Advanced Biomaterials and Biodevicess

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240 Advanced Biomaterials and Biodevices 275. Ngo DN, Lee S-H, Kim MM, Kim SK. Production of chitin oligosaccharides with different molecular weights and their antioxidant effect in RAW 264.7 cells. J. Funct. Foods 2009; 1: 188–198. 276. Anraku M, Kabashima M, Namura H, Maruyama T, Otagiri M, Gebicki JM, Furutani N, Tomida H. Antioxidant protection of human serum albumin by chitosan. Int. J. Biol. Macromol. 2008; 43: 159–164. 277. Anraku M, Fujii T, Furutani N, Kadowaki D, Maruyama T, Otagiri M, Gebicki JM, Tomida H. Antioxidant effects of a dietary supplement: Reduction of indices of oxidative stress in normal subjects by water-soluble chitosan. Food Cheml. Toxicol. 2009; 47: 104–109. 278. Xie W, Xu P, Liu Q. Antioxidant activity of water-soluble chitosan deriva- tives. Bioorg Med Chem Lett 2001; 11: 1699–1701. 279. Yazdani-Pedram M, Lagos A, Campos N, Retuert J. Comparision of rdox initiators reactivities in the grafting of methyl methacrylate onto chitin. Int. J. Polym. Mater. 1992; 18: 25–37. 280. Chen A-S, Taguchi T, Sakai K, Kikuchi K, Wang M-W, Miwa I. Antioxidant Activities of Chitobiose and Chitotriose. Biol. Pharm. Bull. 2003; 26: 1326–1330. 281. Yang Y, Shu R, Shao J, Xu G, Gu X.Radical scavenging activity of chitooli- gosaccharide with different molecular weights. Eur. Food Res. Technol. 2006; 222: 36–40. 282. Park PJ, Je JY, Kim SK. Free radical scavenging activities of differently deacet- ylated chitosan using ESR spectrometer. Carbohydr. Polym. 2004; 55: 17–22. 283. Je J-Y, Park P-J, Kim S-K. Free radical scavenging properties of hetero-chi- tooligosaccharides using an ESR spectroscopy. Food Cheml. Toxicol. 2004; 42: 381–387. 284. Feng T, Du Y, Li J, Wei Y, Yao P Antioxidant activity of half N-acetylated water-soluble chitosan in vitro Eur. Food Res. Technol. 2007; 225: 133–138. 285. Xing R, Liu S, Guo Z, Yu H, Wang P, Li C, Li Z, Li P. Relevance of molecular weight of chitosan and its derivativesand their antioxidant activities in vitro. Bioorg. Med. Chem. 2005; 13: 1573–1577. 286. Sun T, Zhou D, Xie J, Mao F. Preparation of chitosan oligomers and their antioxidant activity. Eur. Food Res. Technol. 2007; 225: 451–456. 287. Tomida H, Fujii T, Furutani N, Michihara A, Yasufuku T, Akasaki K, Maruyama T, Otagiri M, Gebicki JM, Anraku M. Antioxidant properties of some different molecular weight chitosans. Carbohydr. Res. 2009; 344 1690–1696. 288. Feng T, Gong J, Du Y, Huang Z. Free radical scavenging activity of cellulase- treated chitosan. J. Appl. Polym. Sci. 2009; 111: 545–550. 289. Feng T, Du Y, Li J, Hu, Y Kennedy JF. Enhancement of antioxidant activity of chitosan by irradiation. Carbohydr. Polym. 2008; 73: 126–132. 290. Je JY, Kim SK. Antioxidant activity of novel chitin derivative. Bioorg. Medl. Chem. Lett. 2006; 14: 5989–5994.

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7 Anticipating Behaviour of Advanced Materials in Healthcare Tanvir Arfin1,* and Simin Fatma2 1Department of Chemistry, Uka Tarsadia University, Maliba Campus, Gopal Vidyanagar, Bardoli, India 2Department of Biotechnology, Tilka Manjhi Bhagalpur University, Bhagalpur, India Abstract An advanced material in health care is an important aspect prevailing today. It is necessary to recognize that they are not influenced like older traditional materials, reactive chemicals which are prone to arduously react and corrode in ambient environments. Health care with these advanced materials assure the satisfactory performance within their design for which life is approached and enhanced in a compatible way. Sensors for medical and diagnostic application, light and energy harvesting devices, along with multifunctional architectures for electronics and advance drug-delivery are pursued by countless advanced materials in term of health care for various uses. In addition, drug medicine has recently immerged as an effective means to combine upcoming technology for development towards a clinical application. In vitro analysis and animal studies had lead to aforementioned researches. There is no doubt that their results had given a precious fundamental concept for understanding the disease process, behaviour and interactions of advance materials with live tissue. However, future and next generation researches are now warranted to promote translation of the knowledge into toxic studies, and broaden the researches with human model studies. This is the aspect where tissue engineering and regenerative medicine which manifested is most important. With recent studies, autotranplantation of whole tissue unit had been more possible than ever by the scientists. Moreover in-depth understanding of pathology using engineered tissue as disease model would enable the development of better treatment and help the individual to *Corresponding author: [email protected] Ashutosh Tiwari and Anis N. Nordin (eds.) Advanced Biomaterials and Biodevices, (243–288) 2014 © Scrivener Publishing LLC 243

244 Advanced Biomaterials and Biodevices overcome various circumstances. Abundance of discoveries in advanced tech- nology is already making a tremendous impact, and will fluently change the medicine of the twenty-first century forever. Latest studies had demonstrated that advance material exhibit a noticeable and challengeable in vitro cytotoxic activity, comparable to standard platinum-based drugs, cisplatin, carboplatin and oxaliplatin. Over the decades of years chemists have prepared ligands des- ignating a wide range of features designed to achieve their particular targets and goals. Thus, an advance material has modified electrodes which present unusual advantages over macroelectrodes in electroanalysis such as catalysis, enhance- ment of mass transport, high effective surface area, and control over electrode microenvironment. During recent years, metallic alloy in advance material has been of considerable and manifold interest in the field of catalysis and sensors because they often exhibit better catalytic properties than to do their monome- tallic counterparts.. Keywords: Bio-advance material, health care, enzyme, antibody, biosensor avidity 7.1 Introduction An introduction to materials in Healthcare, generally address to design, fabricating, testing, applications, and performance of synthetic and natural materials that are basically used in a wide variety of Implants, devices, and process of equipments that contact the biological systems. These materi- als are basically referred to as bio-advance material. Bio-advance materials address both therapeutically and diagnostically. It encompasses basic sci- ences, and engineering and medicine. The translation of specific bioma- terial to clinically important medical devices is dependent on: (a) sound engineering design; (b) clinical realities; and (c) the involvement of indus- try permitting product development and commercialization and (d) testing in vitro in animal and in human. The schematic diagram of the scientific development to the clinic is shown in Figure 7.1. The content of this book-chapter are : (a) to focus on the scientific and engineering fundamental aspect behind bio-advance materials and their applications; (b) to provide sufficient background and appreciation of the applications of bio-advance material; and (c) to highlight the opportunities and challenges in the field of healthcare. In healthcare applications, bio-advance materials are rarely used as ingredient of isolated materials, but are commonly integrated into various devices or implants. Chemically pure titanium can be called a bio-advance material, but shaped (machined) titanium is in conjunction with the spe- cific ultrahigh molecule weight polyethylene makes the given device, a hip

Anticipating Behaviour of Advanced Materials in Healthcare 245 Research on bio- Engineering Preclinical to advance materials approach to clinical trial establish a medical device Commercialization Regulatory and clinical confirmation confirmation Figure 7.1 Schematic diagram of the scientific development to the clinic in health care. prosthesis. Although this is a book chapter on bio-advance materials, it will quickly become apparent and impressive that the subject cannot be explored sufficiently without considering biomedical devices and biologi- cal response to them. Indeed, both the material and the device had the great impact on the recipient (patient) and, as we will also see that the patient’s host tissue impacts on the device. These interactions can lead to success of device or, when there is inappropriate choice referring to bio- advance materials or at the poor design, device failure takes place. A bio-advance material is a novel interdisciplinary field which includes the development of bio-advance materials emerging out from the interac- tion of materials science, nanotechnology and biotechnology. In the last few years the investigation on these materials has gained and attained very important attention from researchers with expertise in diverse areas of bio- advance materials [1, 2]. Being the results of the combination of biopoly- mers and inorganic solids at the nanometre scale, bio-advance materials belong to this group of materials. These hybrid organic-inorganic materi- als are extraordinary, versatile as they are formed from a large variety of biopolymers namely polypeptides, nucleic acid, etc and also from different inorganic solid particles such as, hydroxyapatite (HAP), silica, layered sili- cates and other metal oxides. To understand the importance of bio- advance composites it should be taken into account that bio-polymers are biodegradable and biocompat- ible compounds are dealt scientifically and, therefore, their composites are of much interest for advanced biomedical materials, as for instance, artificial bones, gene therapy or tissue engineering are explored. Other possible fields of applications of bio-advance materials are related to their thermal, mechanical and barrier properties, making this class of materials very attractive for potential uses in drug controlling and delivery of pesticides, membranes for food processing, drinking water purification, oxygen barrier films, food package and ingredient for our every-day utility.

246 Advanced Biomaterials and Biodevices Biomaterials have moved merely from interacting with the host body to influencing biological processes towards the goal of tissue regenera- tion. However, a more recent definition has been prescribed. According to Williams [3], “A bio material is a substance that has been engineered to take a form which, alone or as part of a complex system, is used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure, in human or veterinary medi- cine.” Different types of materials can be found depending on the function to be performed or on the tissue to be replaced in our daily life. Biomaterials have been very often originated from materials used in diverse research areas that presented desirable and systematic mechani- cal properties but were not specifically designed to interact with the nearby and surrounding tissues or with blood [4]. Among those materi- als, glasses for skeletal repair and reconstruction and the employment of ceramics has been increased due to increases in both life expectancy and the social obligations to provide a better and advanced quality of life. Depending on the type of ceramics employed, the size and their interac- tion with the host tissue, they can be highly categorized as either bio- active or bio-inert, and the bioactive ceramics can be re-absorbable or non re-absorbable [5]. These materials, which may be produced in both porous and dense forms as well as powders, granulates or coating forms are known as bio-ceramics. [5, 6] From the chemical point of view and the different aspect, bio-ceramics can be prepared from, calcium phosphates, silica-containing compounds alumina, zirconium, carbon and some other chemicals. Among them, phosphates can be used to produce generative biomaterials since they pres- ent bone integration and high biocompatibility, and also represent, sim- plifies a similar composition to the inorganic fraction of bones. In fact, bio-ceramics are now used in a various number of different applications throughout the body generally covering all areas of skeleton. There has been a chronological evolution in the field of research of ceramics as bone substitutes. [7, 8] According to Williams [9], “Biocompatibility is the ability of a material to perform with an appropriate host response in a specific application.” 7.2 The Evolution of the Bio-advance Materials Fields The bio-advance materials community has been the major contribution to the understanding and influencing of the interactions of materials with the physiological environment.

Anticipating Behaviour of Advanced Materials in Healthcare 247 The development and growth of bio-advance material for medical and dental applications has evolved through the three generations, each some- what temporally overlapping, yet each one having a distinct objective. 7.2.1 First Generation First generation bio-advance materials were comprised largely of the-shelf which is widely available industrial materials that were not developed specifically for medical use. They were selected because of the desirable combination of physical properties that is specific to the intended clinical use, and because they were bio-inert, and therefore they were considered biocompatible [10, 11]. 7.2.2 Second Generation The second generation of bio-advance materials includes the develop- ment of re-absorbable bio-advance materials, with the rates of degradation that could be tailored to the requirements of a desired application. Thus, the discrete interface between the implant site and the host tissue could be eliminated and desired in the long-term, because the foreign material would ultimately be degraded to soluble and non-toxic products by the host [12, 13]. 7.2.3 Third Generation In the third generation of bio-advance materials, the logical extension and diversity of the rapidly progressing state of the art has the goal in supporting and stimulating the regeneration of different functional tis- sue. Now, with advances in the regenerative medicine and tissue engi- neering and different technology it seems that true replacement with living tissue will be possible. Bio-advance materials play an important and significant role in the rapid field of tissue engineering and regenera- tive therapeutics [14, 15]. 7.3 Evaluation in Humans Federal law in the USA inquires that the study of new bio-advance materi- als in term of “drugs” in humans be conducted in accordance with certain stringent requirements. Scientifically valid results are not guaranteed sim- ply by conforming to government regulations, however, and the design,

248 Advanced Biomaterials and Biodevices implementation and execution of a good clinical trial requires the efforts of a clinician-scientist, clinical pharmacologist, a statistician, and frequently other professionals as well. The need for careful design, implementation and execution is based on three major factors inherent in the study of any therapeutic measures. 7.4 The Natural History of Diseases A good experimental design must be taken into account for the natu- ral history of the disease under study by evaluating and conducting a large enough population of subjects over a sufficient long period of time. Additional protection against the errors of interpretation caused by fluctuations in severity of the manifestations of disease is provided by utilizing a cross-over design, which mainly consists of alternate periods of administration of test drug, placebo preparation, and standard drug control, if any, in each subject. These sequences are systematically var- ied, so that different subsets of patients receive each and every possible sequences of treatment. 7.4.1 Risk Factors The concentrations of a blood component being monitored as a measure of the effect of the new agent which may be influenced by other diseases or other drugs. Attempt to avoid this hazard usually involves the cross- over technique, proper selection and assignment of patients to each of the respective groups. This requires that careful medical and pharmacological histories are obtained and that statistically valid method of randomization is used in assigning subjects to particular study groups. 7.4.2 Subject and Observer Bias Most patients tend to respond in a positive way to many emphasised and therapeutic intervention by interest, caring, enhancing and enthusiastic medical personnel. The manifestation of this phenomenon in the subject is the placebo response and may involve physiologic objective and bio- chemical changes as well as changes in subjective complaints which are associated with the disease. The placebo response is usually quantified and qualified by administration of an inert material, with exactly the same physical appearance, physical features, consistency, odour etc, as the active dosage form.

Anticipating Behaviour of Advanced Materials in Healthcare 249 7.4.3 Basic Process in Drug Drugs can be used for helping the body to reject an invading pathogenic organism and also for modifying some aspect of the metabolism of the body that is functioning abnormally. In the former case, drugs used are not toxic to the host but toxic to the pathogenic organism, and stimulation of the body’s normal processes for combating invaders may play a part. In the latter case, a modulation of a process as the requirement is more subtle to be approached. 7.5 Enzyme Enzymes are a specialised class of proteins, and, as already mentioned, they act as biocatalysts in the metabolic reactions to carry out various activities. They are responsible for most of the activities that take place within the living system and nature of life. More than two thousand enzymes have been identified and noticed. As such enzymes are important in maintain- ing life; they have many medical, commercial and technical uses also. For example, determining the level of particular and specific enzymes in blood gives a clue to the extent of damage of heart muscle after a heart attack. On the commercial side, enzymes have been used for centuries and decades as in fermentation to make alcoholic beverages. Considerable diversity of structure is seen in all the enzymes. Many enzymes are simple protein mol- ecules and biocatalyst too. This implies that the protein molecule in itself is the true catalyst depicting its nature. However, many enzymes require the presence of additional non-protein molecules for the full expression and specification of their catalytic function, which means that these enzymes are conjugated protein molecules that resemble its utility. In all cases, only by interacting with the molecules of the target organ or organism the drug can have biological activity. These molecules are pro- teins called receptors that transmit signals by interacting with messenger molecules such as hormones and neurotransmitters or the proteins such as enzymes that catalyse reactions essential for the functioning of the organ- ism,. The drug shows their effect by binding to either an active site or to a secondary site that influences the active site on the enzyme or receptor which hereby prevents access by the normal substrate ligand or provokes a signal where none was wanted. Enzymes are the biological catalysts which are prominently responsible for most chemical reactions in living organisms. Their splendid task is to initiate or accelerate reactions that could not take place, or is very slow, at the moderate temperature especially in organisms. They also makes the

250 Advanced Biomaterials and Biodevices reactions slow if necessary, or split them up into separate steps, to con- trol the heat evolution of exothermic reactions, which could lead to cell death. The most commonly used biocatalysts in biosensors are enzymes. The enzymes such as urease are highly specific for one compound [1]. Other enzymes are specific for a whole group of substrates. The structure of enzymes is mainly made up of a single polypeptide chain, where the active site is a separate molecule, embedded in the polypeptide backbone. Only specific molecules are allowed to access through the protein shell and the binding site, but not the active site itself. A reactive intermediate, the enzyme substrate complex (ES) is formed when the substrate (S) binds to the binding site of the enzyme (E). The enzyme substrate complex is con- verted to E and a product (P) by the active site. The interaction of the molecules of drug with the protein molecule is specifically measured in terms of the strength of inhibition of an enzyme reaction or the strength of the drug binding to a receptor. It is the quanti- fied as I50 or IC50, the concentration of inhibitor required reducing the rate of a reaction or the binding of a ligand available by one-half of the reaction. On the basis of simplest case of an enzyme reaction with one substrate, S; if the concentration of substrate is extremely higher than that of enzyme, the initial velocity V0 is given by: V0 Vmax [S] (7.1) K m [S] where Vmax is the maximum velocity, and Km the Michaelis constant of the substrate which equals the concentration of substrate that produces the half-maximal rate. V0 1 Vmax . It is also clear that rate will tend to acquire a maximum value 2 when [S] is very high. Significance of Vmax At Vmax, all the enzyme molecule have formed indigenous enzyme –sub- strate complex (ES) and are continuously catalysing the conversion of sub- strate into specific product. Thus at Vmax value the enzyme is fully saturated and organised. Vmax values can be used to compare the activity of various enzymes doing their function systematically, if they happen to catalyse the same reaction. Significance of Kmax As we have mentioned already and shown above, Kmax is equal to the con- centration of the substrate at V0 1 . Since at Vmax all the enzyme 2 Vmax

Anticipating Behaviour of Advanced Materials in Healthcare 251 molecules have formed generative enzyme-substrate complex, it therefore, follows that the concentration of substrate (Km) which is required to con- vert half of enzyme molecules to ES complex, and is a measure of the affin- ity of the various enzyme for substrate. A small value of Kmax signifies the high affinity of the various enzyme for the substrate, since a low concen- tration of substrate would be needed to explore and saturate the enzyme. Similarly, a large value for Kmax would indicate a relative and significant high concentration of substrate for saturating the enzyme, thus signifying a low affinity of the enzyme for its substrate to be synthesized. There are two types of conditions (a) The substrate and inhibitor complete for the same active site, but with different affinities for it in the most common form of competitive inhibi- tion, described by the following equation : E+S E S E+P (7.2) (7.3) E+P EI where EI is the enzyme-inhibitor complex and P is product. The equation enhancing the rate of reaction is: Vi Vmax [S] Km 1 [I] [S] (7.4) Ki where Ki is the dissociation constant of the enzyme-inhibitor complex and Vi is the initial velocity in the presence of inhibitor at concentration I. If the initial velocity is one-half V0, the inhibitor concentration I by defini- 1 tion is equal to the IC50 with Vi 2 V0 , so after rearrangement it becomes as followed: IC 50 Ki 1 [S] (7.5) Km where IC50 is dependent on substrate concentration. Only if the substrate concentration is low when compared with the Km does IC50 approximate to Ki. (b) In the case of non-competitive inhibitor E + I Kis EI E + I KiI ESI (7.6)

252 Advanced Biomaterials and Biodevices where K for the enzymes-substrate complex and KiS is the affinity of the inhibitor for enzyme. It is shown that: IC 50 K m [S] (7.7) K m [S] K iS K iI If the inhibitor binds equally in a good way to the enzyme like the enzyme- substrate complex, KiS = KiI = Ki and the equation simplifies to: IC50 = Ki (7.8) Alternatively, if as is often the case [S]〉〉 m and m/[S]〈〈 iS/KiI, the equa- tion reduce to: IC 50 K iS (7.9) K m [S] K iS K iI Again, if m/[S]〈〈 iS/KiI, then: IC50 = Ki (7.10) IC50 is independent of substrate concentration and equal to K in the case of uncompetitive inhibition, provided that [S]〉〉 m. It is not always conve- nient to maintain a substrate concentration that is extremely higher than the m because this may vary the effect of weaker inhibitors. 7.5.1 Enzyme Units and Concentrations The molar units and mass per unit volume are the identical manner with nonanalytic species through which enzyme concentration may be repre- sented. The percentage purity of the enzyme preparation, the molecular weight of the enzyme, and the mass and volume of the solution prepared are the information required for the calculation which is minimal. As nothing is noticed about the catalytic power of the solution prepared, the enzyme concentration is rarely represented in this way. The number of International Units (I.U.) per unit volume commonly depicts the concentration of an enzyme solution. The International Units is

Anticipating Behaviour of Advanced Materials in Healthcare 253 defined as the quantity of enzyme required to consume one micromole of substrate per minute at a given temperature and pH, under the conditions of substrate saturation: International Unit (I.U.) = 1-μmol substrate consumed/min (7.11) The concentrations are often given as milliunits (mU) or microunits (μU) per liter (L) or milliliter (mL) as 1 I.U is usually a very large quantity of enzyme. To convert these units to molar concentrations, it is important to know the turn over number, cat, of the particular enzyme which is used. For a simple one-substrate enzyme, cat = 2. This value is are the fundamental characteristic of an enzyme which indicates the maximum rate at which substrate are consumed, and is generally given in reciprocal units of sec- onds. (s–1) under the conditions of substrate saturation. = Vmax = Kcal [E]0 (mol . S . converted × s–1 × L–1 ) (7.12) Vmax is obtained from the I.U. of enzyme activity present in a given vol- ume (VT) of enzyme solution: Vmax = (10–6 mol/ mol) × (1.min/60.s ) × (I.U./VT) (7.13) and Vmax = (1.67 × 10–8) × (I.U./VT) (7.14) Eq. 7.14 has units of the molar concentration of an enzyme solution which is found by combinings Eqs. 7.14 and 7.12 [E]0 (1.67 × 10–8) = (I.U./VT) × (1/kcat) (7.15) while Eq. 7.15 is preferred for the calculation of [E0], which requires the value of kcat to be known for the enzyme of interest under the conditions required for its use.It is also assumed that this value is not always available in the literature form. Specific activity is defined as I.U. of enzyme activity with respect to unit weight ,under the conditions of T and pH), and is considered for the mea- surement of enzyme purity. The purity is greater with the higher specific activity of a enzyme preparation. The I.U per milligram determines the specific activity. The specific activity can be used to estimate through kcat if

254 Advanced Biomaterials and Biodevices the molecular weight of an enzyme is known, and if a given preparation is assumed to be 100 % pure,Eq. 7.16: kcat (1.67 × 10–5) × (SA) × (MW) (7.16) where the constant has been calculated for specific activity (in I.U./mg), molecular weight [in daltons (Da)], and kcat (in s-1). Katal can be defined as an equivalent International system of units because of the nonstandard units associated with the I.U. system for defining enzyme concentrations, an equivalent International system of units has been defined, and is called the katal. One katal (kat) of enzyme activity is the quantity that will con- sume 1 mol substrate/s; 1 μkat = 601.U. 7.5.2 Assay of Enzyme Activity All enzymes so far well known are proteins, yet the specific tests for pro- tein cannot be used for the detection,qualification and quantification of enzymes. Evidently such test cannot make distinction and clasification between enzymes and non-enzymes proteins and between various other enzymes. The amount of enzymes in a given solution,substrate formation or tissue extract can be conveniently and eventually measured or assayed quantitatively by using techiques and tools that can measure their ability and capability to convert the substrate into product. One can detect and identify the presence of an enzyme by using a spe- cific, generative quantification procedure either for the substrate or for the product. Under optimal and formal conditions ,the velocity of enzyme cat- alysed reaction is directly proportional to the concentration of the enzyme. One can therefore easily determine and investigate the concentration of enzyme from the velocity of the disappearance of the substrate or that of the formation of product under optimal conditions. From Figure 7.2 dur- ing the enzyme action, the concentration of substrate decreases, while that of product increases. This quantitative estimation of enzyme activity is generally called as the assay of an enzyme. Activity of enxyme is efficiently represented in enzyme units.According to international convention one unit of enzyme activity is merely defined as that amount which especially transforms one micro mole substrate into product in one minute under optimal conditions. Enzyme assays are dormantly carried out at their opti- mal pH, specific temperature and with a near saturating concentration of substrates. Concentration of different substrates or specified products can be deter- mined and verified using methods such as fluorometric spectrophotometric,

Substrate concentrationAnticipating Behaviour of Advanced Materials in Healthcare 255 Product Substrate Reaction time Figure 7.2 Changes in the substrate concentration vs. reaction time. colorimetric or isotopic labelling procedures. The choice of the proce- dure is determined, identified and depicted by the nature of the substrate or the product. For example, spectrophotometric assays are used when the substrate or product was absorbing light of some specific wavelength. Fluoresecence or radioactive measurements determines the highly sensitive assayy procedure. In many instances substates and casese modified substrateby introduc- ing specific groups for the estimationed of their products. Such substrate modification should not, however, result in the decrease in the rate of its transformation to product by the enzyme. For instance the introduction of p-nitrophenyl phosphate compound in substrate adds for making the sub- strate useful for the assay of phosphatases. The product of enzyme action is an intence, ionised form having p-nitrophenylate ion which is of yellow colour and the intensity of color indicates enormously the activity of the enzyme assay. Enzyme concentrations is generally expressed as activity per unit vol- ume and must specify the temperature at which activity is measured. The simple enzyme-catalyzed reactions consist of at least three stages, which are temperature dependent i.e the formation of the enzyme-substrate com- plex, the conversion of this complex to the enzyme-product complex, and the dissociation of the enzyme-product complex. A combination of the effects produced at each stage is the overall effect of temperature on reaction rates is. In general,the increase in T with 10 °C will leads to double the rate of an enzymatic reaction.To ensure the repro- ducible measurement of reaction rates the temperature control at 0.1 °C is

256 Advanced Biomaterials and Biodevices necessary. Temperatures < 40 °C are generally enhanced to avoid protein denaturation. Products are formed during enzymatic reactions where the substrates are consumed. These compounds can be guided with the suitable trans- ducers. In the case of glucose oxidase, there are compounds such as O2 and H2O2, which are easily detected. Some enzymes have additional active areas referred to as co-factors, e.g. NADH. These co-factors are used for measuring enzyme activity [1]. For the catalytic activity many enzymes also requires metal ions. The enzyme in biosensors has two important applications. It can either be used as markers in biosensors affinity or as catalytic biosensors, such as immunosensors and DNA sensors. The concentration of enzyme (E) is constant and the substrate concentration is much smaller in the cata- lytic enzyme sensors. When the enzymes are used for labelling antibodies or DNA strands, the enzyme concentration (E) is the only limiting fac- tor and the substrate must be used in excess amount. Since the enzymes have a capability to convert hundreds of substrate molecules per second, they behave as highly efficient chemical amplifiers for the detection of other molecules [2]. Various enzymes are used as labels, such as horse- radish peroxidise (HRP), glucose oxidase and alkaline phosphatase (AP). Spectrophotometry and electrochemistry are used for detecting the prod- ucts of these enzymes. The products for luminal or luciferin luminescence, allowing optical detection is delivered through enzyme such as peroxidise and luciferase. Enzymes are very sensitive to temperature changes. Increasing in the temperatures, increase the rate of reaction, but at elevated temperatures, the protein structure (tertiary structure) denatures, mostly irreversible, by leaving the enzyme inactive. For most enzymes, the critical tempera- ture stays between 40 °C and 50 °C, however above 100 °C few enzymes shows high thermal stability. Enzymes consist of amino acids due to which it is sensitive to pH. Various species inhibits the enzymes reaction. Inhibition is reversible, which allows the enzyme to regain full activity after dissociation from the inhibitor. The active sites are being blocked competitively and alter the enzyme activity by other mechanisms. Other inhibitors inhibit the enzyme by deactivating it irreversibly. These irre- versible inhibitors work in different way, for example by blocking the binding site, which react with the central metal ion or the denaturing the enzyme. Enzyme inhibition sensors have been reported for the detection of toxic compounds and heavy metal ions and it is commonly based on the selective inhibition of enzymes [3].

Anticipating Behaviour of Advanced Materials in Healthcare 257 During the last few decade, great attention as an alternative synthetic route with lower environmental impact is due to enzymatic polymeriza- tion of aniline as compared with classical chemical oxidations because it is carried out under milder conditions and reduces the oxidation of by- products to water to treat extent [4-6]. The development of the template- assisted enzymatic polymerization approach was the great advancement in the enzymatic polymerization of aniline [7]. This advancement com- prises the use of an anionic polymeric template to promote the head-to-tail coupling of aniline radicals in order to obtain a water-soluble complex of electrically conductive PANI. The template-assisted polymerization of ani- line is also performed enzymatically in a two-phase system [8], either by chemical oxidation using both synthetic [9] and biological polymers [10] yielding water-soluble poly-electrolyte complexes. However, in addition to of the oxidation method employed in this approach, the separation of the PANI from the polyanion is embedded by the high degree of complexity between those two. Noble metal nanostructures for example gold have gained special interest because of the conductivity, optical properties and the biocom- patibility [11, 12]. Gold nanoparticles were one of the most widely used nanoparticles in the past few years, and have highly been used for immo- bilization of enzymes for the fabrication of biosensors [13, 14]. These nanoparticles act as tiny conduction centres by facilitating the transfer of electrons; resembling the works that have shown enzymes to maintain their enzymatic and electro-chemical activity when immobilized the on gold nanoparticles [15.] Enzyme immobilization has recently gained and caught much inter- est because the quantity and quality of the desired, registered enzyme is often inadequate due to high cost, enzyme instability and the limited potential for the recovery of enzyme [16, 17]. The magnetic nanopar- ticles have recently attracted and invited much attention because of their excellent and extraordinary physical and chemical properties as compared to those of conventional bulk materials [18-22]. Ferrites are a group of important magnetic materials that resembles many efficient technological applications including those in bio sensing [23, 24]. Nickel ferrite (NiFe2O4) is known to be an interesting, voluntary ferrite, due to its technologically important soft inverse spinel structure. Besides this, the nickel ferrite contains improved and investigated ferromagnetic properties that originate specifically from magnetic moment of antipar- allel spins between Fe3+ ions at tetrahedral sites and Ni2+ ions at octahe- dral sites [25]; resulting in lower eddy of current loss, high conductivity,

258 Advanced Biomaterials and Biodevices good catalytic behaviour ,high electrochemical stability, abundance in nature and simplicity [26]. Additionally, nickel ferrite crystallizes as a spinal structure and exhibits turn able conducting behaviour. Because of their large surface to volume ratio, high surface reaction activity, invigi- lating properties, high catalytic efficiency and strong adsorption abil- ity, nano nickel ferrites can perhaps be utilized efficiently for biosensor applications as such [27, 28]. 7.5.3 Enzymes in Health Sciences In the present era, enzymes are synchronously used to diagnose and to treat various diseases. Enzymology is a chief essential requirement of the day to day life of modern clinicians and surgeon. The diagnostic value of certain specific enzymes arises from their differential distribution and distinction between cells of other tissues and the blood plasma. For examples plasma contain the enzymes involved in blood coagulation, very exclusively. On the other hand, many other enzymes required for the pro- cess are present in much higher concentrations in the tissue cells, than in the blood. These are realised, combined and immobilised into the blood and various biological fluids only when there is routine destruction and verification of the cells. Their normal levels in plasma are insignificant, approximately being more than one million times lower than their con- centration in the desired cells. In case of cell destruction or injury influ- ence by such cases as a damaged heart or uncontrolled growth of cancer cells, tumour, damage tissue, the plasma levels of these cellular enzymes are elevated significantly. These changes and verification in plasma con- centration of particular enzymes are estimated and enhanced by the clini- cians or surgeon, and are used not only to detect damaged cell but also to suggest the site of cell damage and tissue repair. A clue to the extent of cellular damage is given degree of elevation of plasma concentrations of these enzymes. These enzyme assays have become a critical diagnostic tool, scientific technique in the detection of pancreas, heart, liver, skeletal muscles, bone and malignant diseases. In Table 7.1 a list of some enzymes, clinical assay of which are used for detecting particular diseased states, cell damage or tissue repairs are given as such. Hence it has been found that enzymes have a diagnostic value and importance in the field of medical science. Numerous of enzyme assays have been employed for confirming, locating even indicating the sever- ity of the diseases in human being. Enzymes are used specific reagents in the laboratory and are basically used treating various conditions of diseases.

Anticipating Behaviour of Advanced Materials in Healthcare 259 Table 7.1 Enzymes assayed in medical diagnostics. Enzyme Used in determination of Lactate dehydrogenase (LDH) skeletal muscle or heart damage Alkaline phosphatise Liver and bone disease Serum glutamate oxaloacetate Liver and heart disease transaminase (SGOT) Creatine phosphokinase (CK) Muscle and myocardial infarction disease Acid phosphatase Cancer of the prostate α-Amylase pancreatitis 7.6 Biosensor A biosensor is described as a compact, composed analytical device, sys- tematically incorporating a biological or biometric sensing element or sub- stance, either closely connected to, or integrated highly within a transducer system in Figure 7.3. The principle of detection is the specific binding and ligands formation of the analyte which is of interest to the complimentary bio recognition element immobilised on a suitable support medium or condition for development. The specific interaction and co-relation results in a change in one or more physico-chemical properties such as heat trans- fer, uptake or release of gases, pH change, electron transfer, mass change or specific ions which are been detected, investigated and may be measured by the transducer system. The usual and specified aim and objective is to produce an electronic signal which is directly proportional in magnitude or frequency to the concentration of a specific analyte or group of analytes to which the bio sensing element binds enormously [29, 30]. The history of biosensor began very early with glucose monitor- ing, dated as far as 1956 through the prominent and amazing work of Professor Leland C. Clark. He used an enzyme as called glucose oxidase in a dialysis membrane over an oxygen probe and finally the device was called as enzyme electrode [31]. The determination of glucose was done by enzyme electrode. Updike et al. [32] developed a specific enzyme by using electrochemical procedure to design a well equipped model that uses glucose oxidase immobilised on a gel for measuring and identifying the concentration of glucose in biological solutions and in the tissues

260 Advanced Biomaterials and Biodevices Bioreceptor Immobilisation layer B Electrode i o Current r Ae T nc r Light ao a lg n Signal yn s ti d et u Impedance i c o e n r Mass Biosenser Figure 7.3 A schematic representation of a biosensor adapted from Ref. [29]. in vitro. The term biosensor has eventually emerged from bio selective sensor [33]. Biosensors are classified on the basis of bio receptor, transduction meth- ods, and sometimes the bio-recognition principle. Bio-receptor provides the key factor to specificity for biosensor technology today. They are highly responsible for the bio-recognition event which may include reduction of the substrate or catalytic oxidation that leaded to or binding of the analyte of interest by the biosensor for the measurement. The common bio-recep- tors used are enzymes [34-38], antibody [39, 40], DNA [41, 42], whole cell [43], and of recent aptamers in biosensor. Transduction methods used in biosensor includes the following surface plasma resonance (SPR), elec- trochemical, thermal, piezoelectric, optical, Quartz crystal microbalance (QCM) [44] and cantilever [45-47]. Electrochemical method of transduc- tion constitutes and comprises more than half of the literature portion on biosensor [33]. The two broad classification of biosensors based on bio-recognition principle are affinity biosensors typical of antibody or DNA and cata- lytic biosensors typically which are of enzyme bio-receptors and aptam- ers. Therefore, a biosensor with an intrinsic electrochemical transduction method and DNA as a bio-receptor is called DNA biosensor which is based on bio-receptor or affinity biosensor likely to be based on bio-recognition principle or electrochemical DNA biosensor such as both the bio-recep- tor and transducer are the name used in this writing form of literature. Other biosensors can be named generally as immune-sensor or antibody

Anticipating Behaviour of Advanced Materials in Healthcare 261 bio-receptor, enzyme biosensor and glucose oxidase sensor that is using the specific name of the enzyme biomaterial. DNA biosensor exploits the specificity, versatility, excellent selectivity and reactivity of deoxyribonucleic acid (DNA) in a very co-relative way. It converts the Watson–Crick base pair recognition event into a readable analytical signal by its specific trends. A basic DNA biosensor is designed and assisted by the immobilisation of a single stranded oligonucleotide (probe) on a surface of transducer to recognize its complementary (target) DNA sequences via hybridisation or using the DNA probe to detect other analytes such carcinogens, drugs, mutagenic pollutants, etc. with binding affinities for the specific structure of DNA sequencing. The DNA duplex which is formed on the surface of electrode is known as a hybrid [48]. With the help of electrochemical transducer, these events are converted into an analytical signal and are referred to as Electrochemical DNA Biosensors [49]. Millan and Mikkelesen in 1993 was the first to give the concept of electrochemical DNA biosensors [50] by using tris(2,2’- bipyridyl) cobalt(III),Co(bpy)33+ as an electro-active intercalator. The use of nucleic acids as a tool in the recognition and monitoring of many compounds of analytical interest has been increased significantly in few year. The enzymes counterpart which is a catalyst has the class of affinity biosensor belonging to EBD. Electrochemical biosensors are becoming more preferable than other transduction method because of such aspect: A. Low cost and direct signal measurement: Since the ‘natu- ral’ and realistic signal during hybridisation is electronic in nature, the EDB lends itself to direct measurement using the low cost electrochemical equipment in biosensor. B. Ease of miniaturisation: The ease and possibilities and effi- ciency of producing different kinds of electrodes through advances microelectronics that allows downscale of EDB to smaller size – miniaturisation and finally it’s of great importance. Electrochemical impedance spectroscopy (EIS) has provided the detailed information on interfacial kinetics as it is related to the capaci- tance and electron transfer resistance changes or changes in electrical properties resembling at the modified surface of electrode. Thus EIS is more suitable and stable for the affinity biosensor. EIS signals are some- times used as label free EDSs are called impedimetric biosensors [51]. Modelling of DNA or DNA hybridisation kinetics is being studied with

262 Advanced Biomaterials and Biodevices the help of EIS [52] which demonstrates that the technique is sensitive to the interfacial electrical changes that accompany DNA bio-recognition event. A ratio of the applied voltage to its current response is given as impedance in the EIS. It is found that there is usually a phase shift ϕ in between current (or voltage) response and the voltage (or current) applied. Faradaic impedance and the charge transfer resistance, Rct, is termed due to interfacial electrochemistry involves an electroactive spe- cie and is mostly used as the reporting impedance element in biosensor. On the other hand, if the interfacial electrochemistry does not involve electro-active specie, it is called as non faradic impedance. There are two ways that is (a) non faradic measurement of impedance or capacitance at single frequency (b) faradic measurement of the charge transfer resis- tance Rct over a wide frequency range in the EIS measurement in EDB. EIS as a characterised tool is used in few biosensor design at different stages but do not report the final DNA target responses [53, 54]. Such biosensor can generally not be termed as impedimetric. The main and systematic types of immune-sensor detection devices are as followed: optical, electrochemical (potentiometric, amperometric or conductometric/capacitive) and microgravimetric. All such types can either be described and emphasized as direct (non- labelled) or as indirect (labelled) immune-sensors. The direct sensors are capable of significantly detecting the physical changes in the immune complex formation, but the indirect sensors uses only signal-generating labels which allow more sensi- tive and versatile detection modes when incorporated into the intervene complex. There is a different variety of identified labels which is applied in indirect immune-sensors as such. The most commonly enzyme labels include enzymes such as catalase (EC1.11.1.6), peroxidase (EC 1.11.1.7), alkaline phosphatase (aP), glucose oxidase (EC 1.1.3.4) or lucifarase (EC 1.13.12.7) which is generally due to their excellent stability and high turn- over number. Electro-active compounds namely ferrocene or In2+ salts, and a series of fluorescent labels such Cy5, asrhodamine, ruthenium diamine complexes, fluorescein and phosphorescent porpyhrin dyes have also been used in the detection [55, 56]. A quantitative polypyrrole based and stable potentiometric immune- sensor that is provided by broad-spectrum assay capability was designed and estimated by Purvis et al. [57]. The biosensor detected an enzyme labelled by immuno-complexes were formed at the surface of a polypyrrole is coated with screen printed with gold electrode. Such detection mode was mediated by a secondary reaction that has produced charged products at the end. A shift in potential was measured at the sensor surface, which has been caused by local changes in properties such as pH, ionic strength,

Anticipating Behaviour of Advanced Materials in Healthcare 263 redox state. The magnitude and the strength of the difference in potential were eventually related to the concentration of the receptor-target complex which was formed. The bio-sensor was applied for detecting hepatitis B surface antigen, Digoxin, Troponin I and tumour necrosis factor in general. This technology and tools used was found to be ultrasensitive, rapid and reproducible and had a wide dynamic range. Feng et al. [58], has reported a potentiometric immune-sensor for detecting the immunoglobin G (IgG). The immune-sensor was commonly based on covalent immobilization of specific anti-immunoglobin G on the silver (Ag) electrode. Before and after the antigen-antibody reaction the change in electric potential was specific noticed which were said to be based on change in the detection. Immune- sensor for detection α-2 interferon which was based on pH sensitive field effect the transistor (pH FET) was fabricated by Sergeyeva et al. [59]. The immobilizing α-2 interferon on the gate of pH FET, fabricated the sens- ing element. The interaction of anti-interferon antibodies which has been labelled with β-lactamase and with interferon- pH-FET (in the presence of specific enzyme substrate) leads to a local pH-change at the surface of transducer and finally produces an electrochemical signal which was pro- portional to the conjugate concentration of the complex. The conventional ELISA assays, accounted for analytical data obtained. Amperometric immune-sensors are designed to measure a current flow indigenously generated by an electrochemical reaction at constant voltage every time. There are very less applications available for direct sensing as such, most (protein) of analytes are not intrinsically and superficially able to act as redox couples in an electrochemical reaction at constant volt- age. Therefore, electrochemically active labels which is directly acting as products of an enzyme reaction) are always needed for the electrochemical reaction of the analyte at the sensing electrode in assay. An antiseptic and disposable amperometric immune-migration sensor for the detecting the triazine pesticides in real samples and specimen using the monoclonal antibodies against atrazine and tertbutylazine as bio-rec- ognition element in the reaction was eventually fabricated by Bäumner and Schmid [60]. Generation and amplification of the signal was greatly achieved and enhanced by using hapten-tagged liposomes entrapping the ascorbic acid as a marker molecule. An amperometric immune-sensor used for the detection of red blood cells which was based on a non-compet- itive sandwich assay and flow injection analysis (FIA) was developed and enhanced by Lu et al. [61]. Specific IgM and non specific IgM were chemi- cally immobilised on two specific electrodes to form the blank electrodes and sensing electrodes, respectively. It has been employed and assisted for the determination of the binding of specific blood cells and non-specific

264 Advanced Biomaterials and Biodevices adsorption. In one determination of specific blood cell, HRP-labelled anti- blood group an IgM was utilized in the ELISA assay. Based on electrode working on screen-printed graphite, an immunosensor allowed for the rapid estimation and calculation of fatty acid-binding protein (FABP) in neat plasma samples was designed by Schreiber et al. [62]. The antibodies that had been captured were bound to the electrode surface by adsorp- tion, conduction and trapped FABP from the plasma samples. A second monoclonal antibody had completed the sandwich by conjugating with alkaline phosphatase. The enzyme has been converted to p-aminophenol from p-aminophenyl phosphate which was then detected amperometri- cally. Ivnitski and Rishpon [63], developed a one-step that is separation of free enzyme immune-sensor in the reaction. The bio-sensor consisted of an antibody electrode which is very important for reaction. The immune- sensor combines enzyme channelling immunoassay, cyclic regeneration of an enzyme (peroxidase) substrate at the polymer (polyethylenenimine)/ electrode interface, accumulation of redox mediators, for controlling of the hydrodynamic conditions at the interface of the antibody electrode in the electrochemical reaction. The immunological reactions were monitored electrochemically every time and at every moment. Biosensors reported for urea detection are specifically based on urease (Ur) which is often present in most biological systems as known [64-70]. Ur catalyzes the decomposition of urea into ammonium ions (NH4+) and hydrogen bicarbonate. NH4+ ions are known to be unstable and can be easily disperse in the environment eventually. Keeping the condition in mind, glutamate dehydrogenase (GLDH) along with Ur has been specifi- cally utilized for urea detection as GLDH immediately catalyzes the reac- tion between α- ketoglutarate (α-KG) ,NH4+ and nicotinamide adenine di-nucleotide (NADH) to produce L-glutamate [68-70] and NAD+. Metal oxide nanoparticles-chitosan (CH) which is on based hybrid composites has attracted much interest for the development of a desired biosensor in biological system [69-71]. Metal oxide nanoparticles such as iron oxide (Fe3O4) [72-74], zinc oxide (ZnO) [75, 76], cerium oxide (CeO2) [77, 78], etc. have been suggested as promising matrices for desired bio- molecules to be immobilzed. These nanomaterials exhibit and enhances large surface to volume ratio, high catalytic efficiency, high surface reac- tion activity and strong adsorption ability that can be helpful to obtain improved sensitivity, specificity and stability of a biosensor. Moreover, nanoparticles have a unique ability to promote and establish fast electron transfer between the active site of an enzyme and electrode. Among vari- ous metal oxide nanoparticles noted such as Fe3O4 nanoparticles due to strong super paramagnetic behaviour, biocompatibility and low toxicity

Anticipating Behaviour of Advanced Materials in Healthcare 265 have been considered very efficiently as interesting for immobilization of desired biomolecules [79-81]. Immobilization of bioactive molecules onto surface charged super paramagnetic nanoparticles (size < 25 nm) is of special interest noticed for bio-sensor, since magnetic behaviour of these bioconjugates results in improved delivery and recovery of biomolecules for desired bio-sensing applications and utility [72, 73, 82]. Besides all these existing problem of aggregation and rapid biodegradation of Fe3O4 nanoparticles onto a given amount of matrix containing biomolecules will definitely be helpful in overcoming for modifying these nanoparticles using CH by preparing hybrid nano biocomposite [82-89]. Fe3O4, the metal oxide nanoparticles have been considered very inter- esting for immobilization of desired biomolecules (GOx) because of low toxicity, strong superparamagnetic property, biocompatibility, etc. [90-92]. Immobilization of bioactive molecules on the surface of magnetic nanopar- ticles is very significant, because magnetic behavior of these bioconjugates is likely to improve delivery and recovery of biomolecules for biomedi- cal applications [90]. With the recent advances in clinical diagnostics that have been stimulately demanded for high sensitive and precise analytical methods for estimation of desired analytes including glucose were as such significantly important. It may be noted that the existing and upcoming problem of estimation, aggregation, decomposition and rapid biodegrada- tion of Fe3O4 nanoparticles onto a desired matrix containing GOx could be overcome by modifying these interesting magnetic nanoparticles using materials such as conducting polymers, inorganic semiconductors and biopolymers (polysaccharides), etc. Sole et al. and Li et al. have reported Fe3O4-based magnetic immuno- sensor for flowing injection and piezoelectric immune-sensor, respectively for detection of immunoglobulin (IgG) [93, 94]. Cao et al. On the basis of heme-proteins immobilized onto Fe3O4 nanoparticles have fabricated electrochemical bio-sensor [92]. Cao et al. have studied direct electron transfer between the molecule of haemoglobin and pyrolytic graphite elec- trodes enhanced and appreciated by Fe3O4 nanoparticles in the layer to layer self-assembly films where applicable [95]. Rossi et al. have studied glucose oxidase – Fe3O4 nanoparticles bio-conjugate for sensing of glucose [72]. Kouassi et al. have investigated and inferred that attachment of ChOx with carbodiimide activated Fe3O4 nanoparticles and its response to cho- lesterol [96]. Li et al. have fabricated and invented a renewable potentio- metric immune-sensor which is based on immobilized anti-IgG into Fe3O4 nanoparticles [97].Wei et al. have utilized Fe3O4 nanoparticles both for glu- cose detection and H2O2 [98]. Chumming et al. had electrochemically syn- thesized the Fe3O4 a prusian blue nanoparticles with core-shell structure

266 Advanced Biomaterials and Biodevices and studied its electrocatalytic reduction towards H2O2 [99]. And the nanobiocomposite of Fe3O4 nanoparticles and chitosan has recently been reported for detecting substances such as mycotoxin, glucose, urea, and phenolic compounds [82, 89, 100]. In spite of these interesting develop- ments, nanostructured Fe3O4 film has not as yet been utilized to fabricate the electrochemical cholesterol biosensor in a systematic way. A promising and influencing material for metal support is the Carbon nanotubes (CNTs) because of its good electrical conductivity, tubular structure and high chemical stability, [101, 102]. The applications of CNTs in amperometric biosensors as a catalyst support or an electrode material have been already reported in the literature in normal sense [103-106]. Several methods and technology have been developed for preparation of Pt nanoparticles on CNTs. The strategies of synthesis of Pt nanoparticles can be generally and significantly classified as electrochemical methods [107] and solution-phase reduction [108]. One-step for electrochemical method is generally considered as an effective procedure when compared with others which are time-consuming where impurity might be involved and found during the preparation [109]. CNTs have been used for modifying electrodes that catalyze and hydro- lysed the electrochemical reaction of some biomolecules, such as cyto- chrome, NADH, dopamine, etc. [110-114]. Jason et al. [115] have shown through his work that the catalytic activi- ties of the immobilized β-lactamase I on or in carbon nanotubes had no drastic conformational change in the field. The carbon nanotubes radically appeared to act as a gracious host in its ability to encapsulate the protein molecules within an environment, which offered some protection to the host. Jason et al. [116] have also reported and emphasised for the applica- tion of carbon nanotubes as an electrode material. Redox proteins such as azurin and cytochrome c were immobilized on and within carbon nano- tubes and gave well-behaved, reproducible voltammetric responses for every process taking place. The performance of carbon nanotubes paste electrode (CNTs-PE) pre- pared by deficit dispersion of multi-wall carbon nanotubes (MWCNTs) within mineral oil was described and emphasized [117]. The resulting elec- trode showed an excellent electrocatalytic activity toward chemical sub- stances such as uric acid, ascorbic acid and dopamine. Gold nanomaterials are always ready to offer a substantial increase of a bio- compatible platform and background for functionalization in biosensing and available surface area or therapeutic applications [118-120]. Gold nanorods, anisotropic and elongated nano-particles are always of good biocompatibility, simple preparation and high stability, versatility which has been widely used

Anticipating Behaviour of Advanced Materials in Healthcare 267 as the immobilization matrix for electrochemical biosensors [121, 122] and bio-electrocatalysis [123, 124]. In addition, the surface chemistry of AuNRs is versatile and mobile, allowing the linking of various bio functional groups, like sugars, nucleic acids, amphiphilic polymers and proteins, through strong Au-S or Au-N bonding or through physical adsorption and other properties [125, 126]. Therefore, AuNRs can improve by adhering ability of the com- posite film on surface of electrode and provide an ideal matrix for enzyme immobilization and biosensor fabrication [123, 127]. Polyaniline (PANI), a conducting polymer with chemical stabil- ity, biocompatibility and good electrochemical activity, has been widely, repository used in the DNA biosensor [128, 129]. Compared with a gold nanoparticle or carbon nanotube-based DNA biosensor, the conducting PANI-based DNA biosensor has some advantages: tunable conductivity, low-temperature synthesis, and there is no need for purification, end open- ing, or catalytic deposition processing. Unfortunately, PANI is usually less favourable condition as the element in biosensor construction because it has relative low conductivity than the carbon nanotube as well as their non oriented nanofibre morphology which leads to low detection sensitivity. However, when a PANI nanotube array of well-organized orientation is fabricated and interchanged on electrodes using the well-designed synthe- sis approach, achievement of enhanced detection sensitivity, which is very similar to the gold nanoparticle- and carbon nanotube-based detecting system for DNA hybridization. 7.7 Platinum Material Used in Medicine The significant similarity between the co-ordination chemistry of Pd(II) and Pt(II) compounds has advocated specific studies of Pd(II) complexes as an antitumor drugs, whereas the higher liability and affinity in ligand exchange at Pd centre (105-fold vs Pt) causes for rapid hydrolysis processes which leads to the dissociation of complex of Pd(II) and the reactive species that is unable to reach their pharmacological targets has been formed [130]. These prob- lems could be overcome and resolved by using the chelating ligands and bulky heterocyclic. A very promising and enhancing antitumor characteristics have been shown by a number of palladium complexes with aromatic N-and N,N-containing ligands[131, 132]. Recent studies when experimented dem- onstrated that some Pd(II) complexes exhibit a noticeable in vitro cytotoxic activity, comparable to standard platinum- based drugs, carboplatin, cispla- tin and oxaliplatin [133]. Metal complexes has received a lot of attentions and attraction that contain Salen type ligands due to their versatility and

268 Advanced Biomaterials and Biodevices wide range of complexing ability, capability and are also the important fac- tor in the development of catalysis, magnetism, inorganic biochemistry and medical imaging, clinical application etc. [134-137]. Study related to some of the salen complexes has also revealed interesting and amazing antioxidant and antitumor properties [138]. 7.8 Antibody In general sense the use of highly specific antibodies is specifically well known. Immunoassay, immune-sensor and immune-affinity columns are the most important analytical applications of antibodies. Immunochemical techniques are found to be highly sensitive, very selective, simple and inexpensive [139]. The ability of antibodies is to form complexes with corresponding antigens at the base for immunochemical techniques. The interactions between each molecule are highly specific and it leads to very selective immunoassays. The greater sensitivity is brought about by the extremely high affinity of the anti- body antigen interaction. The tailoring of the biomolecules for the analyte and matrix requirements, within certain limits is allowed by new antibody technologies such as the production of antibody fragments or recombinant antibodies. The amino acids in the constant regions of both light and heavy chains are found virtually identical among the various antibodies. The amino acids of variable region are different in among the hundreds of thousands of different antibodies. A highly specific, three dimensional structure which gives the specificity for a particular antigen is formed from the variable regions of light and heavy chain combine to form. In contrast words it can relatively recognise only the antigen of its own kind. The arms of antibody are identical referring that a single antibody molecule may combine with the two antigen molecules. The two fragments are hydrolised when antibody is treated with papain, a proteolytic enzyme. The first one is known as Fab frag- ment or ‘fragment-antigen-binding’ which is a protein and is able to com- bine with the antigenic determinant. The second fragment is Fc fragment or ‘fragment and to be crystallised which combines with phagocyte and neu- tralises the viral receptor sites. 7.8.1 Antibodies-Production and Properties 7.8.1.1 The Immune System Antibodies are generally produced by mammals as a part of an immune response of the host to foreign intruders such as micro-organisms, viruses,

Anticipating Behaviour of Advanced Materials in Healthcare 269 bacteria and parasites [140-142]. The immune system acts as recogniser and eliminates the pathogens. The first state of defence is innate immunity which is a non-specific defence reaction. Most important for analytical sci- ence is the second state of defence i.e adaptive immunity which is directed specifically against the intruder and is mediated by cells called lympho- cytes. The lymphocytes secrete proteins which are antibodies that specifi- cally bind to the foreign species (antigen) and have specific cell surface receptor. Among them at least 109 lymphocytes guarantee a quick adap- tive immune response. These cells are omnipresent in the body doing its specific function, but accumulate in organs such as the spleen and lymph nodes. There are many different types of lymphocytes, but only three main classes among those have surface receptors specific for the antigen. The most important lymphocytes for the analytical chemist that is B cell which secrete antibodies. Cytotoxic T cells bind to the antigen through surface receptors and lyse the antigen. Helper T cells generally control and config- ure B cells and T cells specifically. A single cell has only one type of recep- tor for its capability. Mutation and recombination of cells can generate 108 different surface receptors for the cells and therefore 108 antibodies with different receptors (binding sites). A process called tolerance eliminates the Lymphocytes that produce antibodies against molecules of the host system. An autoimmune disease is due to a failure of tolerance system. On the first exposure to a foreign molecule, the immune response becomes relatively slow. On the second exposure, the lymphocytes produced during the first exposure recognise the antigen early and then react in a fast and strong immune reaction (antibody production). The mechanism is as such known as immunological memory. During the immune response, lym- phocytes are produced and after removal of the antigen a few lymphocyte remain in the host system as well (memory). Thus the process of introduc- ing a foreign species (immunogen) into the organism of the host animal is termed as immunisation. 7.8.1.2 Antibody Structure It has been evidently found that antibodies are a large family of glycopro- teins. They are highly classified into five classes namely IgG, IgM, IgA, IgE and IgD. Immunoglobin G (IgG) is the most abundant immunoglobin spe- cies are found in serum and is also commonly used antibody in sensor applications. The IgG molecule that consists of one Y shaped unit which is structural features and is easiest explained (Figure 7.4). The other immu- noglobin classes are also characteristically based on these Y shaped units. An IgG molecule consist of four polypeptide chains, two identical heavy

270 Advanced Biomaterials and Biodevices NH2 NH2 Antigen binding site CarbohIyndtreartechbairinddgiessulphideL Chain Fab fragment Complimentarily determining region Fc fragment H Chain COOH COOH Figure 7.4 Important attributes of an antibody adapted from Ref. [143] (H) chains and two identical light (L) chains (Figure 7.4) [143]. The length of the two chains is derived 450 amino acids for the H-chain (~55,000 Dalton) and 212 amino acids for the L-chain (~25,000 Dalton). The two identical H-chains are connected to each other via disulphide bridges. The connection between the L-chain and the H-chain also consists of disul- phide bonds. Since all these bonds connect two chains they are typically named as interchain disulphide bridges. Both the L chain and H chian also have interchain disulphide bridges. The globular structure of the pro- tein that is responsible for the name immunoglobulin, is a result of these interchain disulphide bonds. The L-chain into two sub domains and the H chain is divided into four sub domain. These sub domains are classified on the basis of the variability of their amino acid sequence, into constant (C) and variable (V) regions. The two sub domains of the L-chain are one C region CL and one V region VL. The sub domains of the H-chain are the three C regions, generally CH1, CH2, CH3 and one V region, VH. The base of the Y shape is called as the Fc fragment (fragment that crystallises) and is said to be formed by the association of the two CH2 and the two CH3 domains at each reason. Each arm of the Y shape is called a Fab (fragment containing antigen binding site) and is formed by recombination of dif- ferent CH1 with CL and VH with VL. The small domain between CH1 and CH2 is called the hinge region that is allowing the lateral and rotational movement of the Fab fragments. Furthermore, the Fc fragment contains the terminal carboxyl group is allocated at the end of the Fab fragment;

Anticipating Behaviour of Advanced Materials in Healthcare 271 the terminal amino group of amino acid is sequenced. For the antibody antigen interaction however, most of the IgG fragments are not as such important. Each arm contains one binding site which is located within the V and L domains. In the variable regions, amino acid sequences can vary from one antibody to another by allowing the specific adaptation to certain antigens. The exact regions within these variable regions having very high amino acid variability are called hypervariable regions, and also known as complimentary determining regions (CDRs). Three CDRs are integrated into the L-chain and three are integrated into the H-chain, resulting in six CDRs for each arm. The variability that is created by the CDRs of each Fab fragment allows the creation of 108 different binding sites and domains. The other sub domains are of functional importance for binding sites. CH1 binds complementary C4b fragment, CH2 contains carbohydrate binding sites and CH3 domains are responsible for the interaction with the rest of the immune system. Different immunoglobulin has different H chain. IgG has a γ-chain, IgM a μ-chain, IgE a ε-chain and IgD a δ-chain. Differences in the chains result in subclasses such as IgG1, IgG2a, IgG2b and IgG3. All these different IgG subclasses mainly differ in the Fc fragment, where they appear and function in different stages of the immune response. The IgG molecule which is analytically important is dominant in the secondary response. 7.8.1.3 Antibody Antigen Interaction-Affinity Epitope is the region of an antigen which interacts with the antibody bind- ing site (paratrope) is called the epitope. It states that epitopes are not intrinsic parts of the molecule. The prominent part of the antigen mol- ecule which acts as an epitope can vary from one antibody to another for exactly the same molecule. Antibody antigen interactions are non cova- lent, reversible and it involve various properties such as hydrogen bonds, van der waals forces, ionic coulombic interactions and hydrophobic bonds. Both, antibody and antigen undergoes substantial conformational changes when interacted, but they also stay unchanged depending on the specific antibody antigen pair. There is an affinity to the measure of the strength of the binding of an epitope to an antibody. The equilibrium of their interac- tion is (Eq7) described with the affinity constant KA (Eq18) Ab represents antibody and Ag antigen. Ab + Ag Ab – Ag (7.17) (7.18) KA = [Ab – Ag]/[Ab].[Ag]

272 Advanced Biomaterials and Biodevices High affinity interactions can be considered almost complete in a sub- stantially shorter time than low affinity interactions even when in theory the time to reach the actual equilibrium is independent from the affin- ity. High affinity complexes are said to be much more stable and indepen- dent. Affinity constants KA range from 105 to 1012 M-1.The tailored binding sites of the epitopes are responsible for achieving high affinities. 1,000 fold decreased affinity is brought about by the loss of one hydrogen bond in an interaction. Whereas the affinity constant for a monoclonal antibody can be determined and known that, KA for polyclonal serum are more dif- ficult to be determined. Monoclonal antibodies consist of identical immu- noglobulin molecules whereas Polyclonal antibodies represent a mixture of antibodies, specific for the antigen. The affinity of their components determines the specifications of immune-sensors are determined to a large extent. High affinity results in sensitive sensors, but very high affinities might result in virtually irreversible sensors. 7.8.1.4 Avidity The term avidity is defined as the measure of the overall stability of the antibody antigen complex [140]. For immunochemical reactions avidity is of more evidential importance than affinity, as it refers to the intrinsic affinity of the paratrope such as for the epitope, the valency of the anti- body and the geometric arrangement of the interacting compounds in the nature. High avidity is reached when all the paratropes has an ability to bind epitopes. For the IgM there can be ten epitopes known. Avidity is also significantly increased by multivalent systems, where many antibodies eventually bind to different epitopes of the same antigen and also crosslink the antigens involved. It is easily achieved by using polyclonal antibodies representing mixture of antibodies and large antigens with multiple epit- opes. Even cross-linking (higher avidity) has been highly achieved by add- ing anti-IgG immunoglobulin, beads of protein A or protein G. Avidity is increased by relatively dense antigen layers by allowing the bivalent bind- ing and also increases the overall stability of the antibody antigen complex for any surface bound antigen. 7.8.1.5 Antibody Production-Polyclonal antibody An immunogen i.e. analyte or analyte conjugate is injected into a host animal for the production of polyclonal antibodies. After immunisation, the host species, e.g. mouse, goat rabbit or sheep reacts specifically with a primary immune response and mainly because of it IgM is produced. In the following days and weeks, the host animal is injected again (boost

Anticipating Behaviour of Advanced Materials in Healthcare 273 injections), and inoculated by provoking a secondary response that pro- duces IgG. The serum is tested by ELISA test for specific antibodies. A high serum of high specific antibody concentration is achieved after multiple boost injections. Polyclonal antibodies are used in purified form as serum. Purification has been including Immuneprecipitation and immune- affinity purification and protein A or protein G affinity purification for every complex formed. B lymphocytes produce antibodies which is a part of immune response. Each lymphocyte produces only one type of serum with exactly same amino acid sequence, but different lymphocyte pro- duce different antibodies. Some antibodies can be specific for one analyte, but it has different affinities at different regions. These different antibod- ies specific for one analyte are called generally as polyclonal antibodies at different region. Phagocytosis is referred to as the first antigen process- ing steps. Antigens are non-specifically engulfed in the antigen present- ing cells (APCs), processed (lysed) and presented in fragments for further other steps. Phagocytosis is not possible for small soluble molecules. They are found as non immunogenic, i.e. they do not trigger immune response. For immunogenicity for eg degradability, binding to virgin B cells to cell communication promotion are very important, they are coupled to carrier proteins, such as bovine serum albumin (BSA) or keyhole limplet haemo- cyanine (KLH). In order to provoke an immune reaction of the highly reac- tive low molecular weight analytes. Antibodies are then raised high against the analyte, the carrier and the analyte carrier complex which include the spacer bridge. For immunoassay conjugates, special precautions have to be taken such as different type conjugates for immunisation and immunoas- say needs to be taken. The problem of polyclonal antibodies is that differ- ent animals, individuals or even exact by same animal at a different time, will likely produce a polyclonal antibody serum of different composition, sensitive and specificity. Monoclonal antibody guarantees a limitless sup- ply of a single antibody of defined specificity. 7.8.1.6 Antibody Production- Monoclonal Antibodies Production of monoclonal antibodies is on the basis of proliferation of a single antibody by producing cell, giving out a uniform population of anti- bodies of the same type with identical immune-affinities and specificities [139]. A method namely called as hybridoma technology guarantees an unlimited production of monoclonal antibodies of the same isotype with constant properties of sensitivity and specificity. B lymphocytes only grow and divide for a short period of time [143]. Myeloma cells are immortalised, to the tumourigenic B lymphocytes, which grow and divide rapidly, but do

274 Advanced Biomaterials and Biodevices not produce any of the antibody molecule which is required for producing cells. For the production of antibody a fusion of myeloma cell with the cell shaving ability of growth. Mice when immunised with the antigen produces polyclonal antibodies. Spleen cells are fused with non-antibody producing myeloma cells using polyethylene glycol by removing the spleen of mouse and as it contain a very high concentration of B lymphocytes. The mem- branes of both cells fuse and merge. The cell nucleus merges, then the chro- mosomes are mixed and the immortalised antibody producing hybridoma cells are obtained from it. Unfused spleen cells die and the myeloma cells which are unfused are terminated on a selective HAT medium. The result- ing mixture of polyclonal hybridoma is grown in a culture in a well manner, divided and is diluted. If the supernatant part of the well contains specific polyclonal antibodies, the well is further diluted, divided and synchronised until the hybridomas in the well are cloned from only a single parent cell, producing one type of antibody only. Later, a monoclonal antibody pro- ducing hybridoma is derived from it. Normally, this procedure yields many polyclonal hybridomas, which produce specific antibodies. The monoclonal antibodies produced are tested for affinity, sensitivity and specificity and a few hybridoma cells are chosen for further development of the cells. Monoclonal antibodies can be grown, in culture flasks or bioreactors. Monoclonal antibodies can also be grown in vivo under composite methods. Hybridoma cell are injected into the peritoneum of mice in a very decent manner and the tumour-like growth rate also produces large amounts of antibodies in ascetic fluid with the help of innoculation. In vitro methods delivers pure antibody which has low yields and the in vivo method results in high yields of antibody, which are contaminated and specified with proteins and other antibodies, and purification is very necessary. 7.8.1.7 Antibody fragments and Recombinant Antibodies A genetic technique namely called as combinatorial phage display allows for the production of the Fab fragment of antibodies, by combining and attributing the genes for these specific regions with phage particles and consequently producing the Fab fragment of antibody in bacteria cells [143]. The two Fab fragments of an antibody are identical, which contain two identical binding sites; it is also possible to create antibodies with two different binding sites. The bi-functional antibodies can be created chemically by cleaving and forming the disulphide bonds and cross linking one of the bi-functional antibodies with another antibody fragment. Biological production is achieved by fusing two hybridoma cells the resultant antibody is generally

Anticipating Behaviour of Advanced Materials in Healthcare 275 called as quadroma. Genetically, it is possible only to connect the vari- able regions of two different antibodies among themselves. The resulting antibodies very often contain two different VH regions and two different VL regions. These bi-functional biomolecules are capable to recognise two different molecules in various regions. It is possible to attach them to one molecule; whereas the second binding site is analyte specific. For Examples for the usefulness of bi-functional antibodies are immune-immobilisation and secondary drug delivery to tumours cells [143]. Instead of using whole antibodies, only the binding sites parts of the binding sites containing fragments which can be obtained either enzymati- cally or genetically can be used. Enzyme scan cleave the important Fab fragments from the Fc fragment which is not much important for most sensor applications. The enzyme papain is said to produces two Fab frag- ments on every antibody, cutting the antibody into two parts that is the hinge region and the Fab fragment. The enzyme pepsin cuts the antibody between the hinge region and the Fc segment, which result in F(ab)2 frag- ments.The Genetic methods are more versatile and specific. Numerous antibody fragments have been produced and used in sensors application. It has also been possible to produce the Fab fragments genetically. Fv and scFv fragments have also been produced genetically by this method. Fv is the smallest possible fragment which still guarantees complete binding of antigen and scFv is cross-linked Fv for increased stability. Furthermore, the Fab fragment has been cleaved and divided into two segments that is H-chain segment (Fd) and the L-chain. Both the fragments have been used eminently and significantly in biosensor applications. It has been also possible to produce single CDRs and use them in affin- ity sensors for long time. These CDRs are useful, as they consist of only a short amino acid chain and can be synthesised easily and cheaply. Recently it is now possible to replace human CDRs in relating human antibodies with the analyte specific mouse CDRs. The resulting antibody produced is specific and of much important for a certain analyte that can be used even in vivo in humans, while the remaining antibody is not recognised as an intruder in the human system. Due to the potentiality of recombinant antibodies, the anti- bodies are produced much faster, with new binding properties such as speci- ficity, sensitivity etc. by which experiments on animals can be reduced [139]. 7.9 Antibody microarrays The antibody microarray technology brings about great promise for pro- tein expression profiling of complex non-fractionated proteomes, having

276 Advanced Biomaterials and Biodevices Labeled analyte Printed antibody Array 3.1 Antibody array Content Array design Antibody array Array assay Data analysis Array Sample Disease fabrication proteomics Figure 7.5 Schematic interpretation of the antibody microarray set-up adapted from Ref. [144]. direct bearing on numerous applications within disease proteomics [144- 148]. In the era the current concept of generating miniaturized (<1 cm2 in size) antibody microarrays is generally on the basis of printing small amounts (fmole range in pL scale drop volumes) of numerous individual antibodies (<500 antibodies/array) with the desired specificities in discrete positions and uniformity (<200 μm sized spots) in an ordered pattern or fragments, an microarray, onto a solid support (Figure 7.5) [144, 149]. The immobilized antibodies will now act as highly specific and selective probes for the targeted protein analytes on the arrays [144, 150]. The microarrays are incubated with minute amounts (μL scale) of clinical sample experi- mental setups, e.g. directly labelled plasma, and after that they are visu- alized by using fluorescence as the main sensing technology in antibody microarrays [151].These rapid assays, typically less than 3 h assay time, commonly display an assay sensitivity in the pM to fM range which are truly enabling low-abundant (pg/ml) protein analytes to be targeted even in complex proteomes [152-154]. This is a key feature and is significant as many of the candidate biomarkers are anticipated to be found in this cohort of low-concentrated protein species by their activity [144, 148, 155- 157]. The generated microarray images are transformed and moulded into protein expression profiles or protein atlases by revealing the composition of the proteome in the chain. In the experiment performed it has been found that one humanized antibody, CAMPATH-1H, and is generated by grafting CDRs of a rat

Anticipating Behaviour of Advanced Materials in Healthcare 277 anti-CD52 antibody onto a human antibody frameworks [158], resolving the non- Hodgkins lymphoma in two patients without eliciting a HAMA response in either of the case [159]. In a more recent clinical trial for the treatment of transplant rejection, none of the 12 patients administered CAMPATH-1H showed anti-Ig responses [160]. Unlike CAMPATH-1H, many other MAbs have not been found and rendered non-immunogenic through any humanization. Several investigators have experimented and concluded that humanized antibodies remain immunogenic in both the sub human primates and humans, where the human response of the host is directed and guided towards the variable region of these MAbs. A strong antibody response to the murine variable regions of MAb B72.3 was dem- onstrated and verified in an animal model as well [161]. In rhesus monkeys the antibody responses against a humanized anti-CD18 MAb have been reported [162] and in cynomolgus monkeys [163, 164] against a human- ized anti-Tac MAb. An evaluation and deduction of the immunogenicity of a humanized anti-TNFα in the cynomolgus monkeys and human vol- unteers has been showed that while the monkeys did not have any specific humoral response against the humanized antibody, and these antibodies response was elicited in the human subjects in a very enhancing way [165]. Immunogenic in patients has revealed a humanized antibody against car- cinoembyronic antigen, humanized MN-14 [166]. An approach to reduce the immunogenicity of antibodies to a minimum has been reported using an anti-carcinoma antibody, MAb CC49, as a prototype. This approach involves genetic manipulation of the antibody variable region based on prior identification of: (1) the residues of the hypervariable regions which are very critical in the antigen-antibody interaction; and (2) those residues that comprise the idiotopes are at potential targets of patients’ immune responses. Recently, antibody therapeutics for cancer treatment have been found successful to some extent in the last decade, and seemingly overwhelming small compounds target to signal the molecules involved in tumor growth and metastasis [167, 168]. The development of novel antibody therapeutics is promoted strongly by the advanced technology of antibody engineering [169, 170]. Especially, threatrend of antibody development is moving from mouse/human chimera to fully human monoclonal antibodies (mAbs) [171, 172] which results in clinical applications in the cancer and auto- immune disease patients [173, 174]. In the recent years a single B cell-based human mAb gene cloning using RT-PCR has been developed [175-178]. The direct PCR- based isola- tion of a specific antibody gene is basically a rapid and simple technol- ogy. These systems are limited in terms of a high throughput selection

278 Advanced Biomaterials and Biodevices of antibody-producing, antigen-specific B cells, owing to the absence of high levels of clonal selection compared to phage display or hybridoma method. Several researchers have utilized antibody-bearing B cells derived from patients infected or immunized with hepatitis B, tetanus antigen, and influenza virus, and auto-immune patients, and has shown a high titer of plasma auto-antibodies and succeeded in isolating a variety clone of human mAbs[179-181]. However, it is very difficult to isolate cancer antigen-specific human mAbs from cancer patient B cells, because of the suppression of antibody production under immune modulated conditions, and generate few opportunities to obtain B cells from vaccinated patients. Levels of natural antibodies to auto-antigens, especially phospholipids, generally increase transiently in the acute phase of most infectious dis- eases [182, 183]. Based on an advancement of studies, it is widely believed that natural antibodies is repressing the first line of defense against numer- ous agents which is responsible for infectious diseases, including bacteria, viruses, and parasites [184-195]. In malaria, component such as glyco- sylphosphatidylinositol (GPI) is been believed to cause numerous of toxic symptoms associated with malaria as well [196]. Natural antibodies to GPI, which are mainly short-lived and diversified igG3 and lower amounts of IgG1 such as in an acutely infected patient [197], it is co-related with dis- ease such as severity and parasitemia [196].On the basis of efficacy dem- onstrated in a model infection, though synthetic GPI has been proposed to candidate antigen for a vaccine for limiting the severity of malaria in the level detected [198]. Although it is eminently believed that natural anti- bodies to lipids can have beneficial effects, such antibodies in general only increase transiently, and the levels become very low to control the infection [194, 195, 199]. Therefore, enhancement of the antibodies by an adjuvant or adjuvant system has become useful as a beneficial effect in vaccines. 7.10 Conclusion Biomaterials are an exciting and rapidly developing field in today’s era. Engineered materials and tools are increasingly used in medical applica- tions in everyday life such as scaffolds for tissue engineering, replacement body parts, drug delivery, gene therapy, and biomedical and surgical devices while the concept and understanding of structure-property relationships in natural biomaterials and may finally lead to improved interventions for the different modes variety of diseases, injuries and health problems. Because it is highly interdisciplinary field involving elements of materials science, engineering, biology, chemistry and medicine, biomaterials as a general

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