Chapter 3. Experimental And Characterization Techniques For LSMO Nanoparticles

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1 Experimental And Characterization Techniques For LSMO Nanoparticles

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3 3.1 Introduction This chapter focuses on the synthesis and experimental techniques employed for characterizations. There are many routes in the synthesis of MNPs. However, synthesis of the sample in single phase is the most imperative part of the research work. According to the literature survey, several methods have been embraced for the synthesis of LSMO which are reviewed in last chapter. In current research work, SPM LSMO NPs are synthesized by combustion method due to enormous advantages which has been discussed in below. The different analytical techniques used for phase analysis, composition study, elemental analysis, structural and morphological analysis of the prepared samples are described with principle and working. 3.2 Experimental Mechanism of combustion method Combustion synthesis (CS) is a versatile, simple and rapid process, which allows efficient synthesis of a variety of nanomaterials. In this process mainly a self-sustained reaction in homogeneous solution of different oxidizers (e.g., metal nitrates) and fuels (e.g., urea, glycine, hydrazides) takes place. It occurs at low temperature which offers a unique mechanism via a highly exothermic redox reaction to produce oxides. Mainly, depending on the type of the precursors, and on conditions used for the process organization, the CS may occurs as either volume or layer-by-layer propagating combustion modes. Several processing factors such as C/H ratio (type of fuel), the fuel to oxidizer ratio (F/O), and the water content of the precursor mixture and the ignition temperature mainly influences the combustion reaction. CS is also recognized as self-propagating high-temperature synthesis (SHS). In CS there is the exothermic oxidation of a fuel. The word combustion itself suggests flaming (gas-phase), smouldering heterogeneous Centre For Interdisciplinary Research, DYPU, Kolhapur 70

4 as well as explosive reaction. In this method, the powder characteristics such as crystallite size, surface area, size distribution and nature of agglomeration are basically controlled by enthalpy or flame temperature generated during combustion which itself dependent on the nature of the fuel and fuel-tooxidizer ratio [1]. Main advantages of CS are (i) Use of relatively simple equipment (ii) Formation of high-purity products (iii) Stabilization of metastable phases (iv) Formation of different sizes such as micro, nano and shape like spherical, hexagonal and rod like products. Uniform distribution of dopants takes place through the host material due to the atomic mixing of the reactants in the initial solution. This method has been proven to be a best technique to achieve various types of oxides at the nanometre scale and is used for a variety of technological applications and this broad range of oxides is prepared with an eye on their magnetic, mechanical, dielectric, catalytic, optical and luminescent characteristics. Out of various combustion sub types the solution combustion (SC) method of preparing oxide materials is a fairly recent development compared to SSC or SHS techniques. In present research SC is employed for synthesis of LSMO NPs. The important parameters of combustion that have been broadly studied are: type of flame, combustion temperature, evolved gases, air-fueloxidant ratio and chemical composition of the precursor reagents. The characteristic of the SC method is, after initiation locally, the self- sustained propagation of a reaction wave through the heterogeneous mixture of reactants. Under controlled conditions, SC reaction, generates a peculiar kind of burning or smoldering type flame, depending on the used fuel and oxidizerfuel ratio. The burning flame may be capable of endure for seconds or even minutes, while the smoldering flame does not rise or is extinguished in a few seconds. The type of flame in the combustion plays a vital role in controlling the particle size of as-synthesized materials. There are four important temperatures which can affect the reaction mechanism and final product Centre For Interdisciplinary Research, DYPU, Kolhapur 71

5 properties during the CS reaction. These are initial temperature, ignition temperature, adiabatic flame temperature and maximum flame temperature. Initial temperature is defined as the average temperature of the reagent solution before the reaction is ignited. Ignition temperature is the point at which the combustion reaction is dynamically activated without an additional supply of external heat. Adiabatic flame temperature represents the maximum combustion temperature achieved under adiabatic conditions. And the maximum flame temperature is the highest temperature reached in the actual configuration, i.e., under conditions that are not adiabatic [2]. Evolution of gases is another important parameter which affects on combustion reaction. Mainly, in CS, the powder morphology, particle size and surface area are directly related to the amount of gases that escape during combustion. The difference in particle size, using different fuels, depends on the number of moles of gaseous products released during combustion. Pores between the particles are produced when gases breaks large clusters. In this process one of the most important parameters in determining the properties of synthesized material is fuel-oxidant ratio. A fuel is a substance capable of breaking and burning the CH bonds (electrons acceptor). An oxidant is a substance which helps in burning, supplying oxygen (electrons donor) [3].The effect of gases on the morphology of the particles is studied by the oxidant fuel ratio. Excellent quality product homogeneity is accomplished by the use of chemical precursors intimately mixed. The type and amount of chemicals used in the reactions influences on the characteristic features of the resultant powders. The solubility of the fuel, the presence of water and type of fuel used, are essential. Mainly, in solution, mixtures of metal nitrates (oxidizers) and urea or glycine (fuel) are broken down quickly via deflagration burning or combustion. To realize the highly exothermic nature of this reaction, concepts used in propellant chemistry were used. A solid propellant contains an oxidizer like ammonium perchlorate and a fuel like carboxyl terminated Centre For Interdisciplinary Research, DYPU, Kolhapur 72

6 polybutadiene together with aluminium powder and some additives. The specific impulse (Is) of a propellant, which is a measure of energy released during combustion, is given by the ratio of thrust produced per pound of the propellant. It is expressed as, Isp k Molecularwtofgaseousproduts T (3.1) The highest heat T (chamber temperature in the rocket motor) is produced when the equivalence ratio (ɸ e = oxidizer/fuel ratio) is unity. The equivalence ratio of an oxidizer and fuel mixture is expressed in terms of the elemental stoichiometric coefficient, (Coefficient of oxidizing element in specific formula) (valency) e ( 1) (Coefficient of reducing element in specific formula) (valency) (3.2) A mixture is said to be stoichiometric when ɸ e = 1, fuel lean when ɸ e > 1, and fuel rich when ɸe < 1. Stoichiometric mixtures produce maximum energy. The oxidizer/fuel molar ratio (O/F) required for a stoichiometric mixture (ɸ e = 1) is estimated by summation of the total oxidizing and reducing valences in the oxidizer compounds and dividing it by the sum of the total oxidizing and reducing valences in the fuel compounds. In this kind of calculation, oxygen is the only oxidizing element; carbon, hydrogen, and metal cations are reducing elements and nitrogen is neutral. The oxidizing elements have positive valences and reducing elements have negative valences Synthesis of LSMO nanoparticles The strontium doped perovskite LSMO NPs were synthesized by solution combustion method by using PVA as a fuel. Our previous research Centre For Interdisciplinary Research, DYPU, Kolhapur 73

7 work reported that PVA fuel as a potential candidate for preparation of LSMO in biomedical field [4-5]. La (NO 3 ) 3 6H 2 O, Sr (NO 3 ) 2.4H 2 O and Mn (NO 3 ) 2 4H 2 O were used as the starting reactant precursors and were obtained from Sigma Aldrich (99.9%). The stoichiometric amounts of all these precursors which act as oxidants were dissolved separately in double distilled water (DDW) under constant stirring for about 10 minute to form the solution of uniform mixture of 0.1 M. The solution of PVA was prepared by same way by dissolving in DDW under constant stirring about an hour. The mixture was then kept in beaker and stirred for 30 min at 100 ºC to achieve the homogenous solution. The solution was then converted in to yellowish gel which was further subjected to hot plate preheated to 300 ºC. During this process, foams of LSMO produced along with sparks. Foam does not initiate ignition only helps to sustain combustion. At the time of ignition CO 2, H 2 O and N 2 gases evolved which were responsible for combustion to escape the foam, yielding a voluminous fluffy black coloured product. The maximum exothermic temperature of the redox reaction was reached between ºC. Assigning the +4, +1, +3, +2 and +2 valencies to the C, H, La 3+, Sr 2+, and Mn 2+, reducing elements, respectively, the -2 valency to O 2- oxidizer and assuming nitrogen with the valence 0, 4 then the ɸ e is determined according to the equation (3.2). For this stoichiometric combustion (ɸ e =1), 2.29% of the PVA solution is required to balance the oxidation and reduction valences in the solution. The resulting black coloured powder crushed with mortar and pestle, annealed at 800 ºC for 5 hour, and then used for further characterization. The possible combustion reaction is as follows: 0.7La (NO ) + 0.3Sr (NO ) + Mn (NO ) (-C H O) + 1.5O La Sr MnO H O N CO (3.3) Centre For Interdisciplinary Research, DYPU, Kolhapur 74

8 3.3 Characterization techniques In order to investigate different properties of LSMO NPs various characterization techniques are required. Different techniques provide different information, some about chemical and physical properties, and others about structure, morphology and geometry. The different techniques are used in the present work which includes X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP OES), Field Emission Scanning Electron Microscopy (FE-SEM), Vibrating sample Magnetometer (VSM), Transmission electron microscopy (TEM), Zeta measurement, Dynamic light scattering (DLS) and Induction Heating system etc Structural and phase analysis XRD XRD is a versatile non-destructive technique which is one of the important fundamental tools employed in solid state chemistry and materials science. This technique is used to identify crystalline phase, chemical composition, strain state, grain size preferred orientation and defect structure. X-rays are the electromagnetic radiations having wavelength 1 A The basic principles of X-ray diffraction are found in classic textbooks, e.g. by Cullity [8] and Guinebretiere [9]. The diffraction of X-rays mainly occurs only when the wavelength of the wave motion is of the same order of magnitude as the repeat distance between scattering centres. The scientist Bragg studied the diffraction from crystalline material and formulated the mathematical expression known as Bragg s law and is expressed as [10], 2dsin n (3.4) Centre For Interdisciplinary Research, DYPU, Kolhapur 75

9 Where, d is interplanar spacing, θ is diffraction angle, λ is wavelength of X- ray and n is order of diffraction. Fig. 3.1 (a) Schematic of X-ray diffractometer [6] (b) Benchtop X-ray diffraction instrument [7]. There are three methods used for the diffraction of X-rays as Laue method, rotating-crystal method and Powder method. Out of these three powder method is employed for whole proposed work, where, λ is fixed and θ is variable. Particularly, in present method, the samples to be studied is crushed to a very fine powder and then put in a rectangular plate of glass or aluminium. X-rays beam of single wavelength (monochromatic) is incident on the sample. Each particle of the sample is a small crystal oriented randomly with respect to the incident X-ray beam. The information about the atomic arrangements can be analysed by detecting the directions of diffracted X-rays beam. Typically, the crystal and phase structure can be confirmed by X-ray diffraction analysis. The schematic representation of XRD system is shown in the Fig (a) and (b). Identification of phases in a sample is studied from the d spacing using the standard JCPDS powder diffraction file and the reflections can be indexed with Miller indices. However, if the size of the diffracting tiny Centre For Interdisciplinary Research, DYPU, Kolhapur 76

10 crystal is small, there is no more complete destructive interference at θ±dθ, which broadens the peak corresponding to diffracted beam in proportion to the size of the tiny crystal and that can be used to calculate the particle size. The relation for the same is given by Debye Scherrer formula, 0.9 D cos (3.5) Where, D is particle size, θ is diffraction angle, λ the wavelength of X-rays and is Full Width at Half Maxima (FWHM). Fig. 3.2 A typical XRD pattern LSMO NPs. The phase composition, lattice parameter and the mean size of the crystallites of LSMO NPs were determined by XRD (RIGAKU Miniflex 600) equipped with a crystal monochromator employing Cu-K α radiation of wavelength 1.54 Å and applied scanning rate of 3ºmin -1, ranged from 20 to 80. The patterns were analysed by X'Pert High score software and compared with standard JCPDS (reference code: ). The average crystallite size was calculated from the broadening of the XRD peaks using the Scherrer s equation. Fig. 3.2 show a typical XRD pattern of LSMO NPs. Centre For Interdisciplinary Research, DYPU, Kolhapur 77

11 FT-IR The main aim of this technique is to find the changes in the intensity of beam of infrared radiation as a function of wavelength or frequency after it interacts with the specimen. Quantitative and qualitative measurements of the organic and inorganic samples can be determined with this technique. The spectra provides information about the identification of the chemical bonds in molecule by recording an infrared absorption spectrum that is like a molecular "fingerprint" [11-13]. In biomedicine the MNPs must be coated with organic or inorganic material in order to enhance stability, dispersibility and biocompatibility. The FT-IR analysis helps to understand the successful attachment of coating agent on the surface off MNPs. The schematic of the FT-IR spectrometer is shown in Fig. 3.3 (a). Fig. 3.3 (a) Block diagram of optical FT-IR Spectrometer [14], (b) FT-IR spectrum of LSMO NPs. Michelson interferometer is generally used in FT-IR spectrometry. The interferometer consists of two perpendicularly plane mirrors, one of which can travel in a direction perpendicular to the plane. A semi-reflecting film, known as beam splitter, bisects the planes of these two mirrors. The beam splitter material is decided according to the region to be examined. For the Centre For Interdisciplinary Research, DYPU, Kolhapur 78

12 mid- or near-infrared regions materials like germanium or iron oxide are covered onto an infrared-transparent substrate as like potassium bromide or caesium iodide to produce beam splitters. In this technique mainly, a chemical substance shows selective absorption in the infrared region. The wavelength of absorption depends on relative masses of the atoms, force constants of the bonds and geometry of atoms. Then the molecules of the chemical substance vibrate at many rates of vibrations, giving rise to close packed absorption bands. The IR absorption spectrum may extend over a wide range of wavelength. In spectrum each band corresponds to the characteristic functional groups and bonds present in a compound. FT-IR spectra of the synthesized LSMO NPs were collected on a Perkin Elmer spectrometer model no. 783 USA. FT-IR spectrum of a LSMO NPs is shown in Fig. 3.3 (b) Elemental analysis ICP OES ICP/OES is one of the most powerful and popular analytical technique for the determination of traces elements. In this method when plasma energy is given to an analysis sample from outside, the component elements (atoms) are excited. When the excited atoms return to low energy position, emission rays (spectrum rays) are released and the emission rays that correspond to the photon wavelength are measured. The element type is determined based on the position of the photon rays, and the content of each element is determined based on the rays intensity. To generate plasmas first, argon gas is supplied to torch coil, and high frequency electric current is applied to the work coil at the tip of the torch tube. Using the electromagnetic field created in the torch tube by the high frequency current, argon gas is ionized and plasma is generated. This plasma has high electron density and temperature (10000K) and this energy is used in Centre For Interdisciplinary Research, DYPU, Kolhapur 79

13 the excitation-emission of the sample. Solution samples are introduced into the plasma in an atomized state through the narrow tube in the center of the torch tube [15]. Fig. 3.4 Schematic of ICP-OES system [16]. The block diagram of ICP/OES system is relatively simple as shown in Fig A portion of the photons emitted by the ICP is accumulated with a lens or a concave mirror. This focusing optic forms an image of the ICP on the entrance aperture of a wavelength selection device such as a monochromator. The particular wavelength exiting the monochromator is translated to an electrical signal by a photo detector. The signal is amplified and processed by the detector electronics, then displayed and stored by a personal computer X-ray photoelectron spectroscopy (XPS) XPS has broadly used technique for studying the properties of atoms, molecules, solids and surfaces. XPS spectra are gained by irradiating a material with a beam of X-rays while simultaneously measuring the kinetic Centre For Interdisciplinary Research, DYPU, Kolhapur 80

14 energy and number of electrons that escape from the top 0 to 10 nm of the material being analyzed. It is based on the principle of photoelectric effect. In the working of XPS, electrons are released from the sample as a result of a photoemission process. An electron is emitted from an atomic energy level by an X-ray photon, mostly from an Al-Kα or Mg-Kα primary source, and its energy is analysed by the spectrometer. The schematic representation XPS process is as shown in Fig. 3.5 (a). Fig. 3.5 (a) Block Diagram of XPS system [17], (b) Typical XPS survey of LSMO NPs. The experimental quantity that is measured is the kinetic energy of the electron, which depends on the energy hν of the primary X-ray source. The typical factor for the electron is its binding energy. The relation between these parameters is expressed by the equation, EB h E W k (3.6) Where, E B and E K are respectively the binding and the kinetic energy of the released photoelectron, hν is the photon energy, and W is the spectrometer Centre For Interdisciplinary Research, DYPU, Kolhapur 81

15 work function. In a first approximation, the work function is the major difference between the energy of the Fermi level E F and the energy of the vacuum level E V, which is the zero point of the electron energy scale, W E E F V (3.7) This quantity has been calculated by calibration for the spectrometer used. From Equation (3.7) it is obvious that only binding energies lower than the exciting radiation ( ev for Al-Kα and ev for Mg-Kα) are probed. Each element has a distinguishing electronic structure and thus a characteristic XPS spectrum. Fig. 3.5 (b) shows the XPS spectrum of LSMO Morphological Study FE-SEM Field emission microscopy (FEM) is a powerful tool used in materials science to investigate molecular surface structures and their electronic properties. In FE-SEM a field-emission cathode in the electron gun provides narrower probing beams at low as well as high electron energy. It consists of a metallic sample in the form of a sharp tip and a conducting fluorescent screen enclosed in ultrahigh vacuum. This typical arrangement over SEM results in to improved spatial resolution and minimized sample charging and damage. In actual working process electrons are liberated from a field emission source and accelerated in a high electrical field gradient. In the high vacuum region these so-called primary electrons are focussed and deflected by electronic lenses to produce a narrow scan beam that bombards the object. As a result secondary electrons are emitted from each spot on the object. The angle and velocity of these secondary electrons communicates to the surface structure of the object. A detector collects the secondary electrons and produces an electronic signal. This signal is Centre For Interdisciplinary Research, DYPU, Kolhapur 82

16 amplified and transformed to a video scan-image. Then images can be displayed on monitor and processed further. Fig. 3.6 (a) represents schematic of the FESEM system. Fig. 3.6 (b) shows FE SEM image of LSMO NPs. Fig. 3.6 (a) Schematic of the FESEM system [18], (b) FE SEM images of LSMO NPs TEM TEM is one of the important technique based on the use of electrons rather than light to examine the structure and behaviour. The TEM image gives more depth knowledge of morphology and direct estimation of its size distribution of the nanomaterials. This method has been used for high resolution imaging as well as chemical and structure study on atomic level. Centre For Interdisciplinary Research, DYPU, Kolhapur 83

17 Fig. 3.7 (a) Ray diagram of Transmission electron microscope [20], (b) TEM images of LSMO NPs. Schematic 3.7 (a) shows ray diagram for transmission electron microscope. In actual working of TEM, an electron gun at the top produces the stream of monochromatic electrons. This stream of electrons is focused to a small, thin, coherent beam by the use of two coherent lenses. When a beam of electrons bombards on the sample, part of it gets transmitted. This transmitted portion is focused by the objective lens into an image. There is a mandatory requisite of sample for TEM analysis is, it must be thin enough to allow the electrons to be transmitted. The recommended thickness of the material for TEM is about 0.5 μm. For Preparation of sample for TEM analysis, the sample to be analyzed is dispersed in some dispersive media (inert to powder) to form colloidal solution, then a drop of solution is kept on a conducting grid of copper or silver (sq. size is ~1 μm) and dried. This dried grid is then act as specimen for analysis using TEM. In present work, the shape, size and uniformity of the NPs were measured by TEM and high resolution TEM (HR-TEM) with TECNAI F20 Philips operated at 200 KV. The typical TEM image of LSMO NPs is shown Fig. 3.7 (b) [19]. Centre For Interdisciplinary Research, DYPU, Kolhapur 84

18 3.3.4 Colloidal stability Study Zeta potential Fig 3.10 (a) Optical configurations of the Zetasizer Nano series for zeta potential measurement [21], (b) Zeta potential measured for LSMO NPs. For biomedical applications MNPs should be stable in an aqueous solution. Therefore, colloidal stability of MNPs is an important issues which can be measured in terms of zeta potential. Zeta potential is defined as the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle. When all the particles in the suspension have a large negative or positive zeta potential, then they will tend to repel each other and there is no tendency to flocculate and vice versa. In present work, zeta potential of the nanofluid was measured using a PSS/NICOMP 380 ZLS particle sizing system (Santa Barbara, CA, USA) with a red He Ne laser diode at Å in a fixed angle 90 plastic cell. Measurements were performed at 25 C after a temperature homogenization time of 5 min with varying ph from 2 to 12. For precise data at least three measurements were conducted for each ph value. The instrument calibration was examined before each experiment using a latex suspension of known zeta potential (i.e. 55 ± 5 mv). Zeta Centre For Interdisciplinary Research, DYPU, Kolhapur 85

19 potential is not measurable directly but it can be estimated using theoretical models and an experimentally- determined electrophoretic mobility or dynamic electrophoretic mobility. Fig (a) shows block diagram for zeta potential measurement. Major application of zeta potential is to determine surface charge of NPs. Derivations show that the zeta potential is the double-layer potential close to the particle surface. The liquid layer of a particle in suspension migrating in an electric field moves at the same velocity as the surface (shear surface). This shear surface occurs well within the double layer, likely at a location roughly equivalent to the Stern surface. Although, the precise location of the surface of shear is not known, it is assumed to be within a couple of molecular diameters of the actual particle surface for smooth particles. This thickness is associated with the zeta potential and determines the ion atmosphere near a surface [22]. Electroacoustic phenomena and electrokinetic phenomena are the typical sources of data for estimation of zeta potential. As the magnitude of zeta potential gives an indication of the potential stability of colloidal system. If all the particles in suspension have a larger negative or positive zeta potential then they will tend to repel each other and there is no tendency to flocculate. However, if the particles have lower zeta potential values then there is no force to prevent the particles aggregation. The general dividing line between stable and unstable suspensions is usually taken at either +30mV or -30mV. Particles with zeta potentials more positive than +30mV or more negative than -30mV are generally considered stable. Fig (b) represents zeta potential for measured for LSMO NPs. Centre For Interdisciplinary Research, DYPU, Kolhapur 86

20 DLS This technique is one of the most popular method in nanotechnology used to determine the size of particles in suspension [23]. Particles suspended in liquids show Brownian motion due to random collisions with solvent molecules. It causes the particles to diffuse through the medium. The diffusion coefficient D is inversely proportional to the particle size. According to the Stokes-Einstein equation, D=k B T/6πηa (3.8) Where, a is the radius of the beads, k B is the Boltzmann constant, T is the temperature in Kelvin degrees (in this experiment it will be considered as if it is taking place at room temperature) and η is the viscosity of the solvent. Fig (a) Block diagram of typical DLS instrument [23], (b) DLS histogram of LSMO NPs. In working principle, radiating a monochromatic light beam, such as a laser, onto a suspension with particles in Brownian motion causes a Doppler Shift when the light falls the moving particle, changing the wavelength of the Centre For Interdisciplinary Research, DYPU, Kolhapur 87

21 incoming light. This change is associated to the size of the particle. It is probable to calculate the size distribution of particles and give a description of the particle s motion in the suspension medium, measuring the diffusion coefficient of the particle and using the autocorrelation function. In present case, DLS of the LSMO NPs were measured using a PSS/NICOMP 380 ZLS particle sizing system (Santa Barbara, CA, USA) with a red He Ne laser diode at Å in a fixed angle 90 plastic cell. All measurements were carried out at ºC using a circulating water bath. Cylindrical cells of 10 mm diameter were used in all of the light scattering experiments. Fig (a) shows block diagram of typical DLS instrument and Fig (b) shows DLS histogram of LSMO NPs Magnetic Characterizations VSM Fig (a) Schematic of VSM showing the overall assembly [24], (b) Magnetic measurement of as prepared LSMO NPs at temperature 27 ºC. VSM is used to measure materials entire magnetic behaviour. Using this method the magnetic characteristics of sample as a function of applied field at different temperatures and as a function of temperature at different applied field strengths can be measured. Operating principle of VSM is based Centre For Interdisciplinary Research, DYPU, Kolhapur 88

22 on Faraday's Law of Induction [25]. It states that a changing magnetic flux through the circuit will produce an electric field in any closed circuits. This can be expressed as, d -N BA cos J dt (3.9) Where, N is the number of turns of wire in the coil, A is area coil, and θ is the angle between the B field and the direction normal to the coil surface. In VSM measurements the sample to be analysed is placed in a constant magnetic field. If the sample is magnetic, this constant magnetic field will magnetize the sample by aligning the magnetic domains or the individual magnetic spins, with the applied field. Due to applied magnetic field the magnetic dipole moment of the sample will create the magnetic field around the sample. The oscillatory motion of the magnetized sample will induce a voltage in the detection coils [26]. This voltage can be used to identify a high resolution and accuracy by means of suitable associated electronics. The induced voltage is proportional to the sample s magnetization, which can be varied by changing the dc magnetic field produced by the electromagnet. For application in biomedicine superparamagnetic behaviour of MNPs at room temperature is one of the essential criteria. Field cooled zero field cooled measurements with the help of Superconducting Quantum Interference Device (SQUID) i. e. SQUID -VSM can gives the accurate data of about the blocking temperature of the sample. The blocking temperature of the material can gives the information about the superparamagnetism. Fig show vibrating sample magnetometer (a) and magnetic measurements of LSMO NPs (b). Centre For Interdisciplinary Research, DYPU, Kolhapur 89

23 3.3.6 Biocompatibility study: Cytotoxicity assays In the biomedical field especially, NPs are being utilized in diagnostic and therapeutic tools to better understand, detect, and treat human diseases. Human exposure to NPs is predictable as NPs become more extensively used and, as a result, nanotoxicology research is recently focusing attention. However, while the number of NPs, types and applications continues to increase, studies to distinguish their effects after exposure and to address their potential toxicity are few in comparison. For medical use exposure to NPs involves intentional contact or administration; therefore, understanding the properties of NPs and their effect on the body is vital before clinical use can occur. For NPs to be used in clinical area, it is necessary that nanotoxicology research uncovers and understands how these multiple factors influence the toxicity of NPs so that their undesirable properties can be avoided [28]. Fig Schematic of cytotoxicity test [27]. Centre For Interdisciplinary Research, DYPU, Kolhapur 90

24 Selection of the appropriate cytotoxicity assay is crucial to the accurate assessment of NPs toxicity. Several assays can be used to study the toxic effects of NPs on cell cultures, including lactate dehydrogenase (LDH) leak-age,3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-yltetrazolium bromide (MTT) assay, Trypan blue Dye exclusion (TBDE) assay (and identification of cytokine/ chemokine production etc. In deciding the correct assay, all potential interferences must be considered to avoid obtaining false-positive and false negative results. Interactions between the NPs and the chosen dye have been cited as a major potential interference leading to inaccurate results. In present investigation MTT and TBDE assays have been used to study cytotoxicity. In TBDE assays cell viability analysis is done with trypan blue dye exclusion staining. Cells were routinely counted manually with a hemocytometer. Now a days advanced programmed instrumentation has been introduced to supplement this traditional technique with the efficiency and reproducibility of automated sample handling, computer control and advanced imaging. The conventional method of performing TBDE analysis involves manual staining and use of a hemocytometer for counting. Recent advances in instrumentation have led to a number of fully automated systems that can enhance the output and accuracy of this technique. The MTT assay is a colorimetric assay for measuring cell viability. The reduction of MTT (tetrazolium salts) is now extensively believed as a reliable way to observe cell proliferation. The yellow tetrazolium MTT (3- (4, 5-dimethylt hiazolyl-2)-2,5-di phenyltetrazoli um bromide) is reduced by metabolically active cells through dehydrogenase enzymes, and make reducing equivalents such as NADH and NADPH. The consequential intracellular purple formazan can be solubilized and measured by spectrophotometrically. The cell proliferation rate can be calculated by using MTT cell proliferation assay. When metabolic events initiate apoptosis or necrosis it results into the reduction in cell viability. The number of stages Centre For Interdisciplinary Research, DYPU, Kolhapur 91

25 involved in assay has been minimized as much as possible to expedite sample dispensation. The MTT reagent give ups low background absorbance values in the absence of cells. Fig 3.13 shows schematic of cytotoxicity tests [27]. Cancer cells are more resilient towards NPs toxicity than normal cells due to an increased rate of proliferation and metabolic activity. The difference in toxic effects is even observed for NPs of the same material. Therefore, selection of the appropriate cell type based on target introduction methods of nanomaterials is an essential issue in cytotoxicity assays [29]. In the case of biomedical applications, NPs are often introduced into the human body through the intravenous, subcutaneous, intra-muscular or intraocular pathway. Based on the variety of affected organs, numerous cell types ranging from endothelium, blood, spleen, liver, nervous system, heart and kidney are all of interest in NPs cytotoxicity studies [30] Induction heating system for caner Hyperthermia study Fig induction heating system for magnetic fluid hyperthermia. Centre For Interdisciplinary Research, DYPU, Kolhapur 92

26 Heating ability of MNPs for cancer hyperthermia therapy has been studied with induction heating system. Induction heating characteristics of samples for hyperthermia application were carried out in Eppendorf tube using instrument (Easy Heat 8310, Ambrell, UK).The coil having 6 cm diameter (4turns) consisted of loops of copper (Cu) pipe was cooled by water circulation in coil to keep the temperature constant. Suspensions of NPs were prepared in DDW as well as different physiological media and placed at the centre of the coil. Particularly, magnetic induction heating system has generated a growing interest in the medical field and oncology. Induction Heating offers a controllable and localized method of heat without contact to the parts (components) being heated [31-32]. It has been studied that the malignant cells are more sensitive and responsive to induced heat than normal cells. Using particular range of frequency and magnetic field strength, a heating coil of a given shape and size in conjunction with other methods can apply a hyperthermia effect on tumour cells. The basic principle of working of induction heating is based on the faradays law, The amount of voltage created is equal to the change in magnetic flux divided by the change in time. The greater the change in the magnetic field, the larger will be amount of voltage. In this system, a source of high frequency electricity is used to drive a large alternating current through a coil. This coil is known as the work coil (Fig. 3.14). The passage of current through this coil generates a very intense and rapidly changing magnetic field in the space within the work coil. The work piece to be heated is placed within this intense alternating magnetic field [33]. The intense alternating magnetic field inside the work coil repeatedly magnetises and de-magnetises the crystals to be analysed. This rapid flipping of the magnetic domains causes considerable friction and heating inside the material. Centre For Interdisciplinary Research, DYPU, Kolhapur 93

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