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INTRODUCTION  

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1.1. Nanoparticle:

In nanotechnology, widely accepted definition of a nanoparticle is "A particle having one or more dimensions in the range of 1-100 nm". Novel properties that differentiate nanoparticles from the bulk material typically develop at a critical length scale of 1-100 nm.

Although generally nanoparticles are considered an invention of modern science, they actually have been used for very long time. Nanoparticles were used as far back as the 9th century in Mesopotamia for generating a glittering effect on the surface of pots. Michael Faraday provided the first description, in scientific terms, of the optical properties of nanometre-scale metals in his classic 1857 paper "Experimental relations of gold (and other metals) to light."[1]

Nanoparticles are of great scientific interest as they are effectively a bridge between bulk materials and atomic or molecular structures. A bulk material should have constant physical properties regardless of its size, but at the nano-scale this is often not the case. Size-dependent properties are observed such as quantum confinement in semiconductor particles, surface plasmon resonance in some metal particles and superparamagnetism in magnetic materials. The properties of materials change as their size approaches the nano-scale and as the percentage of atoms at the surface of a material becomes significant. For bulk materials larger than one micrometre the percentage of atoms at the surface is minuscule relative to the total number of atoms of the material. The interesting and sometimes unexpected properties of nanoparticles are partly due to the aspects of the surface of the material dominating the properties in lieu of the bulk properties. Nanoparticles exhibit a number of special properties relative to bulk material. For example, the bending of bulk copper (wire, ribbon, etc.) occurs with movement of copper atoms/clusters at about the 50 nm scale. The change in properties is not always desirable. Ferroelectric materials smaller than 10 nm can switch their magnetisation direction using room temperature thermal energy, thus making them useless for memory storage. Suspensions of nanoparticles are possible because the interaction of the particle surface with the solvent is strong enough to overcome differences in density, which usually result in a material either sinking or floating in a liquid. Nanoparticles often have unexpected visible properties because they are small enough to confine

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their electrons and produce quantum effects. For example gold nanoparticles appear deep red to black in solution.

Nanoparticles have a very high surface area to volume ratio. Reduction in size provides a tremendous driving force for diffusion, especially at elevated temperatures. Sintering can take place at lower temperatures, over shorter time scales than for larger particles. This theoretically does not affect the density of the final product, though flow difficulties and the tendency of nanoparticles to aggregate complicates matters. The large surface area to volume ratio also reduces the incipient melting temperature of nanoparticles [2]. Moreover nanoparticles have been found to impart some extra properties to various day to day products. Nano Zinc Oxide particles have been found to have superior UV blocking properties compared to its bulk substitute. This is one of the reasons why it is often used in the sunscreen lotions. Clay nanoparticles when incorporated into polymer matrices increase reinforcement, leading to stronger plastics, verified by a higher glass transition temperature and other mechanical property tests. These nanoparticles are hard, and impart their properties to the polymer (plastic). Nanoparticles have also been attached to textile fibres in order to create smart and functional clothing as shown by The Textiles Nanotechnology Laboratory at Cornell University.

1.2. Types of Nanoparticles:

Nanoparticles can be classified based upon their sizes, shapes, and materials, and with various chemical and surface properties. There is a constant and rapid growth in the field of nanotechnology which adds to the basis of classification. The classes of nanoparticles listed below are all very general and multi-functional, however, some of their basic properties and current known uses in biotechnology, and particularly nano-medicine, are described here.

1.2.1. Fullerenes: Buckyballs and Carbon nanotubes

Both members of the fullerene structural class, buckyballs and carbon nanotubes are carbon based, lattice-like, potentially porous molecules. Buckyballs are spherical in shape while carbon nanotubes are cylindrical. The diameter of a carbon nanotube can be several nm but the length can be much greater, up to several mm, depending on the synthesis process and its intended use.

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Carbon nanotubes have many applications in materials science due to their strength and unique electrical properties. However, they have also found use in the field of biomedicine as carriers for vaccines, drugs and other molecules. A single wall carbon nanotube is a one-atom-thick sheet of graphite, resembling chicken wire mesh, rolled seamlessly into a nanotube. There are also multi-walled and other types of nanotubes depending on the shape, diameter and other properties.

1.2.2. Liposomes

Liposomes are lipid-based nanoparticles used extensively in the pharmaceutical and cosmetic industries because of their capacity for breaking down inside cells, once their delivery function has been met. Liposomes were the first engineered nanoparticles used for drug delivery but problems such as their propensity to fuse together in aqueous environments and release their payload, have lead to replacement, or stabilization using newer alternative nanoparticles.

1.2.3. Nanoshells

Also referred to as core-shells, nanoshells are spherical cores of a particular compound surrounded by a shell or outer coating of another, which is a few nanometres thick. One application in biomedicine is to create nanoshells that absorb at biologically useful wavelengths, depending on the shell thickness.

One common formula for the construction of nanoshells is to use silica for the core and another sticky compound to adhere gold particles to the outside surface, creating the shell and vice versa. Nanoshells such as these have been used to kill cancer cells in mice. Once injected into a tumour, radiation is applied and the nanoshells heat up enough to kill the tumour cells.

1.2.4. Dendrimers

Dendrimers are highly branched structures gaining wide use in nano-medicine because of the multiple molecular "hooks" on their surfaces that can be used to attach cell-identification tags, fluorescent dyes, enzymes and other molecules. The first dendritic molecules were produced around 1980, but interest in them has blossomed more recently as biotechnological uses are discovered.

Nanomedical applications for dendrimers are many and include nanoscale catalysts and reaction vessels, micelle mimics, imaging agents and chemical

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sensors, and agents for delivering drugs or genes into cells. There are two basic structural types. One is the globular structure with a central core from which branches radiate. The second type has no central core and consists simply of a series of highly branched polymers.

1.2.5. Quantum dots

Also known as nanocrystals, quantum dots are nanosized semiconductors that, depending on their size, can emit light in all colours. These nanostructures confine conduction band electrons, valence band holes, or excitons in all three spatial directions. Examples of quantum dots are semiconductor nanocrystals and core-shell nanocrystals, where there is an interface between different semiconductor materials. They have been applied in biotechnology for cell labelling and imaging, particularly in cancer imaging studies.

1.2.6. Superparamagnetic nanoparticles

Superparamagnetic molecules are those that are attracted to a magnetic field but do not retain residual magnetism after the field is removed. Nanoparticles of iron oxide with diameters in the range of 5-100 nm have been used for selective magnetic bioseparations. Typical techniques involve coating the particles with antibodies to cell-specific antigens, for separation from the surrounding matrix.

Used in membrane transport studies, superparamagenetic iron oxide nanoparticles (SPION) are applied for drug delivery and gene transfection. Targeted delivery of drugs, bioactive molecules or DNA vectors is dependent on the application of an external magnetic force that accelerates and directs their progress towards the target tissue. They are also useful as MRI contrast agents.

1.2.7. Nanorods

Typically 10-100 nm in length, nanorods are most often made from semiconducting materials and used in nanomedicine as imaging and contrast agents. Nanorods can be made by generating small cylinders of silicon, gold or inorganic phosphate, among other materials.

Current concerns over the safety of nanoparticles have lead to the development of many new facets of research. As a result, our collection of knowledge about nanoparticle interactions within cells is still rapidly growing. As resarch progresses in this exciting new area of biotechnology, new nanoparticles are

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continuously being discovered and new applications to nanomedicine will be found.

1.3. Iron Oxide Nanoparticle:

The iron oxides have been known for millennia. Such minerals were used originally as pigments for paints during the Palaeolithic. Much later, they were used in the magnetic compass when it was invented in China. This was the first application of magnetic iron oxides, also known as lodestones. The most plentiful deposits of these stones were found in the district of Magnesia in Asia Minor, hence the mineral’s name became magnetite. Magnetite can be found naturally occurring in molluscs, bees, pigeons, magnetotactic bacteria, and algae. The biogenic formation of magnetite crystals in Magnetobactericum sp. has received a great deal of attention [3] due the narrow-size distribution of the crystals (magnetosomes) and their magnetic properties.[4] Synthetically, magnetite can be formed by precipitation in alkaline aqueous media of a mixture of Fe3+ and Fe2+, by oxidation of Fe(II) solutions or Fe(OH)2, by interaction of Fe2+ with ferrihydrite, or decomposition of organic precursors, etc.[8, 9]

1.3.1. Crystal Structure of Magnetite:

Magnetite, Fe3O4, is a common magnetic iron oxide that has a cubic inverse spinel crystal structure (space group Fd3m) with lattice parameter of 8.3941Å. The spinal structure can be thought of as made up by alternate stacking of two different cubes in which the oxygen atoms form a fcc sub lattice with cations occupying the interstitial sites[5]. There are two types of interstitial positions, the tetrahedral or A-sites and the octahedral or B-sites. The Bravais lattice is face-centred cubic (fcc) with 8 formula units per unit cell.

The valence of various atoms is described by the formal chemical formula, Fe3+(Fe3+Fe2+)(O2-)4. Fe(III) ions occupying 8 tetrahedral (A-sites) and 8 octahedral positions (B-sites) along with Fe(II) ions occupying other 8 octahedral positions (B-sites) leading to the so-called “inverse” spinal structure.

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Fig.1: Iron polyhedra network Fig.2: Magnetite unit cell. (blue tetrahedra and green Red atoms are oxygen, blue octahedra) in magnetite unit cell. and green represent iron. Fig.1 and 2 are taken from http://wikis.lib.ncsu.edu/index.php/Spinel This inverse spinel structure for magnetite was first suggested to explain the fast electron hopping - continuous exchange of electrons between Fe2+and Fe3+ in the octahedral positions at room temperature rendering magnetite an important class of half metallic material. When magnetite is heated to just above 122K, a threshold called the Verwey temperature, conductivity increases more than two orders of magnitude [6]. The Verwey temperature was originally believed to be the temperature above which the fast electron hopping could occur. More recently it has been discovered that at very similar temperatures ~120K a coordination crossover (CC) transition occurs. Below 120K magnetite is a normal spinel, with Fe2+in the A or tetrahedral sites and Fe3+ in the B or octahedral sites. TCC and TV both show pressure dependence supporting the theory that the locations of the smaller Fe3+ ions and larger Fe2+affect these properties. The distribution of Fe2+and Fe3+ between the tetrahedral and octahedral holes also explains why magnetite has a larger magnetic moment below 120K - the net magnetic moment is a sum of the aligned moments of Fe3+ within one local environment and the smaller magnetic moments of Fe2+ in the opposite direction decrease the magnitude only slightly. Above 120K half the Fe3+ moments are aligned with the Fe2+ magnetic moments, but the remaining Fe3+ magnetic moments in the opposite direction decrease the net magnetic moment. [7]

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Mineral Chemical

Composition

Crystallographic

system

Space

group

Unit cell dimensions (nm)

a b c ß

Z

Goethite α-FeOOH Orthorhombic Pnma 0.9956 0.30215 0.4608 4

Lepidocrocite γ-FeOOH Orthorhombic Bbmm 0.3071 1.2520 0.3873 4

Akaganeite ß-FeOOH Monoclinic I2/m 1.0560 0.3031 1.0483 90.63 8

Schwertmannite Fe16O16 (OH) y (SO)z·nH2O

Tetragonal P4/m 1.066 0.604

Feroxyhyte δ –FeOOH Hexagonal P3ml 0.293 0.456 2

Ferrihydrite Fe5O8·4H2O Hexagonal P31c; P3

0.2955 0.937 4

Bernalite Fe(OH)3 Orthorhombic Immm 0.7544 0.7560 0.7558 8

Haematite α –Fe2O3 Hexagonal R3c 0.5034 1.3752 6

Magnetite Fe3O4 Cubic Fd3m 0.8396 8

Maghemite γ-Fe2O3 Cubic P4332 0.8347 8

Wustite FeO Cubic Fm3m 0.4302

Table 1: Crystallographic information for various iron oxides, from [8]. Z is the number of formula units in the unit cell.

1.3.2. Properties of Iron Oxide nanoparticles:

Magnetite has a greyish black colour with an average (typical) density of 5.15 g/cm3. These oxides are having metallic lustre and are opaque. Along with other properties the most important is the magnetic behaviour of magnetite nanoparticles. Iron oxides are classified by their response to an externally applied magnetic field. Description of orientations of the magnetic moments in a particle helps to identify different types of magnetism observed in nature.

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Fig. 3. Orientation of magnetic dipoles in different forms of magnetic behaviours

The magnetic properties of these particles can be described by the dependence of the magnetic induction B on the magnetic field H. Some materials such as iron exhibit ferromagnetism, in that they can be permanently magnetized. In most materials the relation between B and H is linear: B=μH; where μ is the magnetic permeability of the particles. Iron oxide particles exhibit paramagnetism if μ>1; and diamagnetism if μ0; diamagnetic particles χ

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characteristics of nanoparticles. Specifically, the loss of magnetization may be due to the existence of a magnetically dead layer, ~1 nm thick, caused by an asymmetric environment effect of the surface atoms [11].

A ferrimagnetic material is one in which the magnetic moments of the atoms on different sublattices are opposed, as in antiferromagnetism; however, in ferrimagnetic materials, the opposing moments are unequal and a spontaneous magnetization remains. This happens when the sublattices consist of different materials or ions (such as Fe2+ and Fe3+). Magnetite (Fe3O4) is a ferrimagnetic iron oxide. The magnetic moment of the unit cell comes only from Fe2+ ions with a magnetic moment of 4μB [12].

8Fe3+ (S=5/2)

↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ Tetrahedral sites

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ Octahedral sites

8Fe3+ (S=5/2) 8Fe2+ (S=2)

Magnetite was originally classified as a ferromagnet before Néel's discovery of ferrimagnetism and antiferromagnetism. Néel showed that in the case of inverse spinel magnetite nano particles the metal ions in the sub lattice A are antiparallel with respect to the metal ions in the sub lattice B [13]. The net magnetic moment of the material is the difference in magnetic moments of sub lattices A and B, which explained why the magnetic moment per formula unit was lower than the otherwise expected value. Thus, higher the magnetic moment of the divalent cation, higher the magnetisation is.  Ferrimagnetic materials have high resistivity and have anisotropic properties. The anisotropy is actually induced by an external applied field. When this applied field aligns with the magnetic dipoles it causes a net magnetic dipole moment and causes the magnetic dipoles to process at a frequency controlled by the applied field, called Larmor or precession frequency.

1.3.3. Applications of Iron Oxide Nanoparticle:

In the recent years there has been an increased interest in the application of nanotechnology to biotechnology [14]. Thus, a number of different uses of nano objects have been investigated. In the case of iron oxide-based materials, the focus has been on separations and diagnostics, [15, 16] DNA analysis, [17] and

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recently, on the use of iron oxides as contrast enhancers for magnetic resonance imaging (MRI), [18, 19] among others.

A. Cellular labelling/cell separation

Micrometre-sized magnetic particles have been used as commercial applications in biology, biomedicine and biotechnology for the separation of cells. The separation of cells, or immune-magnetic separation (IMS), is achieved by covalently binding magnetic nanoparticles –or magnetised beads – to the cells. The bead carrying a specific antibody -usually monoclonal antibodies- on the surface binds selectively the target cell which can then be removed from the suspension with the help of a magnet. The IMS has been used for the purging and isolation of cancer cells, studies of HIV and AIDS, isolation of granulocytes, isolation of cells from various tissues, stem cells, etc. [20].

B. DNA analysis

The separation and purification of nucleic acids has become more and more important for the accurate mapping of the genome. Nucleic acids are usually extracted from other cell components after cytolysis and repetitive washing procedures. The use of super paramagnetic particles for the analysis of plasmids and nucleic acids was introduced nearly 20 years ago by Uhl´en et al. [21]. Separation and purification of DNA with magnetisable microspheres (with a typical size range of 200–2000 nm) has been reported by numerous groups. The DNA molecule is able to wrap around nano objects in a similar way it does around the histone core in the nucleosomes [22]. This finding indicates that DNA purification can be achieved even with simple non-functionalised nanoparticles such as silica nanoparticles. However, to improve the efficiency and selectivity of the process the surface of the particles is functionalised with specific molecules, e.g. streptavidin, which can bind non-covalently to biotinylated-DNA [21].

C. MRI contrast agents

Magnetic resonance imaging (MRI) is based on the nuclear magnetic resonance of protons in the molecules, mainly water, that exist in a given tissue. Since the local environment of a given tissue varies depends on its position in the body it is possible to use MRI to identify various types of tissues. The characteristic measure in MRI is the proton relaxation rate (R1 or R2) or its inverse (T1 or

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T2), the proton relaxation time, R1 = 1/T1 and R2 = 1/T2. In this technique there are two different types of proton relaxation times: the longitudinal relaxation time, T1, (spin-lattice relaxation) and the transverse relaxation time, T2 (spin-spin relaxation). Hence, there are two types of contrast agents: positive agents, which act on T1 providing a positive enhancement of the signal, appearing bright on the MRI scan; and the negative agents, which reduce the signal and give a negative enhancement, appearing as dark spots in the scan [23].

Typical positive contrast agents are paramagnetic compounds based on rare earth ions, typically gadolinium-containing chelates. Nevertheless, other alternatives are sought after due to the high toxicity of the heavy metals and the low contrast enhancement. However, negative contrast enhancers (T2 agents) as iron oxide nanoparticles have been used due to a much higher magnetisation per concentration unit [24, 19] reported to be at least one order of magnitude higher than gadolinium-containing molecules [25].

Passive targeting : Insofar there have been two different types of superparamagnetic contrast agents based on iron oxides that are clinically approved: the small particles of iron oxides (SPIO, 40 < DH < 200 nm) and the ultra-small particles or iron oxide (USPIO, DH < 40 nm). Due to their large size, the SPIO agents are rapidly cleared (several minutes) from blood by the reticuloendothelial system organs such as liver and spleen, thus enabling the imaging of lesions in such organs. On the other hand, the smaller USPIO agents have longer residence times (hours), allowing for imaging of the lymphatic system [26]. Furthermore, it has been reported that the use of USPIO permits imaging for more than 24 h, and sometimes as long as five days [27]. USPIO have lower T1/T2 ratio than SPIO, which leadsto a higher contrast on T2-weighted images and enables the use of relatively weak magnetic fields (< 0.5 T) [28].

Active targeting : The surface of USPIO and SPIO agents is functionalised with specific biomolecules to actively target certain tissues to be visualised by MRI technique. Several attempts have been performed to improve their surface properties, e.g. coating with different types of biocompatible materials, such as protein, polysaccharide, DNA fragments, etc.[29, 30, 31]. The visualisation of certain organs needs special functional properties of coating layer [32]. Thus, for instance, Kim et al. reported the use of polysaccharide-coated super

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paramagnetic iron oxide nanoparticles (SPION) for the MR imaging of brain tissue. Also, in case of lesions detection in the brain, e.g., multiple sclerosis, Alzheimer’s disease, etc.; The MRI contrast agent has to pass across the blood brain barrier (BBB) [33, 34]. However, there are limited reports on the penetration of the BBB by nanoparticles [35]. Moreover, after passing the BBB those agents have to be recognised and up-taken by cells. Receptor mediated endocytosis has been proposed as possible way to introduce contrast agent in vivo and several studies have been performed where various types of cells were labelled by SPIO and further visualised by MRI [36].

D. Tissue repair

Tissue repair using iron oxide nanoparticles is accomplished either through welding, apposing two tissue surfaces then heating the tissues sufficiently to join them, or through soldering, where protein or synthetic polymer-coated nanoparticles are placed between two tissue surfaces to enhance joining of the tissues. Temperatures greater than 50 0C are known to induce tissue union [37]. This is believed to be induced by the denaturation of proteins and the subsequent entanglement of adjacent protein chains [37, 38]. Nanoparticles that strongly absorb light corresponding to the output of a laser are also useful for tissue-repairing procedures. Specifically, gold- or silica-coated iron oxide nanoparticles have been designed to strongly absorb light [39, 40]. Stem cells are the body’s master cells and have a unique ability to renew themselves and give rise to other specialized cell types. These cells, therefore, have the potential to be used for transplantation purposes. An obstacle to developing such therapy is a lack of targeting strategies on both neural stem cells and on the signals that determine their behaviour and fate for tissue development. The super paramagnetic nanoparticles could be coupled to the cells and used to target these cells at the desired site in the body. In addition, various proteins, growth factors, etc., could be bound to these nanoparticles that might be delivered at the damaged tissue, where it would play a role in tissue development. While there is no doubt that the use of stem cells in the form of cell-based therapies offers tremendous potential for disease treatment and cures for many common diseases including diabetes, cancer, heart disease, Alzheimer’s and Parkinson’s disease, central to this process would be the ability to target and activate these stem cells at required sites of injury and repair using magnetic particle technology [41].

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E. Drug delivery

Another possible and most promising application of these colloidal magnetic nanoparticles is in site-specific drug delivery as carriers of drug. Ideally, they could bear on their surface a pharmaceutical drug that could be driven to the target organ and released there. For these applications, the size, charge and surface chemistry of the magnetic particles are particularly important and strongly affect both the blood circulation time as well as bioavailability of the particles within the body [44]. In addition, magnetic properties and internalization of particles depend strongly on the size of the magnetic particles [45].  Particles ranging from ca. 10 to 100 nm are optimal for intravenous injection and demonstrate the most prolonged blood circulation times. The particles in this size range are small enough both to evade (reticuloendothelial system) RES of the body as well as penetrate the very small capillaries within the body tissues and therefore may offer the most effective distribution in certain tissues [46]. Superparamagnetic iron oxide nanoparticles of narrow size range are easily produced and coated with various polymers, providing convenient, readily targetable magnetic resonance imaging agents. Because of the large surface area to volume ratio, the magnetic nanoparticles tend to aggregate and adsorb plasma proteins. The body’s RES, mainly the kupffer cells in the liver, usually take up these nanoparticles due to the hydrophobic surface. Surface coverage by amphiphilic polymeric surfactants such as poloxamers, poloxamines and poly(ethylene glycol) (PEG) derivatives over the nanoparticles significantly increases the blood circulation time by minimizing or eliminating the protein adsorption to the nanoparticles [47]. In an attempt to resist the protein adsorption and thus avoid the particle recognition by macrophage cells and to facilitate the intracellular uptake by specific cancer cells for cancer therapy and diagnosis, superparamagnetic magnetite nanoparticles were surface modified with PEG by Zhang et al. [48]. Widder et al. demonstrated the utility of magnetic albumin microspheres (MM-ADR) in animal tumour models [49]. Significantly greater responses, both in terms of tumour size and animal survival, were achieved with MM-ADR than adriamycin alone. Gupta et al. demonstrated that the efficacy of magnetic microspheres in the targeted delivery of incorporated drug is predominantly due to the magnetic effects and not due to the particle’s size or nonmagnetic holding [50]. The ultra structural disposition of adriamycin-associated magnetic albumin microspheres was also demonstrated in normal rats by Gallo et al. [51]. Fine ferromagnetic particles

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have been coated with poly (ethylene glycol)/amino or carboxyl groups to permit the covalent attachment of proteins, glycoproteins, and other ligands with the retention of biological activity. Ferromagnetic particles have also been used for various in vivo applications such as a tracer of blood flow, in radionuclide angiography, and for use in inducing clotting in arteriovenous malformations. Zimmermann et al. were the first to propose that erythrocytes or lymphocytes containing fine ferromagnetic particles could be propelled to a desired site by an external magnetic field [52]. It was demonstrated by Freeman and Geer that iron particles could pass through capillaries when properly conditioned and later confirmed by Meyers et al. who showed that iron particles could be magnetically controlled in the vascular system of experimental animals [53, 54].  A number of authors have described the preparation of particles or liposomes containing a certain amount of magnetite or other ferrites [55–57]. Some of them have focused on the field of drug transport and release. Experiments performed ex vivo on the toxicity of magnetite or magnetite-loaded polymeric particles have demonstrated that the latter have rather low cytotoxicity [58], and magnetite itself has much adverse effects [59].  The attachment of drugs to magnetic nanoparticles can be used to reduce drug doses and potential side effects to healthy tissues and the costs associated with drug treatment. Most iron oxides have a relatively short blood half-life and their primary application is for imaging of liver, spleen and the GI tract. Surface modified iron oxide nanoparticles having long blood circulation times, however, may prove very useful for imaging of the vascular compartment (magnetic resonance angiography), imaging of lymph nodes, perfusion imaging, receptor imaging and target specific imaging [60].

F. Magnetofection

Magnetofection (MF) is a method in which magnetic nanoparticles associated with vector DNA are transfected into cells by the influence of an external magnetic field. For this purpose, magnetic particles might be coated with the polycation polyethylenimine. These complexes readily associate with negatively charged DNA since the magnetic particles are positively charged due to the polyethylenimine. MF has been shown to enhance the efficiency of the vectors up to several thousand times [61]. For magnetically enhanced nucleic acid delivery, MF is universally applicable to viral and non-viral vectors, because it is extraordinarily rapid, simple and yields saturation level transfection at low

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dose in vitro [62]. These magnetic particles do not rely on receptors or other cell membrane-bound proteins for cell uptake, it is possible to transfect cells that normally are non-permissive [61].

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OBJECTIVE

 

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A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component. It consists of 3 parts:

■ The sensitive biological element (biological material (eg. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc), a biologically derived material or bio mimic) The sensitive elements can be created by biological engineering.

■ The transducer or the detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal (i.e., transducers) that can be more easily measured and quantified;

■ Associated electronics or signal processors that is primarily responsible for the display of the results in a user-friendly way.

There are many potential applications of biosensors of various types. The main requirements for a biosensor approach to be valuable in terms of research and commercial applications are the identification of a target molecule, availability of a suitable biological recognition element, and the potential for disposable portable detection systems to be preferred to sensitive laboratory-based techniques in some situations.

Applications of nanomaterials to biosensors have recently aroused much interest. This is because these interesting materials exhibit large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency, and strong adsorption ability that are helpful for the immobilization of desired biosensing

Substrate

Product

Analyte solution

Enzyme

Transducer Amplifier Signal processorBiologicalElement

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molecules. Moreover, nanoparticles have a unique ability to promote fast electron transfer between electrode and the active site of the enzyme [100]. In this context, different types of nanoparticles based on gold (AuNPs) [100 - 102], ZnO [103,104], Fe3O4 [105], etc., have been suggested as promising matrices for enzyme immobilization to improve stability and sensitivity of a biosensor. Among the various nanomaterial, magnetic nanoparticles have recently gained increased interest due to promising applications as drug delivery, hyperthermia treatment, cell separation, biosensors, enzymatic assays, etc. In this context, Fe3O4 nanoparticles have been considered as interesting for immobilization of desired biomolecules because of biocompatibility, strong super paramagnetic property, low toxicity, cost effectiveness, etc.

In view of demanding need of nanoparticle based biosensors, this work intends to characterize Fe3O4 nanoparticle. Many biopolymers have been reported for the modification of surface of Fe3O4 nanoparticles till date. One of the major disadvantages of using large polymers is the increase in the overall size of the nanoparticles. In this work glutathione molecule has been used to coat the surface of Fe3O4 nanoparticles. Glutathione also gives additional advantage of ease in protein or enzyme coupling which is the most essential point of a biosensor. In this work HRP (horseradish peroxidase) has been used to characterize the electrochemical properties of Fe3O4 nanoparticles as biosensors. HRP is a redox enzyme which is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule. So the main objectives of this work are:

1. To synthesize Fe3O4 nanoparticles in milligram quantities and smaller size range.

2. To modify the surface using glutathione.

3. To couple suitable protein/enzyme on the surface modified nanoparticles.

4. To characterize protein/enzyme coupled nanoparticles by various quantitative and qualitative methods like FT/IR, XRD, protein estimation, electrochemical studies, etc.

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METHODOLOGY

 

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3.1. Synthesis of Fe3O4 nanoparticles.

3.2. Surface modification using glutathione.

3.3. Characterization of nanoparticles(GT-) and glutathione attached nanoparticles(GT+) using

3.3.1. FT/IR studies

3.3.2. XRD studies

3.4. Coupling protein onto GT+ nanoparticles.

3.5. Characterization of protein bound GT+ nanoparticles using

3.5.1. Bradford estimation

3.5.2. Confocal Laser microscopy

3.5.3. Enzyme assay

3.5.4. Electrochemical studies

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3.1. Synthesis of Fe3O4 nanoparticles

Numerous schemes have been devised to synthesize magnetite nanoparticles. The different methods of magnetite nanoparticle synthesis can be generally grouped as aqueous or non-aqueous according to the solvents used. Two of the most widely used and explored methods for synthesis are the non-aqueous thermal decomposition method and the aqueous co-precipitation method.

3.1.1. Techniques:

A. Thermal decomposition method:

The thermal decomposition method consists of the high temperature thermal decomposition of an iron-oleate complex derived from an iron precursor in the presence of surfactant in a high boiling point organic solvent under an inert atmosphere. For the many variations of this synthetic method many different solvents and surfactants are used. However, in most every method Fe3O4 is formed through the thermal decomposition of an iron-oleate complex to form highly crystalline Fe3O4 in the 5 to 40 nm range with a very small size distribution. The size of Fe3O4 produced is a function of reaction temperature, the iron to surfactant ratio, and the reaction time, and various methods are used that achieve good size control by manipulation of these parameters. The Fe3O4 synthesized by organic methods is soluble in organic solvents because the Fe3O4 is stabilized by a surfactant surface coating with the polar head group of the surfactant attached to and the hydrophobic tail extending away from the Fe3O4.

Fig. 4. Scheme showing the formation of magnetite nanoparticles in thermal decomposition method [Ezekial Fisher, Andrew R. Barron, Synthesis of Magnetite Nanoparticles, Connexions module: m22167].

B. Microemulsion method:

A microemulsion is defined as a thermodynamically stable isotropic dispersion of two immiscible liquids, since the microdomain of either or both liquids has

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been stabilized by an interfacial film of surface-active molecules [92]. In water-in-oil microemulsions, the aqueous phase is dispersed as microdroplets surrounded by a monolayer of surfactant molecules in the continuous hydrocarbon phase [93]. When a soluble metal salt is incorporated in the aqueous phase of the microemulsion, it will reside in the aqueous microdroplets surrounded by oil. These microdroplets will continuously collide, coalesce, and break again [94]. Conceptually, when reactants A and B are dissolved in two identical water-in-oil microemulsions, they will form an AB precipitate on mixing. The growth of these particles in microemulsions can be conceptualized as a progress of interdroplet exchange and nuclei aggregation [95–97]. The finely dispersed precipitate so produced can be extracted from the surfactants.

Fig. 5. Structure of reverse micelles formed by dissolving AOT, a surfactant, in n-hexane. The inner core of the reverse micelle is hydrophilic and can dissolve water-soluble compounds. The size of these inner aqueous droplets can be modulated by controlling the parameter Wo (Wo ¼ [water]/[surfactant]).[64]

C. Co-precipitation method:

The co-precipitation method consists of precipitation of Fe3O4 by addition of a strong base to a solution of Fe2+ and Fe3+ salts in water [63]. This method is very simple, inexpensive and produces highly crystalline Fe3O4. The general size of Fe3O4 produced by co-precipitation is in the 15 to 50 nm range and can be controlled by reaction conditions; however a large size distribution of nanoparticles is produced by this method. Aggregation of particles is also observed with aqueous methods.

 

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Co-precipitation method has been used for this work.

3.1.2. Materials:

A. Reagent grade iron (II) ammonium sulphate [(NH4)2SO4.FeSO4.6H2O] [392].

Mohr's Salt, or ammonium iron sulfate, is a double salt of iron sulfate and ammonium sulphate. It appears as blue-green powder. Mohr's salt is preferred over iron (II) sulfate for titration purposes as it is much less affected by oxygen in the air than iron (II) sulfate, solutions of which tend to oxidise to iron (III). The oxidation of solutions of iron (II) is very pH dependent, occurring much more readily at high pH. The ammonium ions make solutions of Mohr's salt slightly acidic, which prevents this oxidation from occurring.

B. Anhydrous iron (III) chloride (FeCl3) [162].

The colour of iron (III) chloride crystals depends on the viewing angle: by reflected light the crystals appear dark green, but by transmitted light they appear purple-red. Anhydrous iron (III) chloride is deliquescent, forming hydrated hydrogen chloride mists in moist air. When dissolved in water, iron (III) chloride undergoes hydrolysis and gives off heat in an exothermic reaction. The resulting brown, acidic, and corrosive solution is used as a coagulant in sewage treatment and drinking water production, and as an etchant for copper-based metals in printed circuit boards. Anhydrous iron (III) chloride is a fairly strong Lewis acid, and it is used as a catalyst in organic synthesis. Iron (III) chloride has a relatively low melting point and boils at around 315 °C. The vapour consists of the dimer Fe2Cl6 which increasingly dissociates into the monomeric FeCl3 (D3h point group molecular symmetry) at higher temperature, in competition with its reversible decomposition to give iron (II) chloride and chlorine gas.  Iron (III) chloride adopts the BiI3 structure, which features octahedral Fe(III) centres interconnected by two-coordinate chloride ligands.

C. Potassium hydroxide pellets (KOH) [56].

Potassium hydroxide is usually sold as translucent pellets, which will become tacky in air because KOH is hygroscopic. Consequently, KOH characteristically contains varying amounts of water. Its dissolution in water is strongly exothermic, meaning the process gives off significant heat. Concentrated aqueous solutions are sometimes called potassium lyes. At higher temperatures,

25 

 

solid KOH crystallizes in the NaCl motif. The OH group is either rapidly or randomly disordered so that the OH- group is effectively a spherical anion of radius 1.53 Å (between Cl- and F- in size). At room temperature the OH- groups are ordered and the environment about the K+ centers is distorted with K+---OH- distances ranging from 2.69 to 3.15 Å, depending on the orientation of the OH group. KOH forms a series of crystalline hydrates, namely the monohydrate KOH·H2O, the dihydrate KOH·2H2O, and the tetrahydrate KOH·4H2O. KOH exhibits high thermal stability. KOH sublimes unchanged at 400 °C; the gaseous species is dimeric. Even at high temperatures, dehydration does not occur.

26 

 

D. Hydrochloric Acid (HCl) [12 N].

Hydrochloric acid is the solution of hydrogen chloride (HCl) in water. It is a highly corrosive, strong mineral acid . It is found naturally in gastric acid. Hydrogen chloride (HCl) is a monoprotic acid, which means it can dissociate (i.e., ionize) only once to give up one H+ ion (a single proton). In aqueous hydrochloric acid, the H+ joins a water molecule to form a hydronium ion, H3O+ and liberates Cl-.

HCl + H2O → H3O+ + Cl−

The physical properties of hydrochloric acid, such as boiling and melting points, density, and pH depend on the concentration or molarity of HCl in the acid solution. They range from those of water at very low concentrations approaching 0% HCl to values for fuming hydrochloric acid at over 40% HCl.

E. Cetyl trimethylammonium bromide (CTAB) [  ((C16H33)N(CH3)3)Br] [364].

Cetrimonium bromide is one of the components of the topical antiseptic cetrimide. The cetrimonium (or hexadecyltrimethylammonium) cation is an effective antiseptic agent against bacteria and fungi. It is a cationic surfactant. Its uses include providing a buffer solution for the extraction of DNA. It has been widely used in synthesis of gold nanoparticles (e.g., spheres, rods, bipyramids). It is also widely used in hair conditioning products. As any surfactant, it forms micelles in aqueous solutions. At 303 K (30 °C) it forms micelles with aggregation number 75-120 (depending on method of determination, usually avg. ~95) and degree of ionization α (fractional charge) 0.2 - 0.1 (from low to high concentration). Standard constant of Br- counterion binding to the micelle at 303 K (30 °C), calculated from Br- and CTA+ ion selective electrode measurements and conductometric data by using literature data for micelle size (r = ~3 nm), extrapolated to the critical micelle

27 

 

concentration is K° ≈ 400 (it varies with total surfactant concentration so it is extrapolated to the point at which the concentration of micelles is zero).

F. Sodium borohydride [NaBH4][37].

Sodium borohydride is also known as sodium tetrahydroborate. This white solid, usually encountered as a powder, is a specialty reducing agent used in the manufacture of pharmaceuticals and other organic and inorganic compounds. It is soluble in methanol and water, but reacts with both in the absence of base. Sodium borohydride is an odorless white to gray-white microcrystalline powder which often forms lumps.NaBH4 has three known polymorphs: α, β and γ. The stable phase at room temperature and pressure is α-NaBH4, which is cubic and adopts an NaCl-type structure, in the Fm3m space group. At a pressure of 6.3 GPa, the structure changes to the tetragonal β-NaBH4 (space group P421c) and at 8.9 GPa, the orthorhombic γ-NaBH4 (space group Pnma) becomes the most stable. Sodium borohydride is a particularly dangerous laboratory reagent. It is highly corrosive, and will cause burns upon contact with any area of the body. It is harmful if swallowed, inhaled or absorbed through the skin.

G. Double distilled water.

 

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3.1.3. Protocol:

Step 1: 1.25 ml of 1 M HCl was mixed with 6.25 ml double distilled (dd) water to prepare 7.5 ml aqueous acid solution.

Step 2: 0.41 g FeCl3 having molecular mass 162 (2.5 mmoles) and 0.49 g (NH4)2SO4.FeSO4.6H2O having molecular mass 392 (1.25 mmoles) powders were added to the aqueous acid solution prepared in Step 1.

Step 3: 1.52 g of CTAB was added in the ion solution.

Step 4: The resulting solution (pH ~1.04) was added dropwise into 25 ml of 1 M KOH solution (pH ~13.11) along with 1.25 mg NaBH4 under vigorous stirring.

Upon introduction of the Fe ion solution, the KOH solution turned black, suggesting precipitation of particles had occurred.

Step 5: The reaction mixture was stirred further for 30 min, after addition of all of the Fe ion solution.

Step 6: The colloid solution (pH ~11) obtained was then centrifuged at 5000 rpm for 15-20 minutes, and the supernatant solution removed from the precipitate by decantation.

Step 7: Precipitate was rinsed with dd water 4-5 times.

Step 8: A part of the precipitate was freeze dried to obtain particles for subsequent characterization.

Step 9: The remainder was dispersed in dd H2O (pH ~7).

All the above experimental procedures were carried out at room temperature (~22 0C). The black colloid solution obtained was used for further coating work. It was noticed that the colour of colloid solution slowly changed from black to brown, suggesting that the as-synthesized particles were not chemically stable and oxidized further.

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3.1.4. Chemistry of Synthesis:

The chemical reaction of Fe3O4 precipitation is given in the following figure.

Fig.6. Scheme showing the reaction mechanism of magnetite particle formation from an aqueous mixture of ferrous and ferric ions by addition of a base. The precipitated magnetite is black in colour.[64]

The hydrolysis of iron (III) species proceeds through the formation of low molecular weight species (Eq. (2.1)) and that above OH/Fe ~ 1 these species interact to produce polynuclear species (Eq. (2.2)).

Fe3+ + H2O → FeOH2+ + H+ (2.1a)

FeOH2+ + H2O → Fe(OH)2+ + H+ (2.1b)

2FeOH2+ → Fe2(OH)24+ (2.2)

The formation of the dimer in equation 2.2 is a fast reaction with a kfw = 630 m/s whereas the dissolution is very slow in the absence of protons, which suggest that further polymerisation may be very fast [65].

Similarly, hydrolysis of iron (II) species proceeds and low molecular weight species FeOH+ forms. In the alkaline solution these low molecular weight species of Fe(III) and Fe(II) gets oxidised and dehydrated to form magnetite nanoparticles.

The overall reaction may be written as follows [65, 66]:

Fe2+ + 2Fe3+ + 8OH- → Fe3O4 + 4H2O (1)

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According to the thermodynamics of this reaction, a complete precipitation of Fe3O4 should be expected between pH 9 and 14, while maintaining a molar ratio of Fe3+ : Fe2+ is 2:1 under a reducing environment. Otherwise, Fe3O4 might also be oxidized as

Fe3O4 + 0.25O2 + 4.5H2O → 3Fe(OH)3 (2)

This would critically affect the physical and chemical properties of the nanosized magnetic particles. In order to prevent them from possible oxidation in air as well as from aggregation, NaBH4 (reducing agent) and CTAB (cationic surfactant) were used. NaBH4 liberates hydrogen after hydrolysis in alkaline KOH solution. CTAB forms micelle and entraps nanoparticles within it preventing them to aggregation. It also helps in the crystallization process.

Genesis of the particles in the solution under optimum synthetic conditions takes place by the formation of tiny crystalline nuclei in a supersaturated medium, followed by crystal growth [67]. The latter process is controlled by mass transport and by the surface equilibrium of addition and removal of individual monomers, i.e., atoms, ions, or molecules. Hereby, the driving force for monomer removal (dissolution) increases with decreasing particle size. Thus, within an ensemble of particles with slightly different sizes, the large particles will grow at the cost of the small ones. This mechanism is called Ostwald ripening and is generally believed to be the main path of crystal growth [68]. This is a spontaneous process that occurs because larger crystals are more energetically favoured than smaller crystals. While the formation of many small crystals is kinetically favoured, (i.e. they nucleate more easily) large crystals are thermodynamically favoured. Thus, from a standpoint of kinetics, it is easier to nucleate many small crystals. However, small crystals have a larger surface area to volume ratio than large crystals. Molecules on the surface are energetically less stable than the ones already well ordered and packed in the interior. Large crystals, with their greater volume to surface area ratio, represent a lower energy state. Thus, many small crystals will attain a lower energy state if transformed into large crystals. But Ostwald ripening does not happen all the time. One reason is that the nucleation of many small crystals reduces the amount of super saturation and thus, the thermodynamically favoured large crystals never get a chance to appear.

31 

 

Another view of crystal growth has emerged from recent experiments by Penn and Banfield [69–71]. They observed that anatase and iron oxide nanoparticles having a size of a few nanometers can coalesce under hydrothermal conditions called oriented attachment. In the so-formed aggregates, the crystalline lattice planes may be almost perfectly aligned or dislocated at the contact areas between the adjacent particles, leading to defects in the finally formed bulk crystals. They presented strong evidence that this type of crystal growth plays an important role in earth history during mineral formation. Other authors also proposed oriented attachment during crystal growth of TiO2 [72] and for micrometer-sized ZnO particles during the formation of rod-like ZnO microcrystals [73].

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3.2. Surface Modification:

In the preparation and storage of nanoparticles in colloidal form, the stability of the colloid is of utmost importance.  In the absence of any surface coating, magnetic iron oxide particles have hydrophobic surfaces with a large surface area to volume ratio. Due to hydrophobic interactions between the particles, these particles aggregate and form large clusters, resulting in increased particle size. These clusters exhibit strong magnetic dipole–dipole attractions between them and show ferromagnetic behaviour [74]. When two large-particle clusters approach one another, each of them comes into the magnetic field of the neighbour. Besides the arousal of attractive forces between the particles, each particle is in the magnetic field of the neighbour and gets further magnetized [75]. The adherence of remnant magnetic particles causes a mutual magnetization, resulting in increased aggregation properties. Since particles are attracted magnetically, in addition to the usual flocculation due to Vander Waals force, surface modification is often indispensable. For effective stabilization of iron oxide nanoparticles, often a very high requirement of density for coating is desirable. Some stabilizer such as a surfactant or a polymer is usually added at the time of preparation to prevent aggregation of the nanoscale particulate. Most of these polymers adhere to surfaces in a substrate-specific manner [76]. Scheme showing different strategies for fabrication and surface modification of magnetic iron oxide nanoparticles is shown in figure 6.

Fig. 7. Scheme showing different strategies for fabrication and surface modification of magnetic iron oxide nanoparticles. Smaller and more uniform nanoparticles can be prepared inside the aqueous droplets of reverse micelles. Also, the particles below 100nm can evade RES and have long blood circulation times.[64]

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Nanoparticle coatings may be comprised of several materials.  including both inorganic and polymeric materials[77–79]. Polymeric coating materials can be classified into synthetic and natural. Polymers based on poly(ethylene-co-vinyl acetate), poly(vinylpyrrolidone) (PVP), poly(lactic-co-glycolic acid) (PLGA), poly(ethyleneglycol) (PEG), poly(vinyl alcohol) (PVA), etc. are typical examples of synthetic polymeric systems[80–82]. Natural polymer systems include use of gelatin, dextran, chitosan, pullulan, etc. [83–87]. Various surfactants, e.g. sodium oleate, dodecylamine, sodium carboxymethylcellulose, are also usually used to enhance dispersibility in an aqueous medium [88–90]. Figure 7. provides a list of materials that could be used to stabilize the nanoparticles along with their biomedical applications.

Fig. 8. Different polymers/molecules which can be used for nanoparticle coating to stabilize the ferrofluids and also for other biological applications[64]

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3.2.1. Material:

Glutathione (GSH) [C10H17N3O6S] [307] has been used for the surface modification of magnetite nanoparticles for our work.

Glutathione (GSH) is a tripeptide. It contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. IUPAC name of GSH is γ-L-Glutamyl-L-cysteinylglycine. Glutathione, an antioxidant, protects cells from toxins such as free radicals. Glutathione exists in reduced (GSH) and oxidized (GSSG) states. In the reduced state, the thiol group of cysteine is able to donate a reducing equivalent (H++ e-) to other unstable molecules, such as reactive oxygen species. In donating an electron, glutathione itself becomes reactive, but readily reacts with another reactive glutathione to form glutathione disulfide (GSSG). Such a reaction is possible due to the relatively high concentration of glutathione in cells (up to 5 mM in the liver). GSH can be regenerated from GSSG by the enzyme glutathione reductase. GSH is an essential component of plants and human cell systems, as an organocatalyst. An additional benefit of using glutathione is the presence of the highly reactive thiol group, which can be used for anchoring to the nano-ferrite surfaces, keeping active sites free for catalysis.

3.2.2. Protocol:

Step 1: 150 mg of GSH has been added pinch-by-pinch to the black colloidal solution obtained after the synthesis of magnetite nanoparticles under continuous stirring condition.

Step 2: The colloidal solution was stirred for additional 15 minutes after the addition of all of the GSH

Step 3: The mixture was incubated overnight at 40C.

35 

 

Step 4: The mixture was centrifuged at 5000 rpm for 15 minutes and the supernatant was decanted off to remove any free GSH molecules.

Step 5: Washing was done for 3-4 times.

Step 6: After washing some part of the precipitate was freeze dried and rest was re-suspended in dd water for protein/enzyme coupling work.

3.2.3. Chemistry of surface modification using Glutathione:

Glutathione acts as oxygen scavenger and reducing agent. It has a highly reactive thiol group which liberates H+ on oxidation and thus gets adsorbed on the surface of Fe3O4 nanoparticles. It does so by making thiol bond with the Fe ions present on the surface of the nanoparticles as shown in the figure below.

Fig. 9. Surface modification of magnetite nanoparticle using glutathione.[91]

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3.3. Characterization of nanoparticles(GT-) and glutathione attached nanoparticles(GT+)

Nanoparticles synthesized using the co-precipitation method and glutathione attached nanoparticles were characterized using two methods:

3.3.1. FT/IR spectroscopy

3.3.2. XRD Studies

3.3.1. FT/IR Spectroscopy:

Infrared spectroscopy (IR spectroscopy) is the subset of spectroscopy that deals with the infrared region of the electromagnetic spectrum. It covers a range of techniques, the most common being a form of absorption spectroscopy. As with all spectroscopic techniques, it can be used to identify compounds or investigate sample composition. The infrared portion of the electromagnetic spectrum is divided into three regions; the near-, mid- and far- infrared, named for their relation to the visible spectrum. The far-infrared, approximately 400-10 cm-1 (1000–30 μm), lying adjacent to the microwave region, has low energy and may be used for rotational spectroscopy. The mid-infrared, approximately 4000-400 cm-1 (30–2.5 μm) may be used to study the fundamental vibrations and associated rotational-vibrational structure. The higher energy near-IR, approximately 14000-4000 cm-1 (2.5–0.8 μm) can excite overtone or harmonic vibrations. The names and classifications of these sub regions are merely conventions. They are neither strict division nor based on exact molecular or electromagnetic properties.  Infrared spectroscopy exploits the fact that molecules have specific frequencies at which they rotate or vibrate corresponding to discrete energy levels (vibrational modes). These resonant frequencies are determined by the shape of the molecular potential energy surfaces, the masses of the atoms and, by the associated vibronic coupling. In order for a vibrational mode in a molecule to be IR active, it must be associated with changes in the permanent dipole. The resonant frequencies can be related to the strength of the bond, and the mass of the atoms at either end of it. Thus, the frequency of the vibrations can be associated with a particular bond type. The infrared spectrum of a sample is collected by passing a beam of infrared light through the sample. Examination of the transmitted light reveals how much energy was absorbed at each wavelength. This can be done with a

37 

 

monochromatic beam, which changes in wavelength over time, or by using a Fourier transform instrument to measure all wavelengths at once. From this, a transmittance or absorbance spectrum can be produced, showing at which IR wavelengths the sample absorbs. Analysis of these absorption characteristics reveals details about the molecular structure of the sample.

Fourier transform infrared (FT/IR) spectroscopy is a measurement technique for collecting infrared spectra. Instead of recording the amount of energy absorbed when the frequency of the infra-red light is varied (monochromator), the IR light is guided through an interferometer. After passing through the sample, the measured signal is the interferogram. Performing a mathematical Fourier transform on this signal results in a spectrum identical to that from conventional (dispersive) infrared spectroscopy. FT/IR spectrometers are cheaper than conventional spectrometers because building of interferometers is easier than the fabrication of a monochromator. In addition, measurement of a single spectrum is faster for the FT/IR technique because the information at all frequencies is collected simultaneously. This allows multiple samples to be collected and averaged together resulting in an improvement in sensitivity.

Fig. 10. Typical apparatus for IR spectroscopy.

A beam of infrared light is produced and split into two separate beams. One is passed through the sample, the other passed through a reference which is often the substance the sample is dissolved in or mixed with. The beams are both reflected back towards a detector, however first they pass through a splitter

38 

 

which quickly alternates which of the two beams enters the detector. The two signals are then compared and a printout is obtained. A reference is used for two reasons:

* This prevents fluctuations in the output of the source affecting the data

* This allows the effects of the solvent or material to be cancelled out (the reference is usually a pure form of the solvent or material the sample is in)

Sample preparation:

Solid samples can be prepared in four major ways.

a. The first is to crush the sample with a mulling agent (usually nujol) in a marble or agate mortar, with a pestle. A thin film of the mull is applied onto salt plates and measured.

b. The second method is to grind a quantity of the sample with a specially purified salt (usually potassium bromide) finely (to remove scattering effects from large crystals). This powder mixture is then crushed in a mechanical die press to form a translucent pellet through which the beam of the spectrometer can pass.

c. The third technique is the Cast Film technique, which is used mainly for polymeric materials. The sample is first dissolved in a suitable, non hygroscopic solvent. A drop of this solution is deposited on surface of KBr or NaCl cell. The solution is then evaporated to dryness and the film formed on the cell is analysed directly. Care is important to ensure that the film is not too thick otherwise light cannot pass through. This technique is suitable for qualitative analysis.

d. The final method is to use microtomy to cut a thin (20-100 micrometre) film from a solid sample. This is one of the most important ways of analysing failed plastic products for example because the integrity of the solid is preserved.

In this work the second method was used for both the samples (GT- and GT+). Small quantity of GT- nanoparticles and GT+ nanoparticles were grind with potassium bromide (KBr) to get fine powders. These samples were then crushed in a mechanical die press and translucent pellets were got which were inserted into the JASKO FT/IR-5300 apparatus to get the spectrum.

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3.3.2. XRD Studies:

X-ray diffraction finds the geometry or shape of a molecule using X-rays. X-ray diffraction techniques are based on the elastic scattering of X-rays from structures that have long range order. The most comprehensive description of scattering from crystals is given by the dynamical theory of diffraction.[98]

X-rays primarily interact with electrons in atoms. When X-ray photons collide with electrons, some photons from the incident beam will be deflected away from the direction where they originally travel. If the wavelength of these scattered X-rays did not change (meaning that X-ray photons did not lose any energy), the process is called elastic scattering (Thompson Scattering) in that only momentum has been transferred in the scattering process. These are the X-rays that we measure in diffraction experiments, as the scattered X-rays carry information about the electron distribution in materials. On the other hand, in the inelastic scattering process (Compton Scattering), X-rays transfer some of their energy to the electrons and the scattered X-rays will have different wavelength than the incident X-rays. Diffracted waves from different atoms can interfere with each other and the resultant intensity distribution is strongly modulated by this interaction. If the atoms are arranged in a periodic fashion, as in crystals, the diffracted waves will consist of sharp interference maxima (peaks) with the same symmetry as in the distribution of atoms. Measuring the diffraction pattern therefore allows us to deduce the distribution of atoms in a material. The peaks in a X-ray diffraction pattern are directly related to the atomic distances given by Bragg’s law:

2d • sinθ = n λ

Where d is the inter-plane distance, θ is the scattering angle, n is an integer representing the order of the diffraction peak and λ the wavelength of the X-ray.

Fig. 11. (a) showing different lattice planes (b) showing Bragg’s law schematically.[99]

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Powder diffraction (XRD) is a technique used to characterize the crystallographic structure, crystallite size (grain size), and preferred orientation in polycrystalline or powdered solid samples. It is commonly used to identify unknown substances, by comparing diffraction data against a database maintained by the International Centre for Diffraction Data. It may also be used to characterize heterogeneous solid mixtures to determine relative abundance of crystalline compounds and, when coupled with lattice refinement techniques, such as Rietveld refinement, can provide structural information on unknown materials. It is also a common method for determining strains in crystalline materials. An effect of the finite crystallite sizes is seen as a broadening of the peaks in an X-ray diffraction as explained by the Scherrer Equation.

This technique is perhaps the most widely used X-ray diffraction technique for characterizing materials. As the name suggests, the sample is usually in a powdery form, consisting of fine grains of single crystalline material to be studied. The technique is used also widely for studying particles in liquid suspensions or polycrystalline solids (bulk or thin film materials).The term 'powder' really means that the crystalline domains are randomly oriented in the sample. Therefore when the 2-D diffraction pattern is recorded, it shows concentric rings of scattering peaks corresponding to the various d spacing in the crystal lattice. The positions and the intensities of the peaks are used for identifying the underlying structure (or phase) of the material. For example, the diffraction lines of graphite would be different from diamond even though they both are made of carbon atoms. This phase identification is important because the material properties are highly dependent on structure. Powder diffraction data can be collected using either transmission or reflection geometry, as shown below. Because the particles in the powder sample are randomly oriented, these two methods will yield the same data.

Fig. 12. Two methods of data collection in powder diffraction technique.[99]

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3.4. Coupling protein onto GT+ nanoparticles.

In this work protein has been coupled onto the GT+ nanoparticles using the carbodiimide coupling method. Carbodiimide is a functional group consisting of the formula HN=C=NH. These compounds are formed by dehydration of ureas or thioureas.  In synthetic organic chemistry, compounds containing the carbodiimide functionality are dehydration agents and are often used to activate carboxylic acids towards amide or ester formation. Additives, such as N-hydroxybenzotriazole or N-hydroxysuccinimide, are often added to increase yields and decrease side reactions. The formation of an amide using a carbodiimide is straightforward, but with several side reactions complicating the subject. The acid 1 will react with the carbodiimide to produce the key intermediate: the O-acylisourea 2, which can be viewed as a carboxylic ester with an activated leaving group. The O-acylisourea will react with amines to give the desired amide 3 and urea 4. The side reaction of the O-acylisourea 2 produce both desired and undesired products. The O-acylisourea 2 can react with an additional carboxylic acid 1 to give an acid anhydride 5, which can react further to give the desired amide 3. The main undesired reaction pathway involves the rearrangement of the O-acylisourea 2 to the stable N-acylurea 6. The use of solvents with low-dielectric constants such as dichloromethane or chloroform can minimize this side reaction.

Fig. 13. Mechanism of amide formation using carbodiimide [http://en.wikipedia.org/wiki/Carbodiimide]

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3.4.1. Material:

A. Carbodiimide

There are various types of carbodiimides used for amide formation or protein coupling like DDC (N,N'-dicyclohexylcarbodiimide), DIC (N,N'-diisopropyl carbodiimide), EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide), etc.

For this work EDC [molecular mass 155] coupling method was used. EDC is a water soluble carbodiimide usually obtained as the hydrochloride. It is typically employed in the 4.0-6.0 pH range. It is generally used as a carboxyl activating agent for the coupling of primary amines to yield amide bonds. Additionally, EDC can also be used to activate phosphate groups. Common uses for this carbodiimide include peptide synthesis, protein cross linking to nucleic acids and preparation of immunoconjugates.

B. MES Buffer:

MES is the common name for the compound 2-(N-morpholino)ethanesulfonic acid. Its chemical structure contains a morpholine ring. It has a molecular mass of 195 and the chemical formula is C6H13NO4S.  MES is used as a buffering agent in biology and biochemistry. It was developed as one of Good's buffers in the 1960s, with pKa value of 6.15. These buffers were developed with the following criteria in mind: midrange pKa, maximum water solubility and minimum solubility in all other solvents, minimal salt effects, minimal change in pK with temperature, chemically and enzymatically stable, minimal absorption in visible or UV spectral range and reasonably easily synthesized.

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C. Protein/Enzyme:

Two proteins have been used for the coupling purpose. BSA (Bovine Serum Albumin) and HRP (Horse Radish Peroxidase). BSA was used to check whether protein is getting coupled or not because it is easily available and cheap to be used for standardizing process.

a. BSA:

Bovine serum albumin, bovine albumin, BSA, is a serum albumin protein that has numerous biochemical applications including ELISAs (Enzyme-Linked Immunosorbent Assay), immunoblots, and immunohistochemistry. It is also used as a nutrient in cell and microbial culture. In restriction digests, BSA is used to stabilize some enzymes during digestion of DNA and to prevent adhesion of the enzyme to reaction tubes and other vessels. This protein does not affect other enzymes that do not need it for stabilization. BSA is used because of its stability, its lack of effect in many biochemical reactions, and its low cost since large quantities of it can be readily purified from bovine blood, a by-product of the cattle industry. It is a 69323 Da protein with 607 residues.

b. HRP:

The enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.

Horseradish peroxidase is a 44,173 dalton glycoprotein with 4 lysine residue for conjugation to a labelled molecule. It produces a colour, fluorimetric or luminescent derivative of the labelled molecule allowing it to be detected and quantified. HRP is often used in conjugates (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. For example, an antibody conjugated to HRP may be used to detect a small amount of a specific protein in a western blot. Here, the antibody provides the specificity to locate the protein of interest and the HRP enzyme, in the presence of a substrate, produces a detectable signal. It is also commonly used in techniques such as ELISA and Immunohistochemistry. It is ideal in many respects for these applications because it is smaller, more stable and less expensive than other popular alternatives such as alkaline phosphatase. It also has a high turnover rate that allows generation of strong signals in a relatively

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short time span. Alone, the HRP enzyme, or conjugates thereof, is of little value; its presence must be made visible using a substrate that when oxidized by HRP using hydrogen peroxide as the oxidizing agent, yields a characteristic change that is detectable by spectrophotometric methods.

3.4.2. Protocol:

A. BSA coupling:

Step 1: 150 μl of 26 mg/ml (stock) EDC solution was mixed with 1.5 ml of 10.46 mg/ml GT+ colloidal solution.

Step 2: It was then incubated for 1.5 hours at 4 0C.

Step 3: 500 μl of 10 mg/ml (stock) BSA solution was added to 5 ml of 10 mM MES buffer (pH 5.5).

Step 4: After incubation BSA-MES solution was added to the EDC-GT+ colloidal solution.

Step 5: It was then mixed properly and kept overnight for the coupling to take place.

Step 6: The mixture was centrifuged at 5000 rpm for 15 minutes till the supernatant was free from any protein (tested using Bradford Reagent).

Step 7: Precipitate was then suspended in 2ml of 10 mM MES buffer (pH 5.5) and used for protein estimation.

B. HRP coupling:

Step 1: 0.5 ml of 10.46 mg/ml GT+ colloidal solution was mixed with10 ml of 10 mM MES buffer (pH 5.5).

Step 2: 200 μl of 130 mg/ml (stock) EDC solution was added to the colloidal solution. It was then incubated for 1.5 hours at 4 0C.

Step 3: The mixture was centrifuged at 5000 rpm for 15 minutes 3-4 times.

Step 4: Precipitate was suspended in 5 ml of PO42- buffer (pH 7.0).

Step 5: 400 μl of HRP solution was added to the suspended solution and incubated overnight at -4 0C under continuous stirring condition.

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Step 6: After incubation it was centrifuged at 5000 rpm for 15 min and washed till supernatant was free from protein (tested using H2O2 solution).

Step 7: Precipitate was then suspended in PO42- buffer (pH 7.0) and used for protein assay.

3.5. Characterization of protein bound GT+ nanoparticles

3.5.1. Bradford protein estimation

The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is subjective, i.e. dependent on the amino acid composition of the measured protein.

Bradford reagent: 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol is dissolved and added to 100 ml 85% (w/v) phosphoric acid. Solution is diluted to 1 liter when the dye has completely dissolved, and filtered through Whatman #1 paper just before use.

The Bradford assay, a colorimetric protein assay, is based on an absorbance shift in the dye ‘Coomassie’ when the previously red form coomassie reagent changes and stabilizes into coomassie blue by the binding of protein. During the formation of this complex, two types of bond interaction take place: the red form of coomassie dye first donates its free proton to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure bind non-covalently to the non-polar region of the dye via van der Waals forces, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. Binding of the protein stabilizes the blue form of coomassie dye, thus the amount of complex present in solution is a measure for the protein concentration by use of an absorbance reading. The cationic (unbound) forms are green or red while binding of the dye to protein stabilizes the blue anionic form. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample. Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. An exception of note is elevated concentrations of detergent. Other interference may come from the buffer used when preparing the protein

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sample. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. The Bradford assay is linear over a short range, typically from 2 µg/ml to 120 µg/ml; often making dilutions of a sample necessary before analysis.

Protocol:

Step 1: Stock solution of BSA was prepared by dissolving 10 mg of BSA in 1ml of dd water.

Step 2: Several dilutions of the stock BSA solution were prepared. The series of dilutions were having 50, 100,150, 200, 300, 400 and 500 µg of proteins in 1000 µl volume (made up by dd water).

Step 3: 50 µl solution was taken from each standard BSA dilutions in 7 different appendoffs and 950 µl of Bradford reagent was added in each appendoff to make the volume to 1 ml.

Step 4: 50 µl solution was taken from BSA coupled GT+ nanoparticle suspension and 950 µl of Bradford reagent was added to make the volume to 1 ml.

Step 5: Two blanks were also made using 50 µl of water in two different appendoffs (for calibrations) and 950 µl of Bradford reagent was added to make the volume to 1 ml.

In total there were 10 samples.

Step 6: All the samples were then incubated for 5 minutes at room temperature.

Step 7: After incubation absorbance was measured in UV-160 spectrophotometer at 595 nm.

Step 8: Using the absorbance data of BSA standards a standard curve was plotted for absorbance versus concentration of protein and the unknown concentration of the protein in the BSA bound GT+ nanoparticle suspension was determined.

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3.5.2. Confocal Laser microscopy:

Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning or tomography. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically-complex objects. For opaque specimens, this is useful for surface profiling, while for non-opaque specimens, interior structures can be imaged. For interior imaging, the quality of the image is greatly enhanced over simple microscopy because image information from multiple depths in the specimen is not superimposed. A conventional microscope "sees" as far into the specimen as the light can penetrate, while a confocal microscope only images one depth level at a time. In effect, the CLSM achieves a controlled and highly limited depth of focus. In a confocal laser scanning microscope, a laser beam passes through a light source aperture and then is focused by an objective lens into a small (ideally diffraction limited) focal volume within or on the surface of a specimen. In biological applications especially, the specimen may be fluorescent. Scattered and reflected laser light as well as any fluorescent light from the illuminated spot is then re-collected by the objective lens. A beam splitter separates off some portion of the light into the detection apparatus, which in fluorescence confocal microscopy will also have a filter that selectively passes the fluorescent wavelengths. After passing a pinhole, the light intensity is detected by a photo detection device (usually a photomultiplier tube (PMT) or avalanche photodiode), transforming the light signal into an electrical one that is recorded by a computer. The detector aperture obstructs the light that is not coming from the focal point, as shown by the dotted gray line in the image. The out-of-focus light is suppressed: most of the returning light is blocked by the pinhole, which results in sharper images than those from conventional fluorescence microscopy techniques and permits one to obtain images of planes at various depths within the sample.

 

[http://en.wikipedia.org/wiki/Confocal_laser_scanning_microscopy]

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Confocal microscopy provides the capacity for direct, non-invasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution.[4] Biological samples are often treated with fluorescent dyes to make selected objects visible. However, the actual dye concentration can be low to minimize the disturbance of biological systems: some instruments can track single fluorescent molecules. Also, transgenic techniques can create organisms that produce their own fluorescent chimeric molecules (such as a fusion of GFP, green fluorescent protein with the protein of interest).

Fluorescein [C20H12O5] [332]: Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, in forensics and serology to detect latent blood stains, and in dye tracing. Fluorescein has an absorption maximum at 494 nm and emission maximum of 521 nm (in water). Also, fluorescein has an isosbestic point (equal absorption for all pH values) at 460 nm. Fluorescein is also known as a colour additive (D&C Yellow no. 7). The disodium salt form of fluorescein is known as D&C Yellow no. 8.

Fluorescein has a pKa of 6.4 and its ionization equilibrium leads to pH-dependent absorption and emission over the range of 5 to 9. Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately 3 and 4 ns, which allows for pH determination from non-intensity based measurements. The lifetimes can be recovered using time-correlated single photon counting or phase-modulation fluorimetry. There are many fluorescein derivatives, for example fluorescein isothiocyanate, often abbreviated as FITC. FITC is the original fluorescein molecule functionalized with an isothiocyanate group (-N=C=S), replacing a hydrogen atom on the bottom ring of the structure. This derivative is reactive towards amine groups on proteins inside cells. A succinimidyl-ester functional group attached to the

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fluorescein core, creating NHS-fluorescein, forms another common amine reactive derivative.

Protocol:

Step 1: 5 mg of fluorescein dye was dissolved in 1 ml of water.

Step 2: 750 µl of BSA coupled GT+ nanoparticle suspension was mixed with 250 µl of fluorescein dye solution and incubated for 30 minutes at room temperature.

Step 3: It was then centrifuged at 5000 rpm for 15 minutes to remove unbound dye.

Step 4: After washing 1 drop of the dyed BSA coupled GT+ nanoparticle was taken on microscopic glass slide and dried at room temperature.

Step 5: The sample on the slide was observed under confocal laser scanning microscope.

3.5.3. Enzyme assay:

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.

 Enzyme activity = moles of substrate converted per unit time

= rate × reaction volume.

Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical and commonly-used value is 1 enzyme unit (EU) = 1 μmol min-1. 1 U corresponds to 16.67 nanokatals. The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min-1mg-1). Specific activity gives a measurement of the purity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of enzyme. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of enzyme. The SI unit is katal kg-1, but a more practical

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unit is μmol mg-1 min-1. Specific activity is a measure of enzyme processivity, usually constant for a pure enzyme.

In spectrophotometric assays, the course of the reaction is followed by measuring a change in how much light the assay solution absorbs. If this light is in the visible region a change in the colour of the assay can be seen, these are called colorimetric assays.

Peroxidase Assay using pyragallol:

Pyrogallol or benzene-1,2,3-triol is a white crystalline powder and a powerful reducing agent. When in alkaline solution, it absorbs oxygen from the air, turning purple from a colourless solution. It can be used in this way to calculate the amount of oxygen in air.

It has a molar mass of 126 g and it is harmful if swallowed and repeated or prolonged exposure to this compound is not recommended.

Purpurogallin or 2,3,4,6-tetrahydroxy-5H-benzocyclohepten-5-one.

The method of assay measures the oxidation of pyrogallol to purpurogallin by peroxidase when catalysed by peroxidase at 420 nm and at 20°C.

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Pyrogallol + H202 purpurogallin

Peroxidase breaks H2O2 in H2O and O2. Pyrogallol in the presence of O2 gets oxidised into purpurogallin which produces the colour change of the test solution and thus read in spectrophotometer. One unit of peroxidase is defined as the amount of enzyme required to catalyse the production of 1 mg of purpurogallin from pyrogallol in 20 seconds at 20°C under the assay conditions described.

Materials:

1. 100 mM Phosphate buffer (pH 6.0).

2. 0.5% H2O2 solution.

3. 5% (w/v) pyrogallol solution.

4. HRP coupled GT+ nanoparticle suspension.

Protocol:

Two samples were prepared namely “Blank” and “Test”.

Step 1: In sample Blank 139 µl of 100 mM phosphate buffer (pH 6.0), 53 µl of 0.5% H2O2 solution, 106 µl of 5% (w/v) pyrogallol solution and 700 µl water were added.

Step 2: In sample Test 139 µl of 100 mM phosphate buffer (pH 6.0), 53 µl of 0.5% H2O2 solution, 106 µl of 5% (w/v) pyrogallol solution, 700 µl water were added.

Step 3: 33 µl of the HRP coupled GT+ nanoparticle suspension was added in sample Test just before the start of taking the spectrophotometric reading.

Step 4: Spectrophotometer was set at 420 nm and the time interval between each reading was set as 20 seconds.

Step 5: The data was collected for both the samples for 180 seconds and the activity of enzyme in Test sample was calculated.

Peroxidase 

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3.5.4. Electrochemical studies:

Electrochemistry is the study of the movement and separation of charge in matter. As such, it is the study of the transfer of electrons. It is the presence, absence and movement of these wonderful, little, negatively-charged quanta that provide matter with the ability to hold or transfer charge. Most chemical reactions involve charge transfer, and therefore most chemistry, certainly the most interesting chemistry, is electrochemistry. Matter may hold charge, either positive or negative. The charge can be discrete and measureable or partial and diffuse. The statement that opposites attract either emerged from our understanding of electromagnetism or was an intuitive statement of the phenomenon. Thus, it is also intriguing that charged matter can maintain separations leading to interesting effects. Electrochemistry is the study of these phenomena and their relationship to chemical systems. Electrochemis