Methods of extraction of pectin from various agrowaste compounds.
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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:"66671",title:"Extraction and Purification of Pectin from Agro-Industrial Wastes",doi:"10.5772/intechopen.85585",slug:"extraction-and-purification-of-pectin-from-agro-industrial-wastes",body:'\nPectins are complex branched polysaccharides present in the primary cell wall of plants [1]. It is a highly valued food ingredient commonly used as a gelling agent and stabilizer [2]. It is usually extracted by chemical or enzymatic methods from fruits [3]. Pectin is considered as the most complex macromolecule in nature, since it can be composed of up to 17 different monosaccharides containing more than 20 different linkages [4].
\nPectins are enriched with repeated units of methyl ester galacturonic acid [4]. They form chemically stable and physically strong skeletal tissues of plants when combined with proteins and other polysaccharides [5]. They are usually produced in the initial stages of primary cell wall growth and make one third of the cell wall in both monocots and dicots [6]. Pectin is significantly reduced or absent in non-extendable secondary cell walls and is the only major class of plant polysaccharide largely limited to primary cell walls [7]. Pectin imparts strength and flexibility to the cell wall, apart from number of fundamental biological functions such as signalling, cell proliferation, differentiation, cell adhesion and maintaining turgor pressure of cell [8]. Pectins are involved in regulating mobility of water and plant fluids through the rapidly growing parts [6]. It also influences the texture of fruit and vegetables [9]. Apple pomace and orange peel are the two major sources of commercial pectin due to the poor gelling behaviour of pectin from other sources [6].
\nPectin is one of the most important polysaccharides due to its increasing demand in the global market, reaching a total production capacity of around 45–50 Million tonnes per annum. While the demand in 2011 was approximately 140–160 Million tonnes per annum, earning the interest of industry in this complex polysaccharide processing [10]. Pectins have received considerable attention as a high fibre diet that benefits health by reducing cholesterol and, serum glucose levels and acting as anticancer agents [11]. Pectins have shown promising results as drug carriers for oral drug delivery and are widely used for various bio-medical applications [5]. In addition, pectin has been described as an emerging prebiotic with the ability to modulate colon microbiota [12]. Considering above properties and applications, pectin has gained immense priority in the global biopolymer market with great potential and opportunities for future developments.
\nOne of the most abundant macromolecules present in the primary cell wall of the plants is pectin; their presence is detected in the matrix as well as in the middle lamellae [7]. Pectin is highly rich with galacturonic acid (GalA), that forms the backbone of three more domains found along with pectin that are homogalacturonan (HGA), rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) [13]. About 70% of pectin is mainly composed of galacturonic acid (GA) [14]. Pectin is made of three polysaccharides that are covalently linked together, thus forming pectin networks in the cell wall matrix and the middle lamellae [15, 16].
\nHomogalacturonan (HG) takes up about 60–65% of the total pectin [3, 17], with a backbone of alpha-1,4-linked GalA residues, these GalA residues are methyl esterified which has an important role in the physical properties of pectin [4]. The presence of HG is seen to be present in approximately 100 GalA residues, but there are cases when its detected interspersed within other pectin polysaccharide [14]. On the other hand rhamnogalacturonan-I (RG-I) backbone which contributes 20–35% of pectin is composed of repeated and alternating groups of
Rhamnogalacturonan-II (RG-II) is one of the highly conserved and complex structure which consist of distinct regions within HG, which makes up about 10% of the pectin [3], they have side chains of four different types with a particular sugar residue like aceric acid, apiose-3-deoxy-lyxo-2-heptulosaric acid, and 3-deoxy-manno-2-octulosonic acid. The HG residues along with nine of the GalA residues are attached to these side chains [3, 5]. There are other substituted HG residues that make up pectin such as xylogalacturonan and apiogalacturonan whose expression is restriction. Even a minor mutation in R-II structure can lead to defects in the plant growth like dwarfism, thus suggesting its importance for normal growth of plant [3]. RG-I being highly branched in nature thus, called as the hairy region of pectin on the other hand HGA domain are known as the smooth region [7]. It is generally believed and noticed that there is covalent linkage within the pectin polysaccharides and pectin degrading enzymes are needed to separate and isolate HG, RG-I and RG-II from each other [21, 22]. Due to their similarity in HG and RG-II backbone structure composing of 1-4-linked alpha-D-GalA residues, they are likely to be linked covalently but there are no reports of RG-I to be covalently linked with HG [23].
\nPectin precipitates as a solid gel on treating with a dehydrating agent like alcohol. They are extremely sensitive to dehydration and get effected by any other hydrophilic colloids as well, thus they are known to be insoluble in most of the bio-colloids. The negative charge of pectin depends on the number of free carboxyl group that is mainly responsible for its precipitation [24].
\nBased on solubility pectins are of two types i.e., water soluble and water insoluble. Factors affecting the solubility of pectin are pH, temperature, nature of the solute and concentration of the solute [6, 13]. Pectin attains stability at a pH of 4 [17]. The solubility of pectin also depends on its composition like monovalent cation of pectin are soluble in water whereas di or trivalent are insoluble in water.
\nOne of the most interesting properties of pectin is its ability to form gel in the presence of either acid or calcium or sugar, this enables them to be used in many food industries [15]. Hydrogen bonding and hydrophobic interactions between polymer chains stabilizes the pectin polymer [9].
\nPectin is a high molecular weight polysaccharide that is present in almost all plants and help in maintaining the integrity of cell structure. Pectin is used in food industries to increase the viscosity of food products such as beverages, jams and jellies. It also has implications in pharmaceutical industry, especially in drug formulations, as an excipient due to its characteristics in release kinetics. Due to increased demand for pectin in food, pharmaceutical and therapeutic applications, thus, require efficient extraction processes. In order to increase the yield of pectin, various extraction methods have been adapted to obtain insoluble pectin present in the middle lamellae of plant cells, one of them being heating in acidic medium that makes insoluble pectin as soluble. Ripening of fruits also converts insoluble pectin into soluble pectin. Pectin can be extracted from various kinds of fruits, but the most commercial form of pectin is extracted from the peels of citrus fruits by alcohol precipitation [9, 25]. Citrus fruits contain 0.5–3.5% pectin which is largely present in the peel of fruits [26].
\nPectin has been isolated from various plants such as apple [27], citrus peel, carrots [28], sugar beet pulp [29, 30], sunflower heads [31], papaya [32] and oranges [33]. The most commonly used method for extracting pectin from plant tissue is by heating the plant sample in acidified water. The addition of extra chelating agents such as EDTA to the extraction mixture helps in easy release of pectin from cell wall. Care should be taken not to perform a long period of direct heating as it may lead to the thermal degradation of the polymer. Extraction process of pectin is carried out under reflux using acidified water at 97°C for 30 min. The hot acid extract was then filtered using a cheese cloth to remove the pulp. The filtrate was then cooled to 4°C and precipitated using double the volume of ethanol. The solvent precipitate mixture is then mixed till the pectin floats and removed by using cheese cloth followed by drying [27].
\nPectin is also extracted from dried sugar beet pulp after treating with acidified medium followed by purification through alcohol precipitation. Xin Huang et al., slightly modified the traditional method, where the sample was diluted with deionized water and was made acidic (pH −1.2) by using HCl. The sample was then heated to 90°C for 3 h and cooled to 40°C (pH −4.5) with 25 g/100 g ammonia. The mixture was then filtered using a Buchner funnel and pectin was precipitated using ethanol [29]. The ethanol is removed by squeezing with nylon cloth and washed several times followed by drying.
\nThe carrot pomace is also used for pectin extraction by treating with hot aqueous citric acid solution adjusted to the desired pH. The pectin yield was maximum at the following optimum conditions: pH −1.3; temperature 90°C; heating time 79.8 min. Under these conditions, the extraction yield of carrot pomace pectin was found to be 16.0%. The extract mixture was then allowed to cool, filtered and precipitated by using ethanol in the ratio 2:1 [28]. Dried papaya peel can be used in pectin extraction where the majority of the lipids, and soluble pigments are removed by treating with ethanol and acetone. It is repeatedly homogenised with 95% ethanol and filtered until the filtrate becomes clear. The final filtration was done with the residue homogenised in acetone and dried overnight to obtain the alcohol insoluble residue (AIR). The majority of the pectin in the papaya AIR is present as chelator soluble pectin (CSP) followed by sodium carbonate soluble pectin (SSP) and water-soluble pectin (WSP). The WSP fraction is first obtained from the AIR by boiling it in water and filtering it. The remaining residue is treated with 0.05 M cyclohexane trans-1,2-diamine tetra-acetic acid (CDTA) in 0.1 M potassium acetate (pH 6.5) for 6 h at 28°C and filtered to give the CSP fraction. The residue is then treated with 0.05 M sodium carbonate solution having 0.02 M NaBH4 for 16 h at 4°C, and subsequently for 6 h at 28°C. The solution when filtered gives the SSP fraction of the AIR [32].
\nAgro-industrial wastes can be used as the raw material for the production of industrial low and high methoxy pectin. The alcohol insoluble material (AIM) produced from dried agrowaste by boiling it with 3 volumes of ethanol for 25 min and continuous washing with 70% ethanol to remove impurities such as pigments, free sugars, etc. Sunflower heads also act as potential sources for pectin extraction. The heads are washed by hot distilled water through a mesh or cheese cloth and the pectin was precipitated by addition of 1 M nitric acid at 1:5 acid:filtrate ratio [34]. The mixture was maintained for 1 h at 5°C and was washed six times in ethanol solvent at 1:2 gel:solvent ratio to remove the impurities and to increase pH by removing the acid [31]. The washed pectin gel can be dried in a vacuum oven at 55°C for 16 h. The dried pectin flakes are ground into a powder for further use (Table 1).
\nMaterial | \nExtraction process | \nPectin (%) | \nReferences | \n
---|---|---|---|
Cacao pod husk (Theobroma cacao) | \nAcid extraction | \n3.7–8.6 | \n[34] | \n
Mangosteen rind (Garcinia mangostana) | \nChemical treatment | \n23.5 | \n[35] | \n
Durian rind (Durio zibethinus) | \nAcid extraction | \n2.1–10.25 | \n[36] | \n
Orange peels (Citrus sinensis) | \nAcid extraction | \n0.2–6 | \n[37] | \n
Lemon peels (Citrus limon) | \nAcid extraction | \n0.8–8 | \n[37] | \n
Dragon fruit peels (Hylocereus undatus) | \nUltrasound assisted | \n1.89–7.65 | \n[38] | \n
Banana-stem, leaf, peel (Musa acuminata) | \nAlcohol precipitation | \n4–13.8 | \n[39] | \n
Orange peel | \nAlcohol precipitation | \n7.9 | \n[39] | \n
Cucumis melo | \nAqueous acid extraction alcohol precipitation | \n4.53 | \n[40] | \n
Cocoa peel | \nMicrowave assisted | \n42.3 | \n[41] | \n
Apple Pomace | \nAcid extraction | \n12.9–20.9 | \n[27] | \n
Lime-peel and pulp | \nMicrowave assisted extraction under pressure | \n8–17.9 | \n[42] | \n
Watermelon rind | \nAcid and enzymatic extraction | \n\n | [43] | \n
Orange peels | \nAcid extraction | \n5.4–26.3 | \n[44] | \n
Sweet potato peels | \nAcid extraction | \n2.59–5.08 | \n[45] | \n
Orange peel | \nUltrasound assisted | \n20.92 | \n[46] | \n
Orange peels (Citrus sinensis) | \nAcid extraction | \n29.41 | \n[47] | \n
Kaffir lime peel (Citrus hystrix) | \nChemical and acid extraction | \n61.8 | \n[48] | \n
Punica granatum peels | \nAcid extraction | \n27 | \n[49] | \n
Orange peel (Citrus sinensis) | \nAcid extraction | \n45.5 | \n[50] | \n
Lemon (Citrus limon) | \nAcid extraction | \n2.7–16.7 | \n[51] | \n
Orange (Citrus sinensis) | \nAcid extraction | \n1.6–15.9 | \n[51] | \n
Grape (Citrus paradisi) | \nAcid extraction | \n2.3–15.7 | \n[51] | \n
Orange peel | \nWater-based extraction | \n2.2 | \n[52] | \n
Sweet potato peel (Ipomoea batatas) | \nAlkaline extraction | \n16.78 | \n[45] | \n
Tomato waste | \nUltrasound assisted | \n15.1–35.7 | \n[53] | \n
Pumpkin peels | \nSoxhlet extraction | \n6.8–7.7 | \n[54] | \n
Lemon pomace | \nAcid extraction | \n10.3–13.1 | \n[55] | \n
Jackfruit waste (Artocarpus heterophyllus Lam) | \nAcid and chemical extraction | \n12–15 | \n[27] | \n
Lemon peel wastes | \nAqueous extraction | \n\n | [56] | \n
Citric waste | \nAcid extraction | \n78 | \n[57] | \n
Apple peel waste (Malus pumila. Cv Amri) | \nAcid and chemical extraction | \n1.21 | \n[58] | \n
Horse eye bean peel (Mucuna urens) | \nAcid extraction | \n4.4 | \n[59] | \n
Banana peel | \nAcid extraction | \n11.31 | \n[60] | \n
Mango peel | \nAcid extraction | \n18.5 | \n[60] | \n
Grapefruit peel | \nAlcohol extraction | \n25 | \n[33] | \n
Saba banana peel (Musa acuminata × Musa balbisiana) | \nAcid extraction | \n17.05 | \n[61] | \n
Passion fruit peels | \nAcid extraction | \n2.25–14.6 | \n[62] | \n
Citrus peel | \nAcid extraction | \n25.5 | \n[63] | \n
Pumpkin waste (Cucurbita maxima) | \nAcid hydrolysis | \n2.90 | \n[39] | \n
Mango peel | \nAcid extraction | \n20.8 | \n[64] | \n
Jackfruit wastes (Artocarpus integer) | \nOptimised acid extraction | \n38.42 | \n[65] | \n
Citrus depressa endocarp | \nAcid extraction | \n4.1 | \n[66] | \n
Orange peel | \nAcid extraction | \n7.3–52.9 | \n[67] | \n
Jackfruit waste (Artocarpus heterophyllus) | \nChemical and acid extraction | \n8.9–15.1 | \n[27] | \n
Methods of extraction of pectin from various agrowaste compounds.
Large amounts of fruit wastes that are being generated can be disposed effectively by manufacturing beneficial by-products like pectin. Pectin is used to increase foaming power of gases, as agglutinator, as filler in pharmaceutical preparations and also in food industry. The use of pectin for different purposes depends on its characters like acetyl value, degree of esterification, uronic acid content and methoxyl content, etc. [68]. The amount of anhydrouronic acid gives the purity of pectin which is not less than 65% for pectin that is used commercially [69].
\nColour reader can be used to measure the colour of extracted pectin by placing lens of reader on sample powder. The colour of the extracted samples can be compared with that of commercial pectin. Solubility of pectin in different solvents is measured i.e., solubility in cold and, hot water and alkali like NaOH.
\nAcetyl content and equivalent weight of pectin can be estimated using NaOH whereas methoxyl content can be estimated by saponification and titration. Degree of esterification can be calculated from methoxyl content and anhydrouronic acid content. After acid hydrolysis, sugar separation can be achieved by thin layer chromatography. Intrinsic viscosity of pectin is measured by dissolving pectin in water and by preparation of solutions of various concentrations [27, 32, 60].
\nPectin being a great inert, biodegradable and biocompatible complex, is widely used in various fields such as in textiles, food industries as gelling agents, pharmaceuticals and other products as well [70]. Pectin are used as biomaterials in gene delivery [71], application in oral drug delivery [72], as edible coating for food packaging [25], biomass yield and bio refinery [21, 22]. It also has applications in tissue engineering as scaffolds [73], in paper and textile industries for the preparation of ultracentrifugation membrane [74].
\nFrom the very early period of time, pectin had become one of the major natural constituents of human food, and they have been widely used as a gelling agent for jams and jellies. In jam processing, fruits are cooked properly in order to release juice and pectin which converts the proto-pectin into soluble pectin [19]. Pectins are also used as a substitute of sugar in jams that are made without sugar, using LM (low methoxy) pectin due to its stability in acidic condition. Pectin is widely used for making instant jellies for bakery production these are made with the use of HM (high methoxy) pectin that are thermally stable, the only difference between HM and LM pectin is the amount of pectin in the formula, LM requires a higher amount than that of HM [75]. Other food products like artificial cherries [15], are used to make different kinds of gel puddings that is made of pectin present in the fruit syrup and cold milk [25]. Edible coating of food material is also made of pectin [25]. Pectin is used in beverages as a beverage clouding agent like in diabetic soft drinks [76]. Pectins are also used in the fruit preparation of yogurt to make it more soft and to obtain the partial gel texture [77].
\nThe blending of the natural and synthetic polymers is one of the promising areas of development, this gives new polymeric material with better durability and resistance. Materials like sponge, hydrogels, encapsulating drugs etc. are produced by polymer films [32]. Due to development and discovery of natural polymers scientist have started to form bio-based material rather than synthetic one due to its physiochemical properties like biodegradability, this shift is mainly caused due to the environmental issues and concern regarding the heavy use of plastic [78]. Films of pectin are used to encapsulate and thicken food, and in pharmaceuticals [29]. Hoagland et al. made pectin films with glycerol and lactic acid to prevent fungal contamination on the laminated films [47]. The similar kind of products were made by Fisherman et al., where an edible pectin blend film were plasticized with glycerol, they also suggested that the glass transition at about 50°C was large in case of pectin films, which indicates that the films were fairly flexible at room temperature [79]. Liu et al., made different varieties of biofilms one each with pectin, fish skin gelatin and soybean flour protein which in turn resulted in a composite film that showed an increase in stiffness as well as the strength, whereas decrease in water solubility and water vapour transmission rate when compared to the film that was made with pectin alone. They thus suggested that the tensile strength can be improved by crosslinking the films with methanol or glutaraldehyde [80]. A bio-reactive substance for tissue regeneration was developed by Liu et al., which was composed of pectin or PGLA matrix, which demonstrated that pectin was able to carry signals to molecules, further they suggested that the pectin also helped in the adhesion of the cell and promotes cell proliferation [35]. Some researches have reported the use of pectin membranes as a wound dressing material [81].
\nIn recent years, biomedical application especially in case of drug delivery system, the use of natural polymers is preferred over the other types due to their inert nature and its biocompatibility. Pectin as the natural polymer is a new developed interest for drug delivery application due to its properties of gel formation in acidic condition, its mucoadhesiveness and its ability to dissolve in basic environment [36]. These properties of pectin are applied in different ways such as the mucoadhesiveness helps in targeting and controlling the drug delivery especially in the nasal and gastric environment, where as its ability to dissolve in basic condition helps in the release of colon related drugs and the formation of gel helps in increasing the contact time of drug in gastric condition [35, 36]. Recent studies have shown the use of LM pectin for nasal drug delivery due to its mucoadhesive property they have a tendency to bind to the mucin with the help of hydrogen bond [82]. Its use in the production of fentanyl (painkiller) has also been seen that help in treating cancer pain which needs rapid drug release [83, 84]. An alternative for smoking cessation are the nasal pectin containing nicotine [85]. As pectin have resistance towards proteases and amylases it has been highly preferred as an encapsulating nanoparticle for drug delivery as most of the proteins are easily degraded by our digestive enzymes and thus to protect these drugs the use of pectin as an outer cover that cannot be degraded in the gastrointestinal tract are preferred for colon and oral drugs [86]. Studies have shown that pectin is able to inhibit cancer metastasis and primary tumour growth in many animal related cancer [87, 88]. Gal-3 is one of the important factors controlling cancer progression and metastasis, and pectin has the ability to recognise these Gal3 components [89]. In a study, citrus pectin was used to target Gal3 that could inhibit the metastatic successfully [87, 90].
\nThe treatment of any genetic disorder is called gene therapy as it deals with the defected genes that are responsible for the disorder; these are treated by replacing the defective gene, silencing the unwanted gene expression or by substituting missing genes and these are carried out with the help of viral or non-viral vectors [36]. The use of non-viral vectors is preferred over viral due many reasons like biocompatibility, minimal toxicity and immunogenic reactions of our body [71]. These non-viral vectors are made of polymers of polycationic, chitosan or even pectin. It has been observed that the use of carbohydrate mediated products have better binding capacity, to facilitate the uptake by target cell [91, 92, 93]. Pectins were found to be suitable as a coating substance for b-PEI [94, 95]. Opanasopit et al. has also observed the formation of pectin nanoparticle which in turn helps to entrap the DNA for transfection [96]. Katav et al., modified pectin with the help of three different amine groups and these complexes were able to bind with plasmid DNA and there efficiency to transfect or their potential as a non-viral gene delivery carrier was compared and suggested that modified pectin has a promising role in gene delivery [71]. Similar type of study was conducted by Opanaopit et al., where pectin ability as a nanoparticle for gene delivery were studied and the study suggested the potential use of pectin as delivery vector to be safe [96]. Pectin has also been used as wound dressing material in the form of pectin-chitosan based nanoparticles. It has the ability to create an acidic environment in which the bacteria cannot grow. Burapapadh et al. developed a pectin based nanoparticle to improve and enhance the drug dissolution of ITZ (Itraconazole) [97].
\nScaffolds are 3-D biomaterials that are porous in nature and are designed to be applied in various fields, few of its basic functions are to promote cell adhesion, to allow enough nutrients and gases transportation and mainly for tissue engineering [32]. Tissue engineering mainly involves the use of biocompatible scaffolds materials to act as a support matrix or to be used as a substrate for delivery of some compounds. There has been a great research going on to promote tissue reconstructions. Coimbra et al., prepared pectin based scaffolds to be used for bone tissue engineering [98]. Similar study was performed by Munarin et al., who examined the use of pectin as injectable biomaterial for bone tissue engineering [99]. Ninan et al. were also able to fabricate biopolymer scaffold of pectin and other compounds using the technique of lyophilisation, thus suggested the use of pectin as ideal polymeric matrix for tissue engineering [73, 100, 101].
\nPectin is one of the most extensively studied natural biodegradable polymer. In spite of its availability in a large number of plant species, commercial sources of pectin are very limited. There is, therefore, a need to explore other sources of pectin or modify the existing sources to obtain pectin of desired quality attributes. Current knowledge of the molecular basis of pectin has helped us to understand some aspects of this complex polysaccharide. Extensive studies must be carried out to find out more about the biological pathways to devise various efficient means of pectin extraction that are scalable and can be commercialized. The large variety of applications as well as the increasing number of studies on pectin suggests that the potential of pectin as novel and versatile biomaterial will be even more significant in the future. As the research and development continues in pectin-based products, we expect to see many innovative and exciting applications.
\nThe authors would like to thank Fr. Jobi, Head of the Department of Life Sciences, for providing laboratory facilities and supporting this work.
\nThe authors would like to declare that there was no conflict of interest in this work.
\nMass Spectrometry Imaging (MSI) is an incredibly powerful label-free technique that can determine qualitative and quantitative information of hundreds of compounds in a tissue section in one experiment [1, 2]. Small molecule detection, especially of neurotransmitters (NTs), currently relies heavily on histochemical, immunohistochemical, and ligand-based assays. Antibody-based methods suffer from limitations in cost and availability of antibodies, lack of specificity for target molecules of interest, and low throughput [3, 4]. The development of MSI has overcome many of these challenges and will be discussed throughout. The basic methodology of MSI is to section tissue using a cryostat to approximately 10-20 μm thickness; tissues may or may not be embedded in a cryomatrix such as Shandon™ M-1 (ThermoFisher Scientific). Next, matrix must be deposited on the tissue section, which is most often done by spray-coating the tissue using a pneumatic sprayer. Variations on typical organic matrices, such as inorganic nanoparticles (NPs), have been explored by numerous researchers and will be commented on here. Mass spectral data is collected at discrete locations on the sample surface via a raster pattern, which can then be assembled into a heat-map image of molecule location. Figure 1 depicts the typical MSI scheme [5]. A number of overall reviews of MSI have appeared in recent years [6] that address broad topics like ionization of small molecules [7], clinical applications [2], and high-resolution analyses [5].
General scheme of the mass spectrometry imaging process. (a) The tissue section is covered with matrix and irradiated by a pulsed laser beam. (b) Mass spectrum acquired from one spatial location on the tissue section. (c) MS images of different m/z peaks compiled from all spatial locations. Reprinted with permission from Ref. [5]. Copyright 2013 Springer.
The broader scientific community is not yet fully utilizing MSI as there are still challenges to be overcome [7, 8], including: (i) low ionization efficiency for small molecules, (ii) chemical noise interferents/overlapping signals of small molecules with traditional matrices [9], (iii) reproducibility issues across laboratories which limits universal procedures for MSI in pre-/clinical research, (iv) limits to lateral spatial resolution inherent to the matrix crystallization process which affects the ability to clearly define tissue margins, and (v) delocalization of analyte molecules during sample preparation. This chapter will focus on the analysis of small molecules, specifically neurotransmitters (NTs), due to the complex biological processes that occur in the brain and have broad implications in disease states and overall health. This chapter is broken down in two main categories, as strategies to improve ionization must either focus on (i) the chemical nature of the analyte and changing its properties to better facilitate ionization, or (ii) on utilizing a different mechanism of ionization to favor small molecules of interest.
Small molecule NTs are the chemical messengers of the central nervous system. Having a complete picture of NT location and abundance will aid in understanding of many different disease states and developmental processes. NTs are difficult to detect in situ via mass spectrometry due to their low physiological abundance (e.g., nM to pM concentration) within a complex biological tissue with many different classes of biomolecules, and overlapping low molecular mass range with most traditional matrices. Prior to the analyses discussed here, NTs were localized based on their protein-receptors or transporters, which does not always give an accurate accounting of present location.
Instrumentation used for MSI can vary widely, but most laser-based work is performed by time-of-flight (TOF) instruments. The other common setup is using desorption electrospray ionization (DESI) as an ion source, which is not the focus here, but is another option gaining in popularity [10, 11]. Other hardware configurations can favor small molecules (e.g., ion mobility, triple quadrupole instruments, Fourier transform – ion cyclotron resonance) and so there is no one-size-fits-all set-up for small molecule MSI experiments. In contrast to instrument choice, sample preparation/derivatization and ionization conditions are areas that can be standardized in order to achieve similar results across different platforms. The focus here is not on the many instrument combinations as other reviews have adequately explored this topic [6, 7].
Sample handling and preparation of tissue sections are integral to maintaining sample integrity; after cryo-sectioning, tissue is typically thaw mounted onto a solid surface. The surface must be conductive in order to apply a potential to the sample and accelerate ions out of the instrument source. Common materials include coated metal targets (expensive, cannot be archived, and not histology compatible) or indium-tin oxide (ITO) coated glass slides. Matrix application to the tissue section is ideally a homogenous coating of small crystals that provide optimum extraction conditions of analyte. After application, the key process is the co-crystallization that must occur between matrix and analyte. Spraying parameters affect the “wetness” of the surface of the tissue and are a balance between molecular diffusion and effective extraction. Crystal size is one of the more critical factors for a successful MSI experiment and multiple studies determined the parameters important for optimum crystallization.
Commonly used organic acid matrices for MSI are shown in Table 1 and include sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), and α-cyano-4-hydroxycinnamic acid (CHCA). These matrices work for a broad variety of biomolecules including peptides and lipids, but do not always translate well to small molecule detection. The introduction of N-(1-napthyl)ethylenediamine dihydrochloride (NEDC) and 1,5-diaminonapthalene (DAN) have improved the detection of small molecules, though these matrices have different preparation needs (e.g., sublimation and recrystallization) which increases the time required for sample processing [12, 19]. DAN and NEDC matrices have not been fully explored yet in the literature. Moreover, the propensity for organic matrices to self-ionize and create chemical noise in the low mass range prevents effective analysis of most metabolites. Recently, this has led toward the rational design [35] or selection [36] of matrices that can address this, but the lack of consistency in performance can still be an issue. Matrix applications are notorious for behaving differently across laboratories, and significant research in the past 25 years has been devoted to identifying preparation methods that result in the most consistent data [37, 38, 39].
Common MSI organic acid matrices and their applications.
Sublimation procedures, mentioned vida supra, require that matrix and sample are placed in a vacuum chamber which is evacuated [32]. The sample is cooled while the matrix is heated, resulting in sublimation of the matrix which will condense on the cool sample surface. Recrystallization of the matrix is often coupled with this technique. There are advantages and disadvantages to all of the aforementioned matrix application techniques, which have been discussed thoroughly in the literature [40, 41, 42]. Automated sprayers have become increasingly popular and help with consistency of matrix application, though the size and spacing of the matrix droplets will ultimately affect spatial resolution of the experiment. This topic has been frequently discussed and reviewed in the literature [2, 6], so only a basic introduction is given here.
Despite these challenges, there are a few examples of successful metabolomics imaging experiments, though they have utilized purposefully designed matrices that do not generate interfering signals [35] or have used high-resolution instruments that have the high mass accuracy to distinguish between isobaric signals [43, 44]. These approaches are not an all-encompassing solution, and the next sections explore other strategies to achieve broader success with MSI of small molecules, specifically NTs.
As an alternative to the traditional organic acid matrices, contemporary studies have returned to the inorganic materials that were originally proposed for MALDI-MS by Tanaka et al. [45]. Nanoparticles (NPs) made of gold [46, 47, 48, 49], silver [50, 51, 52, 53], carbon based substrates [54, 55, 56, 57], and silicon surfaces [58, 59] have been demonstrated on the target plate as materials that facilitate ionization of biomolecules. In particular, gold NPs (AuNPs) have the potential to be a more universal material to help facilitate ionization of small molecules and seem to have fewer reproducibility issues across multiple instrument platforms, locations, and organisms/biofluids [46, 60, 61]. Specific advantages for ionization of small molecules using AuNPs on the target plate include: (i) less chemical noise in the range where small molecules are found (below m/z 300), (ii) flexible analyte solution preparation conditions, including tolerances for salts, surfactants, and pH, and (iii) broad applicability across chemical classes [46, 48, 57, 60, 62].
The general success, though not broad usage, of these materials on the target plate have led to several different approaches for using alternative inorganic materials for MSI, such as sputtering of metals or the use of metallic NPs, both of which are described in the paragraphs below.
Sputtering of metals over tissue sections has produced a number of quality articles that have utilized silver [50], platinum [63], and gold [64]. Sputtering deposits highly pure and homogeneous metal or metal oxide nanolayers onto biological tissue sections. Magnetron sputtering systems utilize a plasma gun under high- or ultra-high vacuum and deposits layers of metal onto the substrate of interest. Deposition times range from under a minute to several minutes, with total sample preparation time at least several minutes long because of the need for a vacuum-based system. Sputtered layers of silver or gold are typically reported in the 20-50 nm range [49, 50, 65], which is a narrower size distribution than solution-based NPs. The biggest disadvantages of sputtering are the need for expert users, the time involved for sample preparation, and the equipment cost (e.g., sputtering apparatuses are up to tenfold more expensive than pneumatic sprayers).
Molecules that have been successfully detected using Ag or Au sputtering experiments are largely neutral lipids, with cholesterol being of high interest [49, 50, 52, 67]. Pt sputtering has been demonstrated on lipids in tissue [68] and in leaves where metabolites of interest were detected, including many with molecular features similar to NTs, such as acetamiprid [63]. Rafols et al. showed an Au sputtering MSI experiment that resulted in the potential detection of 25 different compounds, but only 1 of which could be called a small molecule metabolite (i.e., citrulline) [64]. A significant advantage that sputtering demonstrates, compared to organic acid matrices [22], is the lack of analyte delocalization [64]. The largest survey of sputtering materials was done by Hansen et al., where Ag, Au, Cu, Ni, Pt, and Ti were sputtered for varying times on plant tissues [66]. Noble metals (e.g., Ag, Au, Pt) were found to be more effective than transition metals (e.g., Cu, Ni, Ti) for overall ionization in positive- and negative-ion modes. While lipids were the most prevalent biomolecule class examined, this is a rare demonstration of the detection of amine-based structures, including choline, asparagine, glutamic acid, and leucine. DHB was used for comparison in positive-ion mode and DAN in negative-ion mode, with primarily insoluble lipids being effectively ionized with organic matrices. A summary of the molecules detected is shown in Figure 2.
Summary of sputter-coated metal screening for small metabolite analysis in (a) positive and (b) negative ion mode. Asterisks indicate a fragment ion was detected. Reprinted with permission from Ref. [66]. Copyright 2018 American Society for Mass Spectrometry.
Nanomaterials in suspension form, such as colloidal NPs, could potentially be deposited or sprayed onto tissue sections for analysis, yet there are only select demonstrations of this application for MSI, which are described herein. This area of research has again been applied primarily to lipidomics, with successful detection of fatty acids and their derivatives, sterols, phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, etc.), sphingomyelins, and ceramides. Silver NPs (AgNPs), including those that have been surface-derivatized are most popular. Polyvinylpyrrolidone-capped AgNPs have been utilized for analysis of brain [69], and AuNPs with alkylamine surface modifications have been used for imaging of glycosphingolipids in the brain [70]. Fluorinated AuNPs have been shown to detect carbohydrates, lipids, bile acids, sulfur metabolites, amino acids, nucleotide precursors, and more in mouse colon [71].
Small molecule examples, specifically NTs, are notably lacking in the MSI literature. We have been developing a method for the detection of endogenous NTs from biological samples using citrate-capped AuNPs that are pneumatically sprayed on tissue sections [47]. Successful detection of acetylcholine, dopamine/octopamine, epinephrine, glutamine, GABA, norepinephrine, and serotonin was achieved in rabbit brain tissue sections, zebrafish embryos, and neuroblastoma cells [61, 72]. See Figure 3 for an image of seven different NTs in 5 day-post fertilization zebrafish embryos.
MSI of a sagittal zebrafish tissue section at 5 μm lateral spatial resolution with eye (E), forebrain (FB), midbrain (MB), and hindbrain (HB) indicated in (a), (b) is the [GABA + Na]+ image, (c) is the epinephrine image, (d) is the histidine image, (e) is the acetylcholine image, (f) is the glutamine image, (g) is the dopamine/octopamine image, (h) is the norepinephrine image, and (i) is the serotonin image. Reprinted with permission from Ref. [61]. Copyright 2020 American Chemical Society.
With the intention of demonstrating the flexibility of AuNPs in terms of sample preparation, spraying parameters have been explored, including matrix concentration, solvent composition, spray temperature, and linear flow rate (which determines overall spray density of material). Early data and statistical analysis by principal component analysis (PCA) suggests that: (i) AuNP concentration can be varied over 4 orders of magnitude, (ii) a variety of organic:aqueous solvent mixes are possible, and (iii) temperatures from 30 to 60°C can be utilized [73, 74]. The flexibility in spray parameters means that less-experienced users with varying spraying capabilities can still get optimum data from their tissue sections, opening the application of MSI to more areas of study.
As previously mentioned, delocalization is an issue with organic acid matrices, often because of a “wet” matrix spray that results in true molecular diffusion instead of analyte extraction from the tissue. A standard literature method for CHCA has been compared with AuNPs, where CHCA gives only extreme delocalization outside of the tissue margins, and the AuNPs result in distinct anatomical visualization, as well as the ability to see subtle differences in analyte concentration [61]. Another advantage of AuNPs that we have discovered is flexibility in sample storage. For example, AuNP-sprayed tissue sections on slides were stored overnight at −20°C and imaging runs were repeated after 24 hours. Nearly identical data resulted and up to 8 imaging runs were completed on the same tissue section without loss of signal or the need to reapply AuNPs. The ability to archive slides for later examination could allow for follow-up data to be collected as well as the potential to reduce the number of organisms needed in a given research project.
The improvements in delocalization, reproducibility, and long-term stability from pneumatically-sprayed AuNPs warrant further investigation of this method. Finally, the quick and low-cost preparation may enable a broad range of new applications in neuroscience, pharmacology, drug discovery, and pathology.
Chemical derivatization of functional groups is a common strategy to improve detection in MS techniques for a variety of molecular classes. On-tissue derivatization has been explored for many different purposes, including tryptic digestion [75, 76], phospholipid digestion [77], N-terminal peptide derivatization [78], and derivatization of various metabolites/drugs of interest [79, 80, 81, 82]. One particularly attractive advantage of derivatization is that small mass species typically have a change in molecular weight which avoids interferences from low mass matrix peaks.
Some of the aforementioned studies were on small molecular weight species, but derivatization was typically for only one analyte of interest. Examples of NT derivatization have met with varied success in terms of how many different molecular classes are accessible. Coniferyl aldehyde has been used to derivatize primary amines in pig adrenal glands and rat brains [83]. Methods included pre-coating target plates and then incubating after tissue was affixed for several minutes. Spray-coating with an organic matrix followed.
Specific reactions focused on NTs have utilized pyrylium salts (e.g., 2,4-diphenyl pyranylium (DPP)) that are reactive toward primary amines. The reaction scheme with a common NT, dopamine, is shown in Figure 4a. The reaction can proceed at room temperature but requires 30-80 spray passes of the derivatizing agent, followed by drying time, and then application of an organic matrix [84]. Additionally, preparation of the derivatizing agent is required and can be a multi-day process. While smaller crystals than typical organic matrix preparations have been reported, there are still limitations on overall spatial resolution. Figure 4b shows dopamine derivatized with 3 different pyrylium salts and the resulting images that are generated; without derivatization no dopamine was observed.
(a) Reaction of dopamine with pyrylium salts. MALDI-MSI images of dopamine derivatized with DPP (b, c), PBDPP (d, e) or TMP (f, g). Signal intensity is indicated using a rainbow scale. Reprinted with permission from Ref. [84]. Copyright 2015 American Society for Mass Spectrometry.
Derivatization with DPP has been demonstrated in multiple instances, with the generation a 3D mouse brain atlas of dopamine, norepinephrine and serotonin [85] as well as detection of up to 23 amino metabolites [86].
Additional derivatization methods have been developed since the initial report on primary amines only. For example, fluoromethylpyridinium-based materials are reactive with phenolic hydroxyl and/or primary or secondary amines, which expand the potential range of NTs that can be detected [87]. Charge-tagging using 2-(4-bromophenyl)-4,6- diphenylpyranylium (Br-TPP) results in distinctive isotopic distributions in the mass spectrum, making it easier to identify derivatized species from other potential species [88].
The last example here is a laser-induced tissue transfer (LATT) system that enhances on-tissue derivatization of small molecules [89]. An electrosprayer applies the derivatization reagent and matrix solution on tissue and is then irradiated with a 450 nm laser beam in transmission mode, which results in transfer of a thin film of tissue to a second slide. Figure 5 shows the setup and diagram of the LATT system. Chemicals used for derivatization include coniferyl acetate or Girard’s T reagent. Preparation time requires multiple hours (overnight) and additional matrix application. Multiple classes of biomolecules were analyzed, including amino acids, NTs, polyamines, dipeptides, and others. The issue of analyte delocalization is improved in LATT as compared to other derivatization techniques.
(A) LATT setup and (B) schematic diagram of the system. Reprinted with permission from Ref. [89]. Copyright 2020 American Chemical Society.
MSI has been applied to quantitative analysis of drugs [90, 91], metabolites [92], and biomarkers in tissue [93] using pneumatic sprayers and sublimation techniques described in this chapter. Nearly all of these demonstrations have utilized organic acid matrices such as DHB, CHCA, and trihydroxyacetophenone (THAP), with one research group utilizing TiO2 NPs [94, 95]. Methods of quantitation are still being investigated [96], as many of the consistency issues with MALDI-MSI that have been discussed in this chapter are even more relevant with quantitative MSI (qMSI). Figure 6 shows a summary of two of the more common methods used for generation of a calibration curve for qMSI, on-tissue spotting and tissue mimetic models which feature spiking of tissue homogenates [96].
Description of qMSI experiments where on-tissue spotting and homogenate spiking are two popular methods. Reprinted with permission from Ref. [94]. Copyright 2019 Elsevier Ltd.
On-tissue spotting uses either a standard molecule that is chemically similar to the analyte or a stable isotope of the analyte for making the calibration curve. Ion intensities between the analyte and standard are used to estimate the drug concentration in dosed tissue. Disadvantages include difficulty in maintaining uniform application of standards and differences in ionization for sprayed on standards vs. analyte molecules embedded within tissue. Advantages are that this method is fast and straightforward. The tissue mimetic model uses a surrogate tissue that is homogenized and spiked with the analyte of interest, frozen, sectioned, then prepared with matrix. The advantage of this method is that there is better representation of the ionization process for analyte embedded within tissue. However, it is more time consuming, labor intensive, and requires more tissue for the calibration curve. Each method has been correlated with LC-MS data, the current primary method used for quantification [97].
Specific examples with clinical relevancy are briefly described here. First, epertinib and lapatinib were quantified in a metastatic brain cancer mouse model using stable isotope labeling, and with liquid chromatography (LC)-MS validation [91]. The topical drugs roflumilast, tofacitinib, ruxolitinib, and LEO 29102 were examined in human skin explants to determine drug penetration and evaluate lipid markers [90]. qMSI data had a much lower quantitation range than LC-MS data of individual skin layers. Rifampicin in mouse liver tissue was quantified via a fragment ion of the intact molecule. The method used an in-house synthesized stable isotope and correlated the results with LC-MS/MS [98]. Lastly, there is one example that specifically focused on comprehensive mapping of NTs in Parkinson’s disease lesioned mouse brain and demonstrated quantitation of dopamine using a stable isotope [87]. All of the drug molecules listed in this paragraph are above the general size range that NTs and metabolites fall within, ranging from 400 to 800 Da, but present possible future avenues of research for the NT-focused methods discussed in this chapter.
The tissue mimetic model first gained popularity with examination of lapatinib and nevirapine in mouse liver by Groseclose and Castellino [99]. In addition to demonstrating high spatial resolution, they examined reproducibility and drug distribution within the homogenate. Fewer applications of the tissue mimetic model have been done, especially with small molecules as opposed to lipids [100]. A notable example includes the determination of the spatial distribution of gemcitabine, a chemotherapeutic agent, and its metabolites in mouse model pancreatic tumors using AuNPs and a traditional matrix as comparison [101]. Further experiments also work on the computational side of MSI and determining the best ways to normalize spectra [102, 103].
This chapter has introduced the utility of mass spectrometry imaging (MSI) for small molecules, with a specific focus on neurotransmitters (NTs). Methods that have resulted in enhanced signals of NTs were highlighted, with alternative matrix materials and chemical derivatization of analytes the two main points of discussion. Future research is needed in both of these areas to determine optimum conditions and applications, as well as establishing standard procedures so that broad application of MSI can continue. Finally, an area not discussed here that is relevant to these techniques and that will likely be explored in the future is the quantitative determination of small molecules.
KS thanks the University of Scranton and the Chemistry Department for providing facilities, equipment, and financial support for any referenced experiments. KS thanks the Johns Hopkins Applied Imaging Mass Spectrometry (AIMS) Core Facility at the Johns Hopkins University School of Medicine for undertaking the imaging experiments referenced in this chapter. Lastly, KS thanks Nolan McLaughlin and Tyler Bielinski for tagging along for the science journey.
The author declares no conflict of interest.
DAG | diacylglycerol |
DAN | 1,5-diaminonapthalene |
DESI | desorption electrospray ionization |
DHB | 2,5-dihydroxybenzoic acid |
DPP | 2,4-diphenyl pyranylium |
CHCA | α-cyano-4-hydroxy cinnamic acid |
Glucose 6-P | glαucose 6-phosphate |
HTP | high throughput |
ITO | indium tin oxide |
LATT | laser-assisted tissue transfer |
LC-MS | liquid chromatography mass spectrometry |
MALDI | matrix-assisted laser desorption ionization |
MSI | |
qMSI | quantitative mass spectrometry imaging |
NEDC | N-(1-napthyl)ethylenediamine dihydrochloride |
NT | neurotransmitter |
NP | nanoparticle |
PA | phosphatidic acid |
PCA | principal component analysis |
PChol | phosphocholine |
PE | phospatidylethanolamine |
PEP | phosphoenolpyruvic acid |
PG | phosphatidylglycerol |
PI | phosphatidylinositol |
PC | phosphatidylcholine |
SA | sinapic (or sinapinic) acid |
TAG | triacylglycerol |
THAP | trihydroxyacetophenone |
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\\n\\nAuthor - in order to be identified as an Author, three criteria must be met: (i) Substantial contribution to the conception or design of the Work, or the acquisition, analysis, or interpretation of data for the Work; (ii) Participation in drafting or revising the Work; (iii) Approval of the final version of the Work to be published.
\\n\\nWork - a Chapter, including Conference Papers, and any and all text, graphics, images and/or other materials forming part of or accompanying the Chapter/Conference Paper.
\\n\\nMonograph/Compacts - a full manuscript usually written by a single Author, including any and all text, graphics, images and/or other materials.
\\n\\nCompilation - a collection of Works distributed in a Book that IntechOpen has selected, and for which the coordination of the preparation, arrangement and publication has been the responsibility of IntechOpen. Any Work included is accepted in its entirety in unmodified form and is published with one or more other contributions, each constituting a separate and independent Work, but which together are assembled into a collective whole.
\\n\\nIntechOpen - Registered publisher with office at 5 Princes Gate Court, London, SW7 2QJ - UNITED KINGDOM
\\n\\nIntechOpen platform - IntechOpen website www.intechopen.com whose main purpose is to host Monographs in the format of Book Chapters, Long Form Monographs, Compacts, Conference Proceedings and Videos.
\\n\\nVideo Lecture – an audiovisual recording of a lecture or a speech given by a Lecturer, recorded, edited, owned and published by IntechOpen.
\\n\\nTERMS
\\n\\nAll Works published on the IntechOpen platform and in print are licensed under a Creative Commons Attribution 3.0 Unported License, a license which allows for the broadest possible reuse of published material.
\\n\\nCopyright on the individual Works belongs to the specific Author, subject to an agreement with IntechOpen. The Creative Common license is granted to all others to:
\\n\\nAnd for any purpose, provided the following conditions are met:
\\n\\nAll Works are published under the CC BY 3.0 license. However, please note that book Chapters may fall under a different CC license, depending on their publication date as indicated in the table below:
\\n\\n\\n\\n
LICENSE | \\n\\t\\t\\tUSED FROM - | \\n\\t\\t\\tUP TO - | \\n\\t\\t
\\n\\t\\t\\t Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported (CC BY-NC-SA 3.0) \\n\\t\\t\\t | \\n\\t\\t\\t\\n\\t\\t\\t 1 July 2005 (2005-07-01) \\n\\t\\t\\t | \\n\\t\\t\\t\\n\\t\\t\\t 3 October 2011 (2011-10-03) \\n\\t\\t\\t | \\n\\t\\t
Creative Commons Attribution 3.0 Unported (CC BY 3.0) | \\n\\t\\t\\t\\n\\t\\t\\t 5 October 2011 (2011-10-05) \\n\\t\\t\\t | \\n\\t\\t\\tCurrently | \\n\\t\\t
The CC BY 3.0 license permits Works to be freely shared in any medium or format, as well as the reuse and adaptation of the original contents of Works (e.g. figures and tables created by the Authors), as long as the source Work is cited and its Authors are acknowledged in the following manner:
\\n\\nContent reuse:
\\n\\n© {year} {authors' full names}. Originally published in {short citation} under {license version} license. Available from: {DOI}
\\n\\nContent adaptation & reuse:
\\n\\n© {year} {authors' full names}. Adapted from {short citation}; originally published under {license version} license. Available from: {DOI}
\\n\\nReposting & sharing:
\\n\\nOriginally published in {full citation}. Available from: {DOI}
\\n\\nRepublishing – More about Attribution Policy can be found here.
\\n\\nThe same principles apply to Works published under the CC BY-NC-SA 3.0 license, with the caveats that (1) the content may not be used for commercial purposes, and (2) derivative works building on this content must be distributed under the same license. The restrictions contained in these license terms may, however, be waived by the copyright holder(s). Users wishing to circumvent any of the license terms are required to obtain explicit permission to do so from the copyright holder(s).
\\n\\nDISCLAIMER: Neither the CC BY 3.0 license, nor any other license IntechOpen currently uses or has used before, applies to figures and tables reproduced from other works, as they may be subject to different terms of reuse. In such cases, if the copyright holder is not noted in the source of a figure or table, it is the responsibility of the User to investigate and determine the exact copyright status of any information utilised. Users requiring assistance in that regard are welcome to send an inquiry to permissions@intechopen.com.
\\n\\nAll rights to Books and all other compilations published on the IntechOpen platform and in print are reserved by IntechOpen.
\\n\\nThe copyright to Books and other compilations is subject to separate copyright from those that exist in the included Works.
\\n\\nAll Long Form Monographs/Compacts are licensed under the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) license granted to all others.
\\n\\nCopyright to the individual Works (Chapters) belongs to their specific Authors, subject to an agreement with IntechOpen and the Creative Common license granted to all others to:
\\n\\nUnder the following terms:
\\n\\nThere must be an Attribution, giving appropriate credit, provision of a link to the license, and indication if any changes were made.
\\n\\nNonCommercial - The use of the material for commercial purposes is prohibited. Commercial rights are reserved to IntechOpen or its licensees.
\\n\\nNo additional restrictions that apply legal terms or technological measures that restrict others from doing anything the license permits are allowed.
\\n\\nThe CC BY-NC 4.0 license permits Works to be freely shared in any medium or format, as well as reuse and adaptation of the original contents of Works (e.g. figures and tables created by the Authors), as long as it is not used for commercial purposes. The source Work must be cited and its Authors acknowledged in the following manner:
\\n\\nContent reuse:
\\n\\n© {year} {authors' full names}. Originally published in {short citation} under {license version} license. Available from: {DOI}
\\n\\nContent adaptation & reuse:
\\n\\n© {year} {authors' full names}. Adapted from {short citation}; originally published under {license version} license. Available from: {DOI}
\\n\\nReposting & sharing:
\\n\\nOriginally published in {full citation}. Available from: {DOI}
\\n\\nAll Book cover design elements, as well as Video image graphics are subject to copyright by IntechOpen.
\\n\\nEvery reproduction of a front cover image must be accompanied by an appropriate Copyright Notice displayed adjacent to the image. The exact Copyright Notice depends on who the Author of a particular cover image is. Users wishing to reproduce cover images should contact permissions@intechopen.com.
\\n\\nAll Video Lectures under IntechOpen's production are subject to copyright and are property of IntechOpen, unless defined otherwise, and are licensed under the Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license. This grants all others the right to:
\\n\\nShare — copy and redistribute the material in any medium or format
\\n\\nUnder the following terms:
\\n\\nUsers wishing to repost and share the Video Lectures are welcome to do so as long as they acknowledge the source in the following manner:
\\n\\n© {year} IntechOpen. Published under CC BY-NC-ND 4.0 license. Available from: {DOI}
\\n\\nUsers wishing to reuse, modify, or adapt the Video Lectures in a way not permitted by the license are welcome to contact us at permissions@intechopen.com to discuss waiving particular license terms.
\\n\\nAll software used on the IntechOpen platform, any used during the publishing process, and the copyright in the code constituting such software, is the property of IntechOpen or its software suppliers. As such, it may not be downloaded or copied without permission.
\\n\\nUnless otherwise indicated, all IntechOpen websites are the property of IntechOpen.
\\n\\nAll content included on IntechOpen Websites not forming part of contributed materials (such as text, images, logos, graphics, design elements, videos, sounds, pictures, trademarks, etc.), are subject to copyright and are property of, or licensed to, IntechOpen. Any other use, including the reproduction, modification, distribution, transmission, republication, display, or performance of the content on this site is strictly prohibited.
\\n\\nPolicy last updated: 2016-06-08
\\n"}]'},components:[{type:"htmlEditorComponent",content:'Copyright is the term used to describe the rights related to the publication and distribution of original Works. Most importantly from a publisher's perspective, copyright governs how Authors, publishers and the general public can use, publish, and distribute publications.
\n\nIntechOpen only publishes manuscripts for which it has publishing rights. This is governed by a publication agreement between the Author and IntechOpen. This agreement is accepted by the Author when the manuscript is submitted and deals with both the rights of the publisher and Author, as well as any obligations concerning a particular manuscript. However, in accepting this agreement, Authors continue to retain significant rights to use and share their publications.
\n\nHOW COPYRIGHT WORKS WITH OPEN ACCESS LICENSES?
\n\nAgreement samples are listed here for the convenience of prospective Authors:
\n\n\n\nDEFINITIONS
\n\nThe following definitions apply in this Copyright Policy:
\n\nAuthor - in order to be identified as an Author, three criteria must be met: (i) Substantial contribution to the conception or design of the Work, or the acquisition, analysis, or interpretation of data for the Work; (ii) Participation in drafting or revising the Work; (iii) Approval of the final version of the Work to be published.
\n\nWork - a Chapter, including Conference Papers, and any and all text, graphics, images and/or other materials forming part of or accompanying the Chapter/Conference Paper.
\n\nMonograph/Compacts - a full manuscript usually written by a single Author, including any and all text, graphics, images and/or other materials.
\n\nCompilation - a collection of Works distributed in a Book that IntechOpen has selected, and for which the coordination of the preparation, arrangement and publication has been the responsibility of IntechOpen. Any Work included is accepted in its entirety in unmodified form and is published with one or more other contributions, each constituting a separate and independent Work, but which together are assembled into a collective whole.
\n\nIntechOpen - Registered publisher with office at 5 Princes Gate Court, London, SW7 2QJ - UNITED KINGDOM
\n\nIntechOpen platform - IntechOpen website www.intechopen.com whose main purpose is to host Monographs in the format of Book Chapters, Long Form Monographs, Compacts, Conference Proceedings and Videos.
\n\nVideo Lecture – an audiovisual recording of a lecture or a speech given by a Lecturer, recorded, edited, owned and published by IntechOpen.
\n\nTERMS
\n\nAll Works published on the IntechOpen platform and in print are licensed under a Creative Commons Attribution 3.0 Unported License, a license which allows for the broadest possible reuse of published material.
\n\nCopyright on the individual Works belongs to the specific Author, subject to an agreement with IntechOpen. The Creative Common license is granted to all others to:
\n\nAnd for any purpose, provided the following conditions are met:
\n\nAll Works are published under the CC BY 3.0 license. However, please note that book Chapters may fall under a different CC license, depending on their publication date as indicated in the table below:
\n\n\n\n
LICENSE | \n\t\t\tUSED FROM - | \n\t\t\tUP TO - | \n\t\t
\n\t\t\t Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported (CC BY-NC-SA 3.0) \n\t\t\t | \n\t\t\t\n\t\t\t 1 July 2005 (2005-07-01) \n\t\t\t | \n\t\t\t\n\t\t\t 3 October 2011 (2011-10-03) \n\t\t\t | \n\t\t
Creative Commons Attribution 3.0 Unported (CC BY 3.0) | \n\t\t\t\n\t\t\t 5 October 2011 (2011-10-05) \n\t\t\t | \n\t\t\tCurrently | \n\t\t
The CC BY 3.0 license permits Works to be freely shared in any medium or format, as well as the reuse and adaptation of the original contents of Works (e.g. figures and tables created by the Authors), as long as the source Work is cited and its Authors are acknowledged in the following manner:
\n\nContent reuse:
\n\n© {year} {authors' full names}. Originally published in {short citation} under {license version} license. Available from: {DOI}
\n\nContent adaptation & reuse:
\n\n© {year} {authors' full names}. Adapted from {short citation}; originally published under {license version} license. Available from: {DOI}
\n\nReposting & sharing:
\n\nOriginally published in {full citation}. Available from: {DOI}
\n\nRepublishing – More about Attribution Policy can be found here.
\n\nThe same principles apply to Works published under the CC BY-NC-SA 3.0 license, with the caveats that (1) the content may not be used for commercial purposes, and (2) derivative works building on this content must be distributed under the same license. The restrictions contained in these license terms may, however, be waived by the copyright holder(s). Users wishing to circumvent any of the license terms are required to obtain explicit permission to do so from the copyright holder(s).
\n\nDISCLAIMER: Neither the CC BY 3.0 license, nor any other license IntechOpen currently uses or has used before, applies to figures and tables reproduced from other works, as they may be subject to different terms of reuse. In such cases, if the copyright holder is not noted in the source of a figure or table, it is the responsibility of the User to investigate and determine the exact copyright status of any information utilised. Users requiring assistance in that regard are welcome to send an inquiry to permissions@intechopen.com.
\n\nAll rights to Books and all other compilations published on the IntechOpen platform and in print are reserved by IntechOpen.
\n\nThe copyright to Books and other compilations is subject to separate copyright from those that exist in the included Works.
\n\nAll Long Form Monographs/Compacts are licensed under the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) license granted to all others.
\n\nCopyright to the individual Works (Chapters) belongs to their specific Authors, subject to an agreement with IntechOpen and the Creative Common license granted to all others to:
\n\nUnder the following terms:
\n\nThere must be an Attribution, giving appropriate credit, provision of a link to the license, and indication if any changes were made.
\n\nNonCommercial - The use of the material for commercial purposes is prohibited. Commercial rights are reserved to IntechOpen or its licensees.
\n\nNo additional restrictions that apply legal terms or technological measures that restrict others from doing anything the license permits are allowed.
\n\nThe CC BY-NC 4.0 license permits Works to be freely shared in any medium or format, as well as reuse and adaptation of the original contents of Works (e.g. figures and tables created by the Authors), as long as it is not used for commercial purposes. The source Work must be cited and its Authors acknowledged in the following manner:
\n\nContent reuse:
\n\n© {year} {authors' full names}. Originally published in {short citation} under {license version} license. Available from: {DOI}
\n\nContent adaptation & reuse:
\n\n© {year} {authors' full names}. Adapted from {short citation}; originally published under {license version} license. Available from: {DOI}
\n\nReposting & sharing:
\n\nOriginally published in {full citation}. Available from: {DOI}
\n\nAll Book cover design elements, as well as Video image graphics are subject to copyright by IntechOpen.
\n\nEvery reproduction of a front cover image must be accompanied by an appropriate Copyright Notice displayed adjacent to the image. The exact Copyright Notice depends on who the Author of a particular cover image is. Users wishing to reproduce cover images should contact permissions@intechopen.com.
\n\nAll Video Lectures under IntechOpen's production are subject to copyright and are property of IntechOpen, unless defined otherwise, and are licensed under the Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license. This grants all others the right to:
\n\nShare — copy and redistribute the material in any medium or format
\n\nUnder the following terms:
\n\nUsers wishing to repost and share the Video Lectures are welcome to do so as long as they acknowledge the source in the following manner:
\n\n© {year} IntechOpen. Published under CC BY-NC-ND 4.0 license. Available from: {DOI}
\n\nUsers wishing to reuse, modify, or adapt the Video Lectures in a way not permitted by the license are welcome to contact us at permissions@intechopen.com to discuss waiving particular license terms.
\n\nAll software used on the IntechOpen platform, any used during the publishing process, and the copyright in the code constituting such software, is the property of IntechOpen or its software suppliers. As such, it may not be downloaded or copied without permission.
\n\nUnless otherwise indicated, all IntechOpen websites are the property of IntechOpen.
\n\nAll content included on IntechOpen Websites not forming part of contributed materials (such as text, images, logos, graphics, design elements, videos, sounds, pictures, trademarks, etc.), are subject to copyright and are property of, or licensed to, IntechOpen. Any other use, including the reproduction, modification, distribution, transmission, republication, display, or performance of the content on this site is strictly prohibited.
\n\nPolicy last updated: 2016-06-08
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I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. 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After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\r\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. He is an expert in structural, absorptive, catalytic and photocatalytic properties, in structural organization and dynamic features of ionic liquids, in magnetic interactions between paramagnetic centers. The author or co-author of 3 books, over 200 articles and reviews in scientific journals and books. He is an actual member of the International EPR/ESR Society, European Society on Quantum Solar Energy Conversion, Moscow House of Scientists, of the Board of Moscow Physical Society.",institutionString:null,institution:{name:"Semenov Institute of Chemical Physics",country:{name:"Russia"}}},{id:"62389",title:"PhD.",name:"Ali Demir",middleName:null,surname:"Sezer",slug:"ali-demir-sezer",fullName:"Ali Demir Sezer",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62389/images/3413_n.jpg",biography:"Dr. Ali Demir Sezer has a Ph.D. from Pharmaceutical Biotechnology at the Faculty of Pharmacy, University of Marmara (Turkey). He is the member of many Pharmaceutical Associations and acts as a reviewer of scientific journals and European projects under different research areas such as: drug delivery systems, nanotechnology and pharmaceutical biotechnology. Dr. Sezer is the author of many scientific publications in peer-reviewed journals and poster communications. Focus of his research activity is drug delivery, physico-chemical characterization and biological evaluation of biopolymers micro and nanoparticles as modified drug delivery system, and colloidal drug carriers (liposomes, nanoparticles etc.).",institutionString:null,institution:{name:"Marmara University",country:{name:"Turkey"}}},{id:"61051",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"100762",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"St David's Medical Center",country:{name:"United States of America"}}},{id:"107416",title:"Dr.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Texas Cardiac Arrhythmia",country:{name:"United States of America"}}},{id:"64434",title:"Dr.",name:"Angkoon",middleName:null,surname:"Phinyomark",slug:"angkoon-phinyomark",fullName:"Angkoon Phinyomark",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/64434/images/2619_n.jpg",biography:"My name is Angkoon Phinyomark. I received a B.Eng. degree in Computer Engineering with First Class Honors in 2008 from Prince of Songkla University, Songkhla, Thailand, where I received a Ph.D. degree in Electrical Engineering. My research interests are primarily in the area of biomedical signal processing and classification notably EMG (electromyography signal), EOG (electrooculography signal), and EEG (electroencephalography signal), image analysis notably breast cancer analysis and optical coherence tomography, and rehabilitation engineering. I became a student member of IEEE in 2008. During October 2011-March 2012, I had worked at School of Computer Science and Electronic Engineering, University of Essex, Colchester, Essex, United Kingdom. In addition, during a B.Eng. I had been a visiting research student at Faculty of Computer Science, University of Murcia, Murcia, Spain for three months.\n\nI have published over 40 papers during 5 years in refereed journals, books, and conference proceedings in the areas of electro-physiological signals processing and classification, notably EMG and EOG signals, fractal analysis, wavelet analysis, texture analysis, feature extraction and machine learning algorithms, and assistive and rehabilitative devices. I have several computer programming language certificates, i.e. Sun Certified Programmer for the Java 2 Platform 1.4 (SCJP), Microsoft Certified Professional Developer, Web Developer (MCPD), Microsoft Certified Technology Specialist, .NET Framework 2.0 Web (MCTS). 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