that from cyanobacteria and from pea, respectively [21, 22]. The core of PSII is a multi-subunit complex. Most of the chromophores involve light harvesting as well as electron transfer reaction and are bound to four main subunits, that is, D1, D2, CP43 and CP47. When the core of PSII and PSI reaction center structures is compared, the arrangement of the pigments and other electron transfer co-factors is also very similar (Figure 4c and d). Here, first we look at the PSII core reaction center. The core of reaction center of PSII is made from two major polypeptides called D1 and D2; each contains five membrane-spanning α-helices. These two helices clasp each other like two cupped hands holding on to each other. The redox cofactors are arranged into two arms that lie on either side of the point where the two groups of helices interact. This arrangement of the helices and the cofactors introduces a pseudo two-fold symmetry axes that runs through the center of reaction center normal to the plane of the membrane. In Figure 4e, it is seen that the electron transport pathway in PSII begins with a pair of chlorophyll molecules called P680 (PD1 and PD2). Then each arm contains, in order, one monomeric chlorophyll molecule, one pheophytin (a chlorophyll derivative) and one plastoquinone molecule. Here, only the D1 arm is active in electron transport. Upon excitation P680 becomes oxidized and one electron is injected out and passes down the active branch to the quinone QA. P680 is re-reduced by electron transfer from a special tyrosine residue called Z (Tyrz). A second turnover of P680 delivers a second electron to the plastoquinone and the secondary quinone QB is now reduced to QBH2. The hole on Tyrz is filled by electron transfer from the manganese cluster, the oxygen evolving complex. Every four turnovers of P680 stores four positive charges in the manganese cluster that are then used to oxidize water and evolve oxygen. While in CP43 and CP47, there are a total of 49 Chl a molecules that are bound and that function as internal antenna and allow excitation energy transfer from the peripheral antenna system to the reaction center.
\nUnlike PSII, in PS I, the same single polypeptides contain both antenna complexes (Lhca) and the reaction center core. The 3.3 Å resolution crystal structure of PSI from pea showed that plant PSI binds at least 173 Chl a and b molecules [22]. At this resolution of the crystal structure, it is not possible to identify the Chl species, but biochemical analysis of purified PSI indicated that it has a Chl a/b ratio in a range of 8.2–9.7 [23, 24]. A large number of Chl a and b molecules are bound to the Lhca protein, only about 100 Chl a are bound in the core complex, and the rest of Chl a and b are between these moieties. The latter represent the so-called “linker” chlorophylls which are located between Lhca monomers and “gap” chlorophylls (between Lhca and PSI core). The linker chlorophyll molecules probably play an important role in excitation energy transfer between Lhca antennas and from Lhca to the PSI core [20, 25, 26]. Based on biochemical analysis, PSI was reported to bind approximately 33/34 carotenoids, that is, about 12 carotenoid molecules are bound to Lhca, at the interface between Lhca and the core complex, and about 22 β-carotene are bound to the core [20, 23, 26]. Based on these biochemical analysis, it can be estimated that PSI-LHCII supercomplex contains about 215 chlorophyll and 45/46 carotenoid molecules.
\nThe core complex of PSI is composed of smaller number of subunits (15 subunit) than PSII. The large PsaA and PsaB subunit, which contain 11 trans-membrane helices each, forms a hetero-dimer that binds ~80 Chl a and ~20 β-carotene as cofactors for light harvesting as well as 6 Chl a, 2 phylloquinones and a 4Fe-4S cluster as cofactors for electron transfer reaction, with the exception of terminal electron acceptors (Fe-S clusters FA and FB) which are bound to the PsaC subunit [25]. At closer look (Figure 4f), the redox co-factors in the core reaction center are arranged into two arms that are located on either side of the region where two groups of helices interact with each other. Two chlorophylls form P700 and then each arm contains two monomeric chlorophyll molecules (the second one being in the equivalent position to the pheophytin present in photosystem II) followed by one quinone molecule. When P700 is oxidized, both arms of the electron transport pathway are able to work as it was reported that the electron can pass either down the B-branch or the A-branch [27].
\nChlorophyll and carotenoid can be isolated as free pigments, detached from the pigment-protein complexes, by organic solvent extraction. Important aspects such as the choice of organic solvents, light exposure and working temperature should be considered while isolating pigments. Based on the structure, chlorophyll is characterized with polar macrocycle ring with non-polar hydrocarbon tail. The structural difference between Chl b and Chl a is by having an aldehyde group in place of the methyl group at the macrocycle side group. This change is effecting the polarity of Chl b to be more polar in comparison to Chl a. In the case of carotenoid, structural difference can be seen from the number of conjugated double bonds and the presence of oxygen atoms. Considering these characteristics, mixtures of miscible polar and semi/non-polar solvents are used commonly to extract plant pigments. The mixture of solvent has double functions, that is, penetrating tissues/matrixes and extracting pigments from their lipophilic surrounding. During extraction, exposure of light should be avoided to reduce photodamage of the pigments. Temperature is also important. It is recommended to conduct extraction at lower temperatures, for example, on ice or using liquid nitrogen, to minimize activity of enzyme (e.g. chlorophyllase) that will catalyze breakdown. Antioxidant agent can be also added during extraction to avoid any unwanted oxidation.
\nAfter successful isolation, liquid chromatography has been widely used as an effective technique to separate individual type of pigments and for further purification. In this technique, the pigment separation is based on the polarity which depends on the interaction of pigment with the stationary and mobile phases. Elution method either normal phase or reversed phase is chosen according to the type of pigment to be separated. In addition, the choice of liquid chromatographic methods, namely thin layer chromatography (TLC), column chromatography (CC) and high-pressure liquid chromatography (HPLC), is referred to the speed, resolution and quantity of sample [30]. Currently, ultra-fast liquid chromatography (UFLC), a recent development of HPLC, has been used as a standard for liquid chromatography to achieve high-resolution data with low time consumption [31]. Purification with non-chromatographic method has also been developed, that is, purification method using dioxane has been effective to separate chlorophyll from most of the carotenoids and some lipids [32].
\nVarious types of column absorbents used for chromatographic separation of plant pigments have been well reviewed [30]. Here, we used a silica C30 column attached to UFLC analytic to achieve well separation of carotenoids from Pleomele angustifolia leaf using elution gradient program with mixture of water, methanol and methyl tert-butyl ether to separate, at least, 7 dominant pigments within 25 min. (Figure 5). The detailed identification of pigments, based on the chromatographic, spectrophotometric and mass properties, is summarized in Table 1. Chlorophyll a and chlorophyll b, α- and β-carotenes and violaxanthin are found to be the main chlorophylls and carotenoids, respectively, while the presence of lutein and zeaxanthin in this chloroplast is in low amount.
\nUFLC chromatogram of pigment extract from chloroplast of Pleomele angustifolia detected at 430 nm. The UFLC separation condition was as follows: Pigment separation was performed using UFLC equipped with PDA (Shimadzu) on C30 column (150 × 4.6 mm I.D; YMC) with a gradient elution program of water, methanol and methyl tert-butyl ether (MTBE) at the flow rate of 1 mL/min at 30°C.
Peak No | \ntR [min] | \nλmaxs [nm] | \nMolecular ion | \nFragment ions [m/z] | \nIdentification | \n|||
---|---|---|---|---|---|---|---|---|
HPLC eluent | \nHexane | \nEthanol | \nAcetone | \nspecies [m/z] | \n||||
1 | \n7.3 | \n412,436,464 | \n— | \n— | \n— | \n— | \n— | \nViolaxanthin | \n
2 | \n12.8 | \n470,601,650 | \n451,595,642 | \n465,601,649 | \n458,596,646 | \n907.7 [M]+ | \n881.7 [M – COH]+ 855.7 [M – COH – Mg]+ | \nChlorophyll b | \n
3 | \n13.4 | \n422,445,472 | \n422,444,473 | \n−,446,474 | \n−,448,476 | \n568.4 [M]+ | \n551.4 [M – OH]+ 476.4 [M – 92]+ 430.3 [M – 138]+ | \nLutein | \n
4 | \n15.3 | \n−,451,477 | \n425,449,478 | \n425,451, 478 | \n428,454,481 | \n568.6 [M]+ | \n476.4 [M – 92]+ | \nZeaxanthin | \n
5 | \n16.6 | \n431,618,664 | \n427,613,661 | \n430,616,664 | \n431,617,662 | \n893.5 [M]+ | \n871.5 [M – Mg]+ 615.2 [M – phytyl]+ | \nChlorophyll a | \n
6 | \n20.1 | \n421,446,473 | \n421,445,474 | \n421,446,476 | \n422,445,473 | \n536.6 [M]+ | \n445.4 [M + H – 92]+ | \nα-carotene | \n
7 | \n21.2 | \n–,452,478 | \n–,451,479 | \n–,453,480 | \n–,454,482 | \n536.6 [M]+ | \n444.5 [M – 92]+ | \nβ-carotene | \n
Chromatographic, spectrophotometric and mass properties of pigments separated from the chloroplast of Pleomele angustifolia.
Larger-scale separation of Chl a and b can be achieved by CC using Sepharose CL-6B as the stationary phase and a mixture of 2-propanol (IPA) and hexane as the mobile phase. Chl a could be eluted using 1.5% IPA in hexane and Chl b with 10% IPA in hexane [33]. To achieve a pure, free carotenoid, saponification step is sometimes necessary to eliminate contamination of lipids and chlorophylls. Moreover, carotenoid ester can be hydrolyzed to produce parent carotenoid by using this method [34]. CC is usually used for carotenoid isolation in high quantity of pigment extract. Generally, the purpose of CC is to separate mixtures into carotenoid fractions which are either having high purity to be processed to crystallization or low purity to be extensively separated with further chromatography, that is, HPLC [35].
\nSilica and alumina are frequently used as the absorbent in the CC with the normal phase elution to separate the distinct carotenoids; however, it is not easy to use this method to separate carotenoid isomers, that is, geometrical isomers, diastereoisomers, and so on. In this case HPLC/UFLC can be used to overcome the difficulty in the separation of carotenoids by CC. Turcsi et al. (2016) revealed that the polar carotenoids including optical isomers, and region and geometrical isomers as well as non-polar carotenes, could be well separated by HPLC on C18 and C30 columns, respectively [36]. High purity of isolated pigment can be achieved by HPLC and crystallization processes. UFLC analysis of the purified zeaxanthin shows that this carotenoid had a high purity of around 99.3% (Figure 2, left). All purified pigments have purity higher than 95% (Figure 6).
\nPurification of zeaxanthin: (a) chromatogram detected at 450 nm. Insert figure is UV–Vis spectrum measured by UFLC diode array detector in the eluent and (b) ESI-MS/MS spectrum identification. The conditions of UFLC and ESI-MS/MS analysis were as follows: UFLC analysis of the purified zeaxanthin was performed using UFLC equipped with PDA (Shimadzu) on C30 column (150 × 4.6 mm I.D; YMC) with a gradient elution program of water, methanol and MTBE at the flow rate of 1 mL/min at 30°C. The purified zeaxanthin was directly analyzed to LCMS 8030 (Shimadzu) with an isocratic elution of 0.1% formic acid (FA) in water (10%) and 0.1% FA in methanol (90%) at the flow rate of 0.3 mL/min. MS analysis was operated under the following conditions: (1) heat block temperature = 400°C; (2) desolvation line temperature = 250°C; (3) nebulizing N2 gas flow = 3 L/min; (4) drying N2 gas flow = 15 L/min; (5) interface voltage = 4.5 kV; (6) interface current = 0.1 μA; (7) mass range 400–700 m/z; (8) ionization mode = positive and negative.
Chromatographic, spectrophotometric and mass properties of pigment are minimum requirements for pigment identification [35]. These properties for all purified pigments are shown in the Table 1. In Figure 7 (right), absorption spectra of the purified chlorophyll a and the purified β-carotene in acetone have the same maximum absorption wavelength (λmax) and other spectral properties, such as the fine structure and spectrum shape, compared to these pigments in the references [37, 38]. Absorption spectrum of chlorophyll a in acetone shows typical Soret (431 nm), Qx (617 nm) and Qy (662 nm) bands, while two well-defined peaks in the absorption spectrum of β-carotene are found at 454 and 482 nm. This pigment analysis based on the results of spectrophotometer UV–Vis could support the advance pigment analysis using HPLC/UFLC equipped with photodiode array detection and coupled with the mass spectrometry. The LCMS technique has provided a power tool for pigment identification [39, 40]. Tentative identification for zeaxanthin peak separated by HPLC/UFLC analysis with PDA revealed that zeaxanthin has similar retention time (tR), maximum absorption wavelength (λmax) and the shape of absorption spectrum (data not shown) compared to the isolated zeaxanthin from corn which is a well-known source of zeaxanthin [41]. In addition the mass analysis provides the precursor and fragment ions at the specific m/z and characteristic fragmentation pattern for pigment identification. Mass spectrum of Chl a indicated the molecular ion [M]+ detected at m/z 893.6 and a fragment ion [M-Mg]+ at m/z 871.6 related to the loss of magnesium as the central metal of chlorophyll (Figure 1). This mass spectrum of Chl a agrees with the result that was reported [42].
\nPurification of Chl: (a) chromatogram detected at 660 nm. Insert figure is UV–Vis spectrum measured by UFLC diode array detector in the eluent and (b) ESI-MS/MS spectrum. The condition of UFLC and ESI-MS/MS analysis was as follows: UFLC analysis of the purified chlorophyll a was performed using HPLC equipped with PDA (Shimadzu) on C30 column (150 × 4.6 mm I.D; YMC) with a gradient elution program of water, methanol and MTBE at the flow rate of 1 mL/min at 30°C. The purified chlorophyll a was directly analyzed to LCMS 8030 (Shimadzu) with an isocratic elution of 0.1% formic acid (FA) in water (10%) and 0.1% FA in methanol (90%) at the flow rate of 0.3 mL/min. MS analysis was operated under the following conditions: (1) heat block temperature = 400°C; (2) desolvation line temperature = 250°C; (3) nebulizing N2 gas flow = 3 L/min; (4) drying N2 gas flow = 15 L/min; (5) interface voltage = 4.5 kV; (6) interface current = 0.1 μA; (7) mass range 400–1000 m/z; (8) ionization mode = positive and negative.
Chlorophyll and carotenoid are chloroplast pigments which are bound non-covalently to protein as pigment-protein complex and play a vital role in photosynthesis. Their functions include light harvesting, energy transfer, photochemical redox reaction, as well as photoprotection. The exact number and stoichiometry of these pigments in higher plants are varied, but their compositions include Chl a, Chl b, lutein, neoxanthin, violaxanthin, zeaxanthin and β-carotene. Liquid chromatography methods are well developed to separate and purify different types of pigments. Identification and characterization of pigments can be well observed by spectroscopy methods such as UV–Vis absorption, fluorescence and mass spectrometry.
\nTatas Hardo Panintingjati Brotosudarmo (THPB) acknowledges the competence research grant (No. 120/SP2H/LT/DRPM/IV/2017) from Kemenristekdikti for the financial support. We also acknowledge Chandra Ayu Siswanti who helped in preparation of chloroplast isolation, pigment isolation and UFLC separation works. We acknowledge Dr. Hendrik Octendy Lintang for supporting fluorescence measurements of photosystem II and I in chloroplast.
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\\n\\n7.4 Waiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\\n\\n7.5 Variation: No variation of this Publication Agreement shall be effective unless it is in writing and signed by the parties (or their duly authorized representatives).
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\\n\\n7.7 No partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Corresponding Author or any Co-Author, nor authorize any party to make or enter into any commitments for or on behalf of any other party.
\\n\\n7.8 Governing law: This Publication Agreement and any dispute or claim (including non-contractual disputes or claims) arising out of or in connection with it or its subject matter or formation shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of or in connection with this Publication Agreement (including any non-contractual disputes or claims).
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The Corresponding Author (acting on behalf of all Authors) and INTECHOPEN LIMITED, incorporated and registered in England and Wales with company number 11086078 and a registered office at 5 Princes Gate Court, London, United Kingdom, SW7 2QJ conclude the following Agreement regarding the publication of a Book Chapter:
\n\n1. DEFINITIONS
\n\nCorresponding Author: The Author of the Chapter who serves as a Signatory to this Agreement. The Corresponding Author acts on behalf of any other Co-Author.
\n\nCo-Author: All other Authors of the Chapter besides the Corresponding Author.
\n\nIntechOpen: IntechOpen Ltd., the Publisher of the Book.
\n\nBook: The publication as a collection of chapters compiled by IntechOpen including the Chapter. Chapter: The original literary work created by Corresponding Author and any Co-Author that is the subject of this Agreement.
\n\n2. CORRESPONDING AUTHOR'S GRANT OF RIGHTS
\n\n2.1 Subject to the following Article, the Corresponding Author grants and shall ensure that each Co-Author grants, to IntechOpen, during the full term of copyright and any extensions or renewals of that term the following:
\n\nThe aforementioned licenses shall survive the expiry or termination of this Agreement for any reason.
\n\n2.2 The Corresponding Author (on their own behalf and on behalf of any Co-Author) reserves the following rights to the Chapter but agrees not to exercise them in such a way as to adversely affect IntechOpen's ability to utilize the full benefit of this Publication Agreement: (i) reprographic rights worldwide, other than those which subsist in the typographical arrangement of the Chapter as published by IntechOpen; and (ii) public lending rights arising under the Public Lending Right Act 1979, as amended from time to time, and any similar rights arising in any part of the world.
\n\nThe Corresponding Author confirms that they (and any Co-Author) are and will remain a member of any applicable licensing and collecting society and any successor to that body responsible for administering royalties for the reprographic reproduction of copyright works.
\n\nSubject to the license granted above, copyright in the Chapter and all versions of it created during IntechOpen's editing process (including the published version) is retained by the Corresponding Author and any Co-Author.
\n\nSubject to the license granted above, the Corresponding Author and any Co-Author retains patent, trademark and other intellectual property rights to the Chapter.
\n\n2.3 All rights granted to IntechOpen in this Article are assignable, sublicensable or otherwise transferrable to third parties without the Corresponding Author's or any Co-Author’s specific approval.
\n\n2.4 The Corresponding Author (on their own behalf and on behalf of each Co-Author) will not assert any rights under the Copyright, Designs and Patents Act 1988 to object to derogatory treatment of the Chapter as a consequence of IntechOpen's changes to the Chapter arising from translation of it, corrections and edits for house style, removal of problematic material and other reasonable edits.
\n\n3. CORRESPONDING AUTHOR'S DUTIES
\n\n3.1 When distributing or re-publishing the Chapter, the Corresponding Author agrees to credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen. The Corresponding Author warrants that each Co-Author will also credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen, when they are distributing or re-publishing the Chapter.
\n\n3.2 When submitting the Chapter, the Corresponding Author agrees to:
\n\nThe Corresponding Author will be held responsible for the payment of the Open Access Publishing Fees.
\n\nAll payments shall be due 30 days from the date of the issued invoice. The Corresponding Author or the payer on the Corresponding Author's and Co-Authors' behalf will bear all banking and similar charges incurred.
\n\n3.3 The Corresponding Author shall obtain in writing all consents necessary for the reproduction of any material in which a third-party right exists, including quotations, photographs and illustrations, in all editions of the Chapter worldwide for the full term of the above licenses, and shall provide to IntechOpen upon request the original copies of such consents for inspection (at IntechOpen's option) or photocopies of such consents.
\n\nThe Corresponding Author shall obtain written informed consent for publication from people who might recognize themselves or be identified by others (e.g. from case reports or photographs).
\n\n3.4 The Corresponding Author and any Co-Author shall respect confidentiality rights during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Corresponding Author and any Co-Author are confidential and are intended only for the recipient. The contents may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\n\n4. CORRESPONDING AUTHOR'S WARRANTY
\n\n4.1 The Corresponding Author represents and warrants that the Chapter does not and will not breach any applicable law or the rights of any third party and, specifically, that the Chapter contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy. The Corresponding Author warrants and represents that: (i) the Chapter is the original work of themselves and any Co-Author and is not copied wholly or substantially from any other work or material or any other source; (ii) the Chapter has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) they themselves and any Co-Author are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) they themselves and any Co-Author have not assigned and will not during the term of this Publication Agreement purport to assign any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\n\nThe Corresponding Author also warrants and represents that: (i) they have the full power to enter into this Publication Agreement on their own behalf and on behalf of each Co-Author; and (ii) they have the necessary rights and/or title in and to the Chapter to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licenses expressed to be granted in this Publication Agreement. If the Chapter was prepared jointly by the Corresponding Author and any Co-Author, the Corresponding Author warrants and represents that: (i) each Co-Author agrees to the submission, license and publication of the Chapter on the terms of this Publication Agreement; and (ii) they have the authority to enter into this Publication Agreement on behalf of and bind each Co-Author. The Corresponding Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each such Co-Author.
\n\nThe Corresponding Author agrees to indemnify and hold IntechOpen harmless against all liabilities, costs, expenses, damages and losses and all reasonable legal costs and expenses suffered or incurred by IntechOpen arising out of or in connection with any breach of the aforementioned representations and warranties. This indemnity shall not cover IntechOpen to the extent that a claim under it results from IntechOpen's negligence or willful misconduct.
\n\n4.2 Nothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\n\n5. TERMINATION
\n\n5.1 IntechOpen has a right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Corresponding Author or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Corresponding Author or any Co-Author (being an individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Corresponding Author or any Co-Author (being a company) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for or enters into any compromise or arrangement with any of its creditors.
\n\nIn case of termination, IntechOpen will notify the Corresponding Author, in writing, of the decision.
\n\n6. INTECHOPEN’S DUTIES AND RIGHTS
\n\n6.1 Unless prevented from doing so by events outside its reasonable control, IntechOpen, in its discretion, agrees to publish the Chapter attributing it to the Corresponding Author and any Co-Author.
\n\n6.2 IntechOpen has the right to use the Corresponding Author’s and any Co-Author’s names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Chapter and has the right to contact the Corresponding Author and any Co-Author until the Chapter is publicly available on any platform owned and/or operated by IntechOpen.
\n\n6.3 IntechOpen is granted the authority to enforce the rights from this Publication Agreement, on behalf of the Corresponding Author and any Co-Author, against third parties (for example in cases of plagiarism or copyright infringements). In respect of any such infringement or suspected infringement of the copyright in the Chapter, IntechOpen shall have absolute discretion in addressing any such infringement which is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\n\n7. MISCELLANEOUS
\n\n7.1 Further Assurance: The Corresponding Author shall and will ensure that any relevant third party (including any Co-Author) shall, execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\n\n7.2 Third Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\n\n7.3 Entire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces and extinguishes all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by or on behalf of the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (together "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of its pre-contract fraudulent misrepresentation or fraudulent concealment.
\n\n7.4 Waiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\n\n7.5 Variation: No variation of this Publication Agreement shall be effective unless it is in writing and signed by the parties (or their duly authorized representatives).
\n\n7.6 Severance: If any provision or part-provision of this Publication Agreement is or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted.
\n\nAny modification to or deletion of a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\n\n7.7 No partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Corresponding Author or any Co-Author, nor authorize any party to make or enter into any commitments for or on behalf of any other party.
\n\n7.8 Governing law: This Publication Agreement and any dispute or claim (including non-contractual disputes or claims) arising out of or in connection with it or its subject matter or formation shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of or in connection with this Publication Agreement (including any non-contractual disputes or claims).
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