Synthesis of New Magnetic Nanoparticles and Study their Effect on Prolactin Structure

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Chapter One
Introduction
I-1Nanotechnology
I-2 Nanoparticle
I-3 Properties
I-3-A-Shape
I-3-B- Physical properties
I-3-C-Chemical Properties
I-3-D- Optical properties
I-3-E- Ferromagnetic Properties
I-4 Surface coating for biological applications
I-5 Applications of NPs in biology and medicine
I-6 Magnetic nanoparticles
I-6 A-Definition
I-6 B-Properties
I-6 C-Types of magnetic nanoparticles
1-Oxides: ferrite1
2- Metallic
3- Metallic with a shell
Preparation Methods of Magnetic
1- Co-precipitation
2- Thermal decomposition
3- Microemulsion
4- Flame spray synthesis
I -7Applications Of Magnetic NPs
A) Medical diagnostics and treatments
B) Magnetic immunoassay
C) Waste water treatment
D) Chemistry
E) Biomedical imaging
F) Information storage
G) Genetic engineering
H) Industrial applications
I) Biomedical applications
J) In vivo applications
K) Removal of organic pollutants
L) Removal of Inorganic pollutants
M) Analytical applications
I-8-Preperation of the modified magnetic NPs
I-9 Identification of NP
I-9-1 Scanning electron microscope (SEM)
I-9-2 Transmission electron microscopy (TEM )
I-9-3 Wide angle x-ray scattering (WAXS) or wide-angle X-ray diffraction (WAXD )
I-9-4- FTIR
I-9-5- Dynamic Light Scattering
I-9-6-Zeta potential analysis
I-9-7 Magnetic property (magnetic behavior)
I-10- Protein-NPs Interaction
I-10- 1- Covalent Protein-NP Conjugation هل الترقيم صحيح ؟
I-10- 2 - Non-Covalent Protein-NP Conjugation
I-10-3 Application of protein-NPs conjugates.
I-11 - Prolactin(PRL)
I-12 Folic acid
I-13 - Palmitic acid
I-14 - Adsorption of Proteins
I -11 -Adsorption isotherms
a-Ionic or Electrostatic Interactions
b-Hydrogen Bonding
c-Hydrophobic Interactions
d-Charge-Transfer Interactions
Aim of the study

Chapter two
II- Method And Materials
II-1 Chemicals
II-2 Instruments
II-3-Methods
II-3-1- Synthesis of the Fe3O4nanoparticales
II-3-2 Synthesis of magnetic nanoparticales-palmatic acid
II-3-3 Synthesis of magnetic nanoparticales-folic acid
II-3-4 Preparation of Phosphate Buffer Saline (pH=7.4)
II-4 Identification of the prepared NPs
II-4-1 Identification of the Prepared MNPs by TEM
II-4-2 Identification of the Prepared MNPs by SEM
II-4-3 FTIR charts
II-4-4 Dynamic light scattering (DLS) method
II-4-5 TEM study for MNPs- Prolactincomplexes
II-4-6 TGA Characters.
II-5 Estimation of Prolactine Concentration by ELISA
A-Principle:
B- Reagents of the kit
C-Reagent Preperation
D-Test Procedure
II-6 Estimation of Equilibrium Time of Adsorption
II-7 Adsorption Isotherms
II-8 Thermodynamicsof the Adsorption of PRL on MNPs
II-9 Desorption process

Chapter three
Result & discussion
III.1 Characterization of the synthesized NP.
III.1.1 MNPs
III.1.2 MNP@Folic
III.1.2 MNP@Palmitic
III-2 Prolactin-MNP compounds Interaction
III-2-1-Equilibrium Time
III-2-2-Properties of NP after interaction with prolactin hormone
III-2-3 Adsorption Process
III-2-4 Applicability of Langmuir and Freundlisch Adsorption Isotherms in the NP-Prolactin systems.
III-2-5 Thermodynamics of the adsorption process:
III-2-6 Desorption Processes:
Application of protein-NPs conjugates.
Conclusions
Recommendations
References

Table of Abbreviations

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List of Table

II-1 Chemicals Used in the Study

II-2 Instruments used in the study

III-1 Correlation coefficient (r-values) of the Langmuir and Freundlisch lines.

III-2 Thermodynamic parameters of the adsorption of PRL on MNPs, MNP@Folic, and MNP@Palmitic.

List of Figures

I-1 Different shapes of NPs

I-2 Structure of PRL molecule

I-3 Structure of folic acid

II-1 A schematic illustration of the formation mechanism of magnetite nanoparticles

II-2 shows the preparation of MNPs

II-3 TEM (Oxford, JEOL, Model: JEM2010F)

II4 SEM (Hitashi , Model: S 4300 )

II-5 DLS (90 Plus Particle Sizer, Brookhaven Instruments)

II-6 TGA (TGA 7, perkin elmer )

III-1 Figure (1): TEM images of the prepared MNP under very high resolution.

III-2 SEM images of the prepared MNP under very high resolution.

III-3 IR spectrum of the prepared MNP.

III-4 Weight loss of MNP as a function of increasing temperature.

III-5 Weight loss percentage per degree of MNP as a function of increasing temperature.

III-6 TEM images of the prepared MNP@Folic under very high resolution.

III-7 SEM images of the prepared MNP@Folic under very high resolution.

III-8 IR spectrum of the prepared MNP prepared MNP@Folic acid (below) in comparing with the spectrum of folic acid alone (above).

III-9 Weight loss of MNP@Folic as a function of increasing temperature

III-10 Weight loss percentage per degree of MNP@Folic as a function of increasing temperature.

III-11 TEM images of the prepared MNP@Palmitic under very high resolution.

III-12 SEM images of the prepared MNP@Palmitic under very high resolution.

III-13 IR spectrum of the prepared MNP@Palmitic acid (below) in comparing with the spectrum of folic acid alone (above).

III-14 Weight loss of MNP@Palmitic as a function of increasing temperature.

III-15 Weight loss percentage per degree of MNP@Palmitic as a function of increasing temperature.

III-16 Calibration curve of prolactin solution

III-17 Prolactin-MNP composite

III-18 Prolactin-MNP@Folic composite

III-19 Prolactin-MNP@Palmitic composite

III-20 Adsorption isotherms of prolactin hormone on the surface of MNP compounds at 25°C, 35°C, and 45°C.

III-21 Adsorption isotherms of prolactin hormone on the surface of MNP@Folic acid at 25°C, 35°C, and 45°C.

III-22 Adsorption isotherms of prolactin hormone on the surface of MNP@Palmitic at 25°C, 35°C, and 45°C.

III-23 Adsorption isotherms according to Giles Classification

III-24 Langmuir line of the adsorption of prolactine on MNP.

III-25 Langmuir line of the adsorption of prolactine on MNP@Folic acid.

III-26 Langmuir line of the adsorption of prolactine on MNP@Palmitic acid.

III-27 Langmuir line of the adsorption of prolactine on MNP.

III-28 Langmuir line of the adsorption of prolactine on MNP@Folic acid.

III-29 Langmuir line of the adsorption of prolactine on MNP@Palmitic acid.

III-30 Vant-Hoff's equation of the adsorption of prolactin on MNP, MNP@Folic, and MNP@Palmitic acid at 25°C, 35°C, and 45°C.

III-31 Desorption percentages of prolactin from the surface of MNP, MNP@Folic, and MNP@Palmitic acid at Red 25°C, Yellow 35°C, and Blue 45°C.

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Chapter One

1-1 Nanotechnology:

TheNational Nanotechnology Initiativedefines nanotechnology as the manipulation of matter to at least one dimension sized from 1 to 100nm(Timothy, 2011). This definition shifted from a particular technological goal to a research category inclusive of all types of research and technologies that deal with the special properties of matter that occur below the given size threshold(Lydia et al.,2014). These unique effects often provide nanoscale materials the desired chemical, physical, and biological properties that differ from those of their larger or bulk counterparts (Yeet al.,2011)

Nanotechnology allows scientists, engineers, chemists, and physicians to work at the molecular and cellular levels to produce important advances in life sciences and healthcare.Defined by size, nanotechnology has very broad application, including fields of science as diverse assurface science,organic and inorganic chemistry,molecular biology,semiconductor physics, andmicrofabrication(Saini et al.,2010)

It is one of the most important research and development frontiers in modern science, and now widely used throughout pharmaceutical, medicine, electronics, robotics, and tissue engineering industries. The use of nanoparticle (NP) materials offers many advantages because of their unique size and physical properties (Freitas, 2005; Faraji et al., 2010).

However, nanotechnology is presented with many of the same issues on any new technology, includingtoxicityconcerns and environmental effects of nanomaterials(Cristina et al.,2007), as well as their potential effects on global economics .

Essentially, nanotechnology is the engineering of functional systems at the molecular scale. It refers to the projected ability to construct items from bottom to top using techniques and tools being developed today to produce complete and efficient products.(Allhoff et al.,2010).

1-2 Nanoparticle (NP):

In nanotechnology, a particle is a small object that behaves as a whole unit with respect to its transport and properties. Based on their diameter, particles are classified as coarse (2500–10000 nm), fine (100–2500 nm), and ultrafine (1–100 nm) particles (Singh et al., 2013). NPs may or may not exhibit size-related properties that significantly differ from those observed in fine particles or bulk materials (Buzea et al., 2007). Nanoclusters have at least a dimension between 1 and 10 nm and a narrow size distribution. Nanopowders are agglomerates of ultrafine particles, NPs, or nanoclusters. Nanometer-sized single crystals or single-domain ultrafine particles are often referred to as nanocrystals (Fahlman, 2007).

1-2-1-Properties

1-2-1 - A Shape:

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Figure (1-1): Different shapes of NPs

Scientists name NPs after the shapes that they resemble. Nanospheres, (Agam et al., 2007), nanoreefs (Choy et al., 2004), nanoboxes (Sun et al., 2002), and more have been reported in literatures. Amorphous particles usually adopt a spherical shape because of their microstructural isotropy, whereas the shape of anisotropic microcrystalline whiskers corresponds to their particular crystal habit. At the smallest-size range, NPs are often referred to as clusters. Spheres, rods, fibers, and cups are only a few of the shapes that have been grown. Micromeritics is the study of fine particles. Nanorods are one morphology of nanoscale objects with dimensions ranging from 1–100 nm and standard aspect ratios (length/width) of 3 to 5. Nanorods are produced by direct chemical synthesis from metals or semiconducting materials. Examples of nanorods are zinc oxide nanorods, also known as nanowire (Gyu-Chul et al., 2005), and gold nanorods (Huang et al., 2009).

1-2-1 -B Physical properties

NPs are great scientific interests as they link bulk materials to their atomic or molecular structures. A bulk material should have constant physical properties regardless of size; however, nanoscale size-dependent properties are often observed. Thus, the properties of materials change as their sizes approach the nanoscale and as the percentage of atoms at the surface of a material becomes more significant. Surface physical properties are important in regulating the interaction between biomaterials and biological systems (Mitragotri et al., 2009). For bulk materials larger than 1 µm (or micron), the atomic percentage at the surface is insignificant with respect to the number of atoms at the bulk of the material. Therefore, NP properties are largely due to the large surface area of the material that dominates the contributions of the small bulk of the material. Some NPs have self-cleaning effects and superior UV-blocking properties that can be used in preparations of sunscreen lotions that are completely photostable (Mitchnick et al., 1999). Other NPs like clay NPs when incorporated into polymer matrices increase reinforcements, leading to stronger plastics, as confirmed by a high glass transition temperature and other mechanical property tests. These NPs are relatively hard and impart their properties to the polymer (plastic). Metal, dielectric, and semiconductor NPs have been formed also, as well as hybrid structures (e.g., core–shell NPs) (Taylor et al., 2013). NPs made of semiconducting material may also be labeled quantum dots (QDs) if they are small enough (typically sub 10nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents. Semi-solid and soft NPs have also been manufactured. A prototype of semi-solid NP is the liposome. Various types of liposome NPs are currently clinically used as delivery systems for anticancer drugs and vaccines. Colloid is used primarily to describe a broad range of solid–liquid (and/or liquid–liquid) mixtures, all of which containing distinct solid (and/or liquid) particles dispersed at various degrees in liquid medium. However, the term is specific to the size of individual particles that are larger than atomic dimensions, but small enough to exhibit Brownian motion. The dynamic behavior of large colloid particles have their governed by forces of gravity and sedimentation at any given period of time. However, colloid particles that are relatively small exhibit irregular motions that can be attributed to the collective bombardment of a myriad of thermally agitated molecules in the liquid suspending medium (Pais, 2005).

1-2-1 -C Chemical Properties

- are mainly characterized based on shape (including aspect ratios where appropriate), size, and morphological substructure. In the presence of chemical agents (surfactants), the surface and interfacial properties of NPs may be modified. Indirectly, such agents inhibit coagulation or aggregation by stabilizing particlechargesand modifying the outmost layer of particles. However, this could result to very complex compositions, possibly with complex mixtures of adsorbate, depending on growth history and lifetime of NP. Complex-surface chemical processes also occur and have been identified only on a number of particulate-model systems (Kamat, 2002). At the NP–liquid interface, polyelectrolytes have been utilized to modify surface properties and interactions between particles and their environment (Liufu et al., 2005). Atnanoscale level, particle–particle interactions are either dominated by weak van der Waals (vDW) forces, strong polar and electrostatic interactions, or covalent interactions. Depending on the viscosity and polarizability of fluid, particle aggregation is determined by interparticle interactions. The tendency of a colloid to coagulate can be enhanced or hindered by surface-layer modification. ForNPssuspended in air, charges can be accumulated by physical processes such as glow discharge or photoemission. In liquids, particlechargecan be stabilized by electrochemical processes at surfaces. Knowledge on NP–NP forces of interaction and NP–fluid interactions are important in describing the physical and chemical processes they undergo and the temporal evolution of free NPs (Penn et al., 2003; Schins et al., 2004).

1-2-1 -D Optical properties

NPs often demonstrate surprising optical properties because of their small structures that confine electrons and produce quantum effects (Hewakuruppu et al., 2013). For example gold NPs appear deep-red to black in solution. NPs of yellow gold and grey silicon are red. Gold NPs melt at relatively lower temperatures (»300 °C for 2.5nm size) than gold slabs (1064 °C) (Buffat et al., 1976). Absorption of solar radiation is higher in materials composed of NPs than thin films of continuous sheets of material (Taylor et al., 2012; Taylor et al., 2013).

1-2-1 -E Ferromagnetic Properties

Another important property of NPs is their magnetic behavior. For biomedical uses, application of superparamagnetic particles at room temperature is preferred (Cabuil, 2004; Morcos, 2007; Ersoy et al., 2007). Furthermore, applications in therapy, biology, and medical diagnosis require magnetic particles that are stable in water at pH 7 and in physiological environments. The colloidal stability of this fluid depends on charge and surface chemistry, which results to steric and Coulombic repulsions, and on the dimensions of the particles, which should be sufficiently small to prevent precipitation because of gravitational force (Thakral et al., 2007). Ferromagnetic materials that are smaller than 10nm can switch their magnetization direction using room-temperature thermal energy, making them unsuitable for memory storage (Gubin et al., 2009).

1-3 Surface coating for biological applications

For biological applications, surface coatings should be polar to obtain high solubility in aqueous medium and prevent NP aggregation. In serum or on cell surface, highly charged coatings promote non-specific binding, whereaspolyethylene glycollinked to terminal hydroxyl or methoxy groups repel non-specific interactions (Liu et al., 2010). NP functionalization is typically achieved by different linkage methods, such as covalent linkages (e.g., amide linkage, disulfide linkage), encapsulation, and entrapment (Wagner et al., 2006; Larocque et al., 2009). NPs can be linked to biological molecules that can act as tags to direct NPs to specific sites within the body (Akerman et al., 2002), specific organelles within the cell (Hoshino et al., 2004)or to specifically follow the movement of individual protein or RNA molecules in living cells (Suzuki et al., 2007).

1-4 Applications of NPs in biology and medicine

At present, nanomaterials are most advanced both in scientific knowledge and commercial applications(Mazzola, 2003; Murray et al.,2000).Additional restrictions on NPs could be used for biomedical in vivo and in vitro applications. Forin vivoapplications, MNPs must be encapsulated with a biocompatible polymer during or after preparation to prevent changes on the original structure, formation of large aggregates, and biodegradation when exposed to the biological system. The polymer-coated NP also facilitate binding of drugs by entrapment on the particles, adsorption, or covalent interactions (Muldoon et al., 2005; Moghimi et al., 2001; Sosnovik et al., 2007) . The major factors that determine the toxicity and biocompatibility of MNPs are the nature of the magnetically responsive components, such as magnetite, iron, nickel, and cobalt, final particle size, core, and coatings. Iron oxide NPs, such as magnetite (Fe3O4) or its oxidized form maghemite (γ-Fe2O3), are by far the most commonly used NPs for biomedical applications. Highly magnetic materials such as Co and Ni are susceptible to oxidation and are toxic; hence, they are of little interest for in vivo biomedical applications (Choi et al., 2007; Murray et al., 1993; Peng et al., 2000). MNPs must be produced from non-toxic and non-immunogenic materials and exhibit relatively small particle sizes to remain in the circulation after injection and to pass through capillary systems of organs and tissues, avoiding vessel embolism. They must also have a high magnetization so their movement in the blood can be controlled with a magnetic field and they can be immobilized close to target pathological tissue/s(Lopez et al.,2004; Thorek et al., 2006). As stated earlier,semi-solid and soft NPs such as liposome NPs are already manufactured and currently clinically used as delivery systems for anticancer drugs and vaccines.NPs that are half hydrophilic and half hydrophobic are referred to asJanus particlesand are particularly effective for stabilizing emulsions. Janus particles can self-assemble at water–oil interfaces and act as solid surfactants.

1-5 Magnetic nanoparticles :

1-5-1 Definition:

MNPs are a class of NPs that is manipulated using magnetic field. MNPs commonly consist of magnetic elements, such as Fe, Ni, and Co and their chemical compounds. NPs are smaller than 1 µm in diameter (typically 5–500 nm) and large microbeads are 0.5–500 µm in diameter. MNPs have been the focus of much research recently because of their interesting properties, which could be potentially used in catalysis, including nanomaterial-based catalysts, (Lu et al., 2004) biomedicine (Gupta et al., 2005), magnetic resonance imaging (MRI) (Mornet et al., (2006), magnetic particle imaging (Gleich et al., 2005), data storage (Hyeon, 2003), environmental remediation, (Elliott et al., (2001), nanofluids (Philip et al., 2006), optical filters (Philip et al., 2003), defect sensors (Mahendran et al., 2012), and cation sensors (Philip et al., 2013).

1-5-2 Properties:

The physical and chemical properties of MNPs largely depend on their method of synthesis and chemical structure. In most cases, particles may display superparamagnetism with sizes ranging from 1 nm to 100nm (Lu et al., 2007). Magnetic effects are caused by movements of particles that have both mass and electric charges, such as electrons, holes, and protons, as well as positive and negative ions. A spinning electric-charged particle creates a magnetic dipole called magneton. In ferromagnetic materials, magnetons are associated in groups. A magnetic domain (Weiss domain) is the volume of ferromagnetic material in which all magnetons are aligned in the same direction by opposing forces. This concept of domains distinguishes ferromagnetism from paramagnetism. The domain structure of a ferromagnetic material determines the size dependence of its magnetic behavior. At a size below a critical value, a ferromagnetic material becomes a single domain. Fine-particle magnetism results from size effects, which are based on the magnetic domain structure of ferromagnetic materials. It assumes that the lowest free energy state of ferromagnetic particles has uniform magnetization for particles smaller than a certain critical size and has non-uniform magnetization for larger particles. The former ones are referred to as single domain particles, whereas the latter are called multidomain particles (Hines et al., 1996; Qu et al.,2000).

1-5-3 Types of magnetic nanoparticles:

Currently, three different types of MNPs are being produced and used, namely, oxides, metallic, and metallic with a shell.FerriteMNP, an oxide MNP, is the most explored MNP to date. Ferrite particles that are smaller than 128 nm(An-Hui, et al.,2007)becomesuperparamagnetic,which prevents self-agglomeration because they only exhibit magnetic behavior on application of external magnetic field(Kim et al.,2003).Switching off the external magnetic field results inremanencefalls back to zero. Similar to non-magnetic oxide NPs, the surface of ferrite NPs is often modified bysurfactants,siliconesorphosphoric acidderivatives to increase their stability in solution (Kim et al., 2003). Conversely,metallic NPs are a disadvantage for beingpyrophoricand reactive tooxidizing agentsat various degrees, making them difficult to handle and enabling unwanted side reactions. Thus, metallic core of MNPs may be passivated by gentle oxidation, surfactants, polymers, and precious metals(An-Hui et al., 2007).Under aerobic environment, Co-NPs form an anti-ferromagnetic CoO layer on Co-NP surface. A recent study has explored the synthesis and exchange bias effect in the Co-core CoO-shell NPs with a gold outer shell(Grass et al., 2006).Moreover, NPs with a magnetic cores consisting of either elementaryFeor Co and a nonreactive shell made ofgraphenehave been synthesized recently.(Grass et al., 2006).

1-6 Preparation Methods of Magnetic NPs

Two main approaches are used in nanotechnology, the bottom-up and top-up approaches. In the bottom-up approach, materials and devices are built from molecular components whichassemble themselveschemically by principles ofmolecular recognition. In the "top-down" approach, nano-objects are constructed from larger entities without atomic-level control(Rodgers,2006). Duringthe last few years, a large portion of published studies about MNPs have described efficient routes to attain shape-controlled, highly stable, and narrow size distribution of MNPs. To date, several popular methods including co-precipitation, microemulsion, thermal decomposition, solvothermal, sonochemical, microwave assisted, chemical vapor deposition, combustion, carbon arc, and laser pyrolysis have been reported for synthesis of MNPs.The established methods for MNPsynthesis include co-precipitation,thermal decomposition, microemulsion, and flame-spray.

1-6-1 Co-precipitation

Co-precipitation is a facile and convenient method of synthesizing iron oxides (Fe3O4 or γ-Fe2O3) from aqueous Fe2+/Fe3+ salt solutions by addition of a base under inert atmosphere at room or elevated temperatures. The size, shape, and composition of the MNPs greatly depend on the type of salts used (e.g., chlorides, sulfates, nitrates), Fe2+/Fe3+ ratio, reaction temperature, pH, and ionic strength of the medium (An-Hui et al., 2007). Co-precipitation approach has been extensively used to produce ferrite NPs with controlled sizes and magnetic properties (Gnanaprakash et al., 2006; Gnanaprakash et al., 2007; Ayyappan et al., 2009; Ayyappan et al., 2010). A variety of experimental arrangements have been reported to facilitate continuous and large-scale co-precipitation of MNPs by rapid mixing (Chin, 2008).

1-6-2 Thermal decomposition :

Thermal decomposition or thermolysis is achemical decompositioncaused by heat. Thedecomposition temperatureof a substance is thetemperatureat which it chemically decomposes. The reaction is usuallyendothermicas heat is required to breakchemical bondswithin the compound undergoing decomposition. If decomposition is sufficientlyexothermic, apositive feedback loopis created producing athermal runawayand possibly an explosion.Magnetic nanocrystals with smaller size can be essentially synthesized by thermal decomposition of organometallic compounds in high-boiling organic solvents containing stabilizing surfactants(An-Hui et al., 2007).

1-6-3 Microemulsion

Microemulsionsare clear, thermodynamically stable, isotropicliquid mixtures of oil, water, andsurfactant that are frequently mixed with aco-surfactant. The aqueousphasemay containsalt(s) and/or other ingredients, and the oil-phase may be a complex mixture of differenthydrocarbonsandolefins. In contrast to ordinaryemulsions, microemulsions form upon simple mixing of the components and do not require the high-shearconditions, which are generally used in the formation of ordinary emulsions. The three basic types of microemulsions are direct (oil dispersed in water, o/w), reversed (water dispersed in oil, w/o), and bicontinuous. In ternary systems, such as microemulsions, where two immiscible phases (water and oil) are present with a surfactant,surfactantmoleculesform amonolayerat the oil–water interface with thehydrophobictails of the surfactant molecules dissolved in the oil phase and the hydrophilic head groups in the aqueous phase. Using microemulsion, metallic Co, Co/Pt alloys, and gold-coated Co/Pt NPs have been synthesized in reverse micelles of cetyltrimethlyammonium bromide with 1-butanol as the co-surfactant and octane as the oil phase (An-Hui et al., 2007; Rana et al., 2010).

1-6-4 Flame spray synthesis:

Using flame-spray pyrolysis (Grass et al., 2006; Athanassiou et al., 2010) at varied reaction conditions, oxides, metal- or carbon-coated NPs are produced at > 30 g/h.

1-7 Applications Of MNPs:

NP research is currently an area of intense scientific interest because of a wide variety of potential applications of NPs in biomedical, optical, and electronic fields (Taylor et al., 2012, 2013; Hewakuruppu et al., 2013). A wide variety of applications have been envisaged for this class of particles, which include medical diagnostics and treatments, magnetic immunoassay, wastewater treatment, Chemistry, biomedical imaging, information storage, genetic engineering, and removal of organic and Inorganic pollutants, as well as industrial, biomedical, in vivo applications, and M) analytical applications.

1-7-1 Medical diagnostics and treatments

MNPs are used in an experimental cancer treatment called magnetic hyperthermia (Rabias et al., 2010), which utilizes the heat that NPs heat under alternating magnetic fields. Another potential treatment of cancer includes the attachment of MNPs to free-floating cancer cells, capturing and carrying them out of the body. The treatment has been tested already on mice in the laboratory and will be evaluated survival studies (Scarberry et al., 2008). Moreover, MNPs can be used for the detection of cancer. Blood can be inserted onto a microfluidic chip with MNPs in it, and these MNPs are trapped inside through an externally applied magnetic field with the blood freely flowing through. The MNPs are coated with antibodies targeting cancer cells or proteins.

1-7- 2 Magnetic immunoassay

Magnetic immunoassay is a novel type of diagnostic immunoassay which utilizes magnetic beads as labels in lieu of conventional enzymes, radioisotopes, or fluorescent moieties. This assay involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of the pair. The presence of magnetic beads is then detected by a magnetic reader (magnetometer), which measures the magnitude of magnetic-field changes induced by the beads. The signal measured by the magnetometer is proportional to the quantity of analyte (virus, toxin, bacteria, cardiac marker, etc.) in the initial sample (Yousafa et al., 2013).

1-7-3 Waste water treatment

MNPs have potentials for treatment of contaminated water because they can be easily removed by applying magnetic field and they have very large surface to volume ratios (Koehler et al., 2009). In this method, attachment of EDTA-like chelators to carbon-coated metal nanomagnets results in a magnetic reagent that rapidly removes heavy metals from contaminated water by three orders of magnitude to concentrations as low as µg/L.

1-7-4 Chemistry .

Magnetic NPs have potentials as catalysts or catalyst supports (Schätz et al., 2010). In chemistry, a catalyst support is a material, which is usually a solid, with high surface area to which a catalyst is affixed. The reactivity of heterogeneous catalysts occurs at the surface atoms.

1-7-5 Biomedical imaging

iron-oxide-based NPs in concert with magnetic resonance imaging have many applications (Colombo et al., 2012). Magnetic Co-Pt NPs are used as MRI contrast agents for transplanted neural stem-cell detection (Xiaoting et al., 2011).

1-7-6 Information storag e

Studies are currently being directed on the use MNPs for magnetic media recording. The most promising candidate for high-density storage is the face-centered tetragonal-phase Fe-Pt alloy. Grain sizes can be as small as 3 nm. Possible modification of MNPs at this scale could easily surpass 1 terabyte per square inch (Service, 2006).

1-7-7 Genetic engineering.

MNPs can be used on a variety of applications in genetics. One application is the isolation of mRNA that can be done quickly, usually within 15 minutes. In this particular application, the magnetic bead is attached to a poly T tail. When mixed with mRNA, the poly A tail of the mRNA attaches to the bead’s poly T tail and the isolation is conducted by simply placing a magnet on the side of the tube and pouring out the liquid solution (Elaissari et al., 2010).

1-7- 8 Industrial applications.

Magnetic iron oxides are commonly used as synthetic pigments in ceramics, paints, and porcelains (Jun et al., 2006; Nunez et al., 2003). Hematite and magnetite have been also applied as catalysts for a number of important reactions, such as in the preparation of NH3, desulfurization of natural gas, and high-temperature water-gas shift reaction. Other reactions include the Fishere-Tropsch synthesis of hydrocarbons, the dehydrogenation of ethylbenzene to styrene, the oxidation of alcohols, and the large-scale synthesis of butadiene (Park et al., 2000; Dumestre, 2002; 2004).

1-7- 9 Biomedical applications

Biomedical applications of MNPs are classified based on their application inside (in vivo) or outside (in vitro) the body. In vitroapplications are mainly on diagnostic separation, selection, and magnetorelaxometry, whereasin vivoapplications could be further classified into therapeutic (hyperthermia and drug-targeting) and diagnostic applications (i.e., nuclear magnetic resonance imaging) (Park et al., 2001; Liu et al., 2005; Piao et al.,2008).

1-7- 10 In vivo applications

Two major factors play an important role for in vivo application of MNPs, size and surface functionality . The diameters of superparamagnetic iron oxide NP (SPIOs) greatly affecttheir in vivobiodistribution, even without targeting surface ligands. Particles with 10 nm to 40 nm diameters including ultra-small SPIOs are important for prolonged blood circulation because they can cross capillary walls and are often phagocytosed by macrophages which traffic to the lymph nodes and bone marrow (An-Hui et al., 2007). MNPs are used also in therapeutic applications (Mikhaylova et al., 2004; Kim et al., 2006) and drug delivery (Casula et al., 2006). )

1-7- 11 Removal of organic pollutants.

A few studies have reported the removal of high concentrations of organic compounds which are mostly related to the removal of dyes. MNPs have high capacity of removing high concentrations of organic compounds (Caruntu et al., 2005; Lyon et al., 2004)

1-7- 12 Removal of Inorganic pollutants.

A very important aspect in metal-toxin removal is the preparation of functionalized sorbents for affinity or selective removal of hazardous metal ions from complex matrices. MNPs are used as sorbents for the removal of metal ions. Their high and efficient removal of different metal ions are attributed to their large surface area with respect to micron-sized sorbents (Zambaux et al., 1999; Stolnik et al., 1995; van der Veen et al., 1998). These findings can be used to design an appropriate adsorption-treatment plan for the removal and recovery of metal ions from wastewaters.

1-7- 13 Analytical applications

Given their small size, magnetic luminescent NPs offer a larger surface area-to-volume ratio than currently used microbeads, which result in good reaction homogeneity and faster reaction kinetics. Thus, preparation of magnetic fluorescent particles, such as polystyrene magnetic beads with entrapped organic dyes, QDs, or shells of QDs (Savva et al., 1999), iron oxide particles coated with dye-doped silica shells, and silica NPs embedded with iron oxide and QDs, is easier.

1-8 Preperation of the modified magnetic NPs

Inorganic and hybrid coatings (or shells) on colloidal templates have been prepared by precipitation and surface reactions (Yuyama et al., 2000). This method can give uniform and smooth coatings with adequate selection of the experimental conditions, such as mainly the nature of the precursors, temperature, and pH, which lead to monodispersed spherical composites. Using this technique, submicrometer-sized anionic-polystyrene lattices have been coated with uniform layers of iron compounds (Allemann et al., 1993 ) by aging at elevated temperature and by dispersion of the colloid polymer in aqueous solutions of ferric chloride, urea, hydrochloric acid, and polyvinyl pyrrolidone. The iron oxide NPs are formed from the coprecipitation of ferrous and ferric salts with inorganic bases. A strong base, NaOH, and a comparatively mild base, NH4OH, have been used with each surfactant to observe the influence of basicity in crystallization during particle formation. These systems show magnetic behavior close to that of superparamagnetic materials. Using this method, uniformly sized MNPs as small as 1 nm to 2 nm (standard deviation < 10%) have been synthesized. A uniform silica coating as thin as 1 nm encapsulating the bare NPs is formed the base-catalyzed hydrolysis and polymerization reaction of tetraethoxysilane in the microemulsion. Remarkably, the small particle size of the composite suggest the potential of MNPs in in vivo applications and several other uses of silica-coated MNPs. For example, pathogen-detection kits are prepared from amino-modified silica-coated MNPs (Baia et al., 2013).

1-9 Identification of NP

1-9-1 Scanning electron microscope (SEM)

SEM is a type of electron microscope that produces sample images by scanning the sample using a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that can be detected and that contain information about the surface topography and composition of the sample. The electron beam is generally scanned in a raster scan pattern, and the beam’s position is combined with the detected signal to produce an image. SEM can achieve better resolution at 1 nm scale. Specimens can be observed at high and low vacuums and in wet conditions (environmental SEM). The most common mode of detection is by the emission of secondary electrons from atoms excited by the electron beam. The number of secondary electrons is a function of the angle between the surface and the beam. On a flat surface, the plume of secondary electrons is mostly contained in the sample; however, on a tilted surface, the plume is partially exposed and more electrons are emitted. By scanning the sample and detecting the secondary electrons, an image displaying the tilt of the surface is created.

1-9-2 Transmission electron microscopy (TEM)

TEM is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen. The image is then magnified and focused onto an imaging device such as a fluorescent screen, on a layer of photographic film, or detected by a sensor such as a charge-coupled camera. TEMs are capable of imaging at significantly higher resolution than light microscopes, owing to the small de Broglie wavelength of electrons.

1-9-3 Wide angle X-ray scattering (WAXS) or wide-angle X-ray diffraction (WAXD)

an X-ray-diffractionHYPERLINK "http://en.wikipedia.org/wiki/X-ray_diffraction"HYPERLINK "http://en.wikipedia.org/wiki/X-ray_diffraction" technique often used to determine the crystalline structure of polymers. This technique specifically refers to the analysis of Bragg peaks scattered to wide angles, which (by Bragg’s law) implies that they are caused by subnanometer-sized structures (Podoroz et al., 2006; 2009). The technique is a old but somewhat unpopular in investigating the degree of crystallinity of polymer samples. The diffraction pattern generated allows the analysis of the chemical or phase composition of the film, the texture of the film (preferred alignment of crystallites), the crystallite size, and presence of film stress. According to this method the sample is scanned in a wide-angle x-ray goniometer, and the scattering intensity is plotted as a function of the 2θ angle. X-ray diffraction is a nondestructive method for characterization of solid materials. X-rays that are directed in solids scatter in predictable patterns based upon the internal structure of the solid. A crystalline solid consists of regularly spaced atoms (electrons) that can be described by imaginary planes.

1-9-4 FTIR

FTIR is the preferred method of infrared spectroscopy. In IR spectroscopy, IR radiation is passed through a sample, which absorbed or transmits some of the IR radiation. The resulting spectrum represents molecular absorption and transmission, which is a molecular fingerprint of the sample. Like a fingerprint no two unique molecular structures produce the same IR spectrum, which makes IR spectroscopy useful for several types of analysis. IR spectrometry is based on molecular vibrations and rotations. These motions are accompanied by a change in the dipole moment of a molecule. The radiation from an IR source is collimated by the mirror, and the resultant beam is passes through the cell. The radiation is then focused on the detector, which measures the amount of energy that has passed through the sample at each frequency, resulting in a spectrum that plots intensity against frequency (Skoog et al., 2007; Baravkar et al., 2011; Manjusha et al., 2011).

1-9-5 Dynamic Light Scattering

DLS is one of the methods used to determine the size of particles. Its experimental theory is essentially based on two assumptions. The first assumption is that the particles are in Brownian motion. The second assumption is that the particles observed in the experiment are spherical with smaller diameter compared to the molecular dimensions. Brownian motion causes a Doppler shift when the light hits the moving particle, changing the wavelength of the incoming light. This change is related to the size of the particle. The spherical size distribution can be calculated and provide a description of the particle’s motion in the medium, which measures the diffusion coefficient of the particle. Considering that from the light scattering, information about the position of the particles can be obtained, the formulas above the radius of the particles can be easily determined. DLS determinations are performed using a zetasizer (Viktoriya, 2006).

1-9-6 Zeta potential analysis .

Theoretically, zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle. The zeta potential is a key indicator of the stability of colloidal dispersions. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent and similarly charged particles in dispersion. For molecules and particles that are small enough, a high zeta potential confers stability, with the solution or dispersion resisting aggregation. At small potentials, attractive forces may exceed repulsion and the dispersion may break and flocculate. Thus, colloids with high zeta potential (negative or positive) are electrically stabilized, whereas colloids with low zeta potentials tend to coagulate or flocculate (Hanaor et al., 2012).

1-10 Magnetic property (magnetic behavior)

Materials are classified by their response to an externally applied magnetic field. Orientation of magnetic moments in a material help identify the different forms of magnetism observed in nature. Five basic types of magnetism can be described: diamagnetism, paramagnetism, ferromagnetism, antiferromagnetism, and ferrimagnetism. All other types of magnetic behaviors are observed in materials that are at least partially attributed to unpaired electrons in their atomic shells, often in the 3dor 4fshells of each atom. Materials with uncoupled atomic magnetic moments display paramagnetism; thus, paramagnetic materials have moments with no long-range orders and have small positive magnetic susceptibility (χ≈ 0), such as pyrite (Sun et al., 2000; Chen et al., 2004; Sun et al., 2004). Given its fourunpaired electronsin3d shell, Fe atom has a strongmagnetic moment. Fe2+ also have four unpaired electrons in 3d shell and Fe3+ have five unpaired electrons in 3d shell. Therefore, when crystals are formed from iron atoms or ions Fe2+ and Fe3+ can beferromagnetic,antiferromagnetic, orferrimagnetic. The individual atomicmagnetic momentsare randomly oriented inparamagneticmaterials, which have a zero net magnetic moment in the absence of amagnetic field. In aferromagneticmaterial, all the atomic moments are aligned even without an external field. Aferrimagneticmaterial is similar to a ferromagnet but has two different types of atoms with opposite magnetic moments of different strengths, resulting in a magnetic moment. Similar magnitude of the opposite magnetic moments results inantiferromagnetism, which does not exhibit net magnetic moment (Teja et al., 2009). Asingle domainmagnetic material (e.g., MNPs) that has no hysteresis loop is consideredsuperparamagnetic. The order of magnetic moments inferromagnetic,antiferromagnetic, andferrimagneticmaterials decreases at increasing temperature.Magnetiteis ferrimagnetic at room temperature (Teja et al., 2009).

1-11 Protein-NPs Interaction

The interaction of specific proteins with NPs led to a vast improvement of in vivogene delivery, clinical diagnosis, medical/cancer imaging, and receptor-targeted delivery. The development of biocompatible nanomaterials for enhancing or modifying the bioproperties is the new challenge in biotechnology. These applications can be classified into four main categories, namely, biomolecular interactions, drug and gene delivery application, biosensing, and bio-imaging(Buddai et al., 2002; Fischer et al., 2003; Filfil et al., 2003). The interactions between proteins and NP surfaces lead to the formation of protein corona around NPs that largely define their biological identities as well their potential toxicities (Lynch et al., 2006; Lynch et al., 2007).Ligands (groups) are the most common NPs used for biological system interactions(Subinoy et al., 2010).Proteins and other biomolecules compete for the NP surface when NPs enter a biological fluid, leading to the formation of a protein corona that defines the biological identity of the particle(Cedervall et al., 2007).This concept has generated new research approaches, wherein the control of surface nanostructure is used as a biomaterial design parameter to regulate cell functions, such as stem cell differentiation for in vitroand in vivo tissue engineering(Lipski et al., 2007; Ferreira et al., 2008).

The solubility and stability of NPs mainly depend on the surface properties of the particle. More importantly, bio-interfacial interactions of NPs depend on the chemical and topological nature of the NP coverage. A common motif used for NP stabilization/solubilization and biocompatibility features a ligand shell with a hydrophobic interior for micelle-like stabilization of the shell and an oligo(ethylene glycol) spacer to minimize protein denaturation(Subinoy et al., 2010). The NP surface is immediately occupied by proteins with high concentrations and high association rate constants, and subsequently by proteins with lower concentrations but higher affinity(Cedervall et al., 2007).

NPs have significant adsorption capacities because of their relatively large surface areas; therefore, they can bind or carry other molecules such as chemical compounds, drugs, and proteins attached to the surface by covalent bonds or by adsorption. Hence, the physicochemical properties of NPs, such as charge and hydrophobicity, can be modified by attaching specific chemical compounds, peptides, or proteins onto the NP surface(Aili et al., 2008; De et al., 2008). Considering that the protein corona could affect NP behavior including its biological effect, NP could also affect protein behavior. Some NPs promote in vitroprotein assembly into amyloid fibrils by assisting in the nucleation process(Linse et al., 2007).

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