Optimization of multifunctional nanoparticles for biosensor application

In addition, by coating silica on a cluster of MNPs instead of single nanoparticles, the ratio of silica/magnetite would be reduced while the thickness of silica layer can be still the s[r]

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY

LO TUAN SON

OPTIMIZATION OF MULTIFUNCTIONAL NANOPARTICLES FOR BIOSENSOR

APPLICATION

MASTER'S THESIS ……….

MASTER OF NANOTECHNOLOGY

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY

LO TUAN SON

OPTIMIZATION OF MULTIFUNCTIONAL NANOPARTICLES FOR BIOSENSOR

APPLICATION MAJOR: Nanotechnology

CODE: Pilot

RESEARCH SUPERVISOR:

Associate Prof Dr NGUYEN HOANG NAM

ACKNOWLEDGEMENT

At first, I would like to express my acknowledgement to my supervisor, Associate Prof Dr Nguyen Hoang Nam, for his advice, instructions, for supplying researching environment in laboratory and for giving motivation during my research

I would like to express my gratefulness to Professor Tamiya, my supervisor in Osaka during this internship for supplying working environment, all of group meeting, seminars, discussion and suggestion for my research and my future plans

I sincerely thank all professors, staff, and friends in Vietnam Japan University and VNU - University of Science for supplying me the best condition for my research

TABLE OF CONTENT LIST OF FIGURES LIST OF TABLES

LIST OF ABBREVIATION

CHAPTER 1: GENERAL INTRODUCTION

1.1 Targeted nanoparticles and biosensors for disease therapy in biomedicine1 1.2 Multi - functional magnetite-silica-amine-gold nanoparticles (MSAANPs)

1.2.1 Magnetite nanoparticles (MNPs) 3

1.2.1.1 Introduction 3

1.2.1.2 Magnetic property 4

1.2.1.3 Synthesis of magnetite nanoparticles 5

a Co-precipitation method 5

b Thermal decomposition of iron organic precursor method 6

1.2.2 Core-shell structure magnetite-silica nanoparticles 7

1.1.2.1 Roles of silica shell 7

1.1.2.2 Coating silica shell on magnetite nanoparticles 7

a Stöber method 7

b Inverse microemulsion 10

1.2.3 Multifunctional magnetite-silica nanoparticles 11

1.2.3.1 Introduction 11

1.1.3.2 Application of multifunctional magnetite - silica nanoparticles 12

a Drug delivery system 12

b Hyperthermia 13

c MRI imaging 14

1.3 Multi - functional MSAANPs applied for biosensor 14

1.4.2 Synthesis of MSNPs 17

CHAPTER 2: PRINCIPLES OF MEASUREMENT METHODS 19

2.1 Dynamic Light Scattering (DLS) measurement 19

2.2 Zeta Potential measurement 20

2.3 Transmission Electron Microscope (TEM) measurement 21

2.4 Ultraviolet - visible spectroscopy (UV-VIS) 22

2.5 X-ray Diffraction (XRD) 23

2.6 Vibrating sample Magnetometer (VSM) 23

2.7 Fourier Transform - Infrared Spectroscopy (FT-IR) 24

CHAPTER 3: EXPERIMENTAL PROCEDURE 26

3.1 Synthesis and characterization of MNPs, MSNPs, MSANPs and MSAANPs 26

3.1.1 Magnetite nanoparticles (MNPs) 26

3.1.2 Magnetite/silica nanoparticles (MSNPs) 26

3.1.3 Synthesis of magnetite-silica nanoparticles functionalized by amine groups (MSANPs) 27

3.1.4 Magnetite/silica/amine/gold nanoparticles (MSAANPs) 28

3.2 Investigation and optimization of synthesis procedure 28

3.2.1 Investigation of effect of pH on PSD and zeta potential of MNPs 28 3.2.2 Investigation of effect of surfactant on stability of MNPs 29

3.2.3 Investigation of effect of temperature on silica coating reaction 29

3.2.4 Investigation of effect of TEOS on magnetic properties of MNPs in silica coating reaction 30

3.2.5 Investigation of mechanism of silica coating reaction 30

CHAPTER 4: RESULTS AND DISCUSSION 31

4.1 Characterization of MNPs, MSNPs, MSANPs and MSAANPs 31

4.1.1 TEM and DLS results 31

4.1.2 UV-VIS results 33

4.1.5 XRD results 39

4.2 Investigation and optimization of experimental procedure 42

4.2.1 Effect of pH on stability of MNPs 42

4.2.2 Effect of surfactant on preventing aggregation of MNPs during silica coating reaction 45

4.2.3 Effect of temperature on silica coating reaction 47

4.2.4 Effect of silica precursor on the magnetic properties of magnetite core 48

4.2.5 Effect of silica precursor on the mechanism of silica coating reaction 51

CONCLUSION 55

FUTURE PLAN 56

LIST OF FIGURES

Figure 1.1 Some application of nanoparticles as targeted agent in medical

diagnosis

Figure 1.2 Working principles of biosensor using combined CCD camera and fluorescence

Figure 1.3 Working principle of biosensor measuring the change in electrical impedance

Figure 1.4 Crystal structure of magnetite

Figure 1.5 Vibrating sample Magnetometer (VSM) spectrum of MNPs proves their superparamagnetic property

Figure 1.6 Chemical formula of tetraethyl orthosilicate

Figure 1.7 Possible processes in silica coating reaction

Figure 1.8 Competitive reactions between silica growing on silica nanoparticles and silica seeds 10

Figure 1.9 Synthesis of MNPs by inverse microemulsion method 11

Figure 1.10 Some branch of functionalizing silica layer on magnetite - silica nanoparticles 12

Figure 1.11 Principle of hyperthermia method using MNPs 13

Figure 1.12 MRI images of human brain without using (left) and using (right) MNPs 14

Figure 1.13 Structure of magnetite - silica - amine - gold nanoparticles 15

Figure 1.14 Procedure of synthesizing MSAANPs 15

Figure 1.15 Criteria of MSAANPs needed to be optimized in this research.18 Figure 2.1 Working principles of DLS measurement 19

Figure 2.2 Description of zeta potential 20

Figure 2.3 Instrumental components of TEM 21

Figure 2.4 Instrumental components of UV-VIS measurement 23

Figure 2.5 Working components of VSM measurement 24

Figure 4.1 (left) TEM image of magnetite nanoparticles 31

Figure 4.2 (right) Particles size distribution of magnetite nanoparticles calculated from TEM measurement 31

Figure 4.3 TEM image of magnetite-silica nanoparticles 31

Figure 4.4 TEM image of MNAANPs 32

Figure 4.5 Particles size distribution (PSD) of MNPs, MSNPs, MSANPs and MSAANPs 33

Figure 4.6 UV-VIS spectra of MNPs, MSNPs (sample MS5) and MSAANPs34 Figure 4.7 FT-IR spectra of MNPs, MSNPs and MSANPs 35

Figure 4.8 VSM spectra of MNPs and MSNPs (sample MS4) 37

Figure 4.9 VSM spectra of samples MNPs, MS6 and MSAANPs (1000/H versus Ms) 38

Figure 4.10 XRD spectra of MNPs and MSAANPs 39

Figure 4.11 PSD and zeta potential of MNPs under different pH 42

Figure 4.12 Sedimentation of MNPs under different pH 43

Figure 4.13 Effect of sodium citrate on sedimentation of MNPs 44

Figure 4.14 Description of PVP playing a role on the stabilization of MNPs45 Figure 4.15 PSD and zeta - potential of MNPs under different concentration of PVP 46

Figure 4.16 Sedimentation experiment of MNPs under different concentration of PVP 47

Figure 4.17 TEM images of (a): sample MS5 and (b): sample MS5.1 47

Figure 4.18 DLS results of (a): sample MS5 and (b): sample MS5.1 48

Figure 4.19 VSM results of sample MNPs, MS1, MS2, MS3, MS4 and MS549 Figure 4.20 Hydrodynamic diameter of MNPs during silica coating reaction51 Figure 4.21 The change (Δd) of hydrodynamic diameter of sample MNPs, MS4, MS5, MS6, MS7 during silica coating reaction 52

LIST OF TABLES

Table 3.1 Reacting condition from sample MS1 to MS7 27 Table 4.1 Positions and corresponding type of vibration of MNPs, MSNPs

and MSANPs 36 Table 4.2 Magnetic parameters of samples MNP, MS6 and MSAANPs

calculated from their VSM spectra 39 Table 4.3 Position of diffraction peaks of magnetite in sample MNPs and their crystal parameters 41 Table 4.4 Position of diffraction peaks of gold nanoparticles in sample

MSAANPs and their crystal parameters 41 Table 4.5 Comparison between some magnetic parameters of sample MNPs,

LIST OF ABBREVIATION

Abbreviation Description

APTES (3-Amino propyl) Triothoxysilane DLS Polyvinyl pyrrolidone FT-IR Tetraethyl orthosilicate MNPs Magnetite nanoparticles MSNPs Magnetite - silica nanoparticles MSANPs Magnetite - silica - amine nanoparticles MSAANPs Magnetite - silica - amine - gold nanoparticles

PSD Particles size distribution PVP Poly vinylpyrrolidone TEM Transmission electron microscope TEOS Tetraethyl orthosilicate UV -VIS Ultra violet - visible light

CHAPTER 1: GENERAL INTRODUCTION

1.1 Targeted nanoparticles and biosensors for disease therapy in biomedicine

The current development of Nanotechnology is promising for application in biomedicine Nanoparticles are kind of material that owns many specical properties such as high surface area, great biocompatibility and potential abilities to be modified [26] Research about nanoparticles, as shown in figure 1.1, applied in medicine are currently focusing on disease through imaging, detection and therapeutics with various products being approved in clinical[12].

Figure 1.1 Some application of nanoparticles as targeted agent in medical diagnosis

developed algorithm to count the number of targeted cells in a chamber microflora[45] However, the image sensor and bio-chips in this technique is just one time - used[35].

Figure 1.2 Working principles of biosensor using combined CCD camera and fluorescence

Fluorescent technology have been developed and combined with CCD camera in order to increase the accuracy of measurement and simultaneous detection of targeted cells, as illustrated in figure 1.2 The principle of this method is similar to the biosensors that just use single CCD camera, except that the system uses two color LEDs and a color image sensor to identify fluorescently marked cells However, the type of device is still quite bulky and its accuracy depends on the specificity of antibodies against used in the device[20].

Figure 1.3 Working principle of biosensor measuring the change in electrical impedance

Developing targeted nanoparticles is a currently promising branch for the disease diagnosis using biosensor Some measurement can be applied for detecting disease such as measuring concentration of cancer cell or simultaneously observing them in human body [23] Magnetic nanoparticles is very appropriate for applying in targeted diagnosis Since it owns very high ratio of surface area to volume and ease to be functionlized, nanoparticles can be modified by attaching with functional groups such as amine, carboxylic acid to connect with biological molecules [25] Moreover, some metal nanoparticles such as gold, silver or zinc also can be attached for detection by photo luminescence or localized surface plasmon resonance (LSPR)

1.2 Multi - functional magnetite-silica-amine-gold nanoparticles (MSAANPs)

1.2.1 Magnetite nanoparticles (MNPs)

1.2.1.1 Introduction

Magnetite(iron (II,III) oxide or ferrous-ferric oxide) is one kind of iron oxide that show ferrimagnetic property [24,58] The empirical formula of magnetite, which is Fe3O4, is usually considered as a combination of one ferrous oxide and

one ferric oxide Magnetite shows inverse spinel lattice structure, as illustrated in figure 1.4 Each unit cell consists of 32 O2- anions occupying and forming face

monoclinic to cubic structure when temperature decreases to a certain point Research have found out that this transition of magnetite occurs at 120 K[19]

Figure 1.4 Crystal structure of magnetite

1.2.1.2 Magnetic property

The Curie temperature of magnetite is 850K Below this temperature, magnetite show ferrimagnetic properties due to alignment of magnetic moments in its crystal structure The tetrahedral sites, where are occupied by Fe3+ cations

express ferromagnetic moments, whereas it is anti-ferromagnetic in octahedral sites occupied by both Fe2+ and Fe3+ cations Therefore, they cancel each other

and lead to the ferrimagnetic property of magnetite[16]

When the diameter of MNPs reduces to below 30 nm, the number of exchange - coupling spins that resist magnetic reorientation decrease That leads to the appearance of superparamagnetic property of MNPs [17] This superparamagnetic property of MNPs can be verified by the absence of hysteresis loop in its magnetization spectrum, also the value of coercivity and saturation remanence (can be determined by taking intercept of VSM spectrum to the Ox and Oy axes respectively) are approximately zero, as shown in figure 1.5

Superparamagnetic property of MNPs plays important role in controlling its magnetic behaviour MNPs can be easily become magnetically saturated at low magnetic field and after its removal, there is almost no magnetic remanence That makes MNPs can be used in application that requires separating process for micro and nano - subjects

1.2.1.3 Synthesis of magnetite nanoparticles a Co-precipitation method

One of the most popular method to synthesize MNPs is co-precipitation method, which is discovered 30 years ago by Massart [26] This reaction bases on the co-precipitation of Fe2+and Fe3+in basic aqueous solution.

Fe2++ 2Fe3+ 8OH-→ Fe3O4+ 4H2O

The mechanism of this reaction is complicated and not direct Some complexes of iron was formed as intermediates[33].

(Fe(H2O)6)3+ → FeOOH + 3H++4H2O

Fe2++2OH-→ Fe(OH)2

2FeOOH + Fe(OH)2→ Fe3O4+ 2H2O

Co-precipitation method shows its advantage in requiring simple reacting condition and equipment In addition, this method is applicable for producing a large amount of MNPs However, adjustments should be applied to obtain MNPs with narrow size distribution, suitable size of single nanoparticles and good dispersion In details, narrow size distribution of MNPs can be obtained if the nucleation and growth process can be proceeded separately [47] Hence, high (1.1)

obtained single MNPs can be smaller and narrower when some salt is added to reacting solution For examples, addition of M NaCl can minimizes the average diameter of the nanoparticles for 1.5 nm[5].

Another drawback of co-precipitation method is that MNPs tend to aggregate during reaction to form very big cluster that can not be called nanoparticles and would not able to be applied in biomedicine Their superparamagnetic property make sure that they can not attach with each other by magnetic remanence, however their crystal surface with large surface area/volume ratio lead to very high surface energy and very sensitively and easily to be aggregated To overcome this problem, some surfactant, such as polyvinylpyrrolidone (PVP) are used to cover the surface of MNPs and minimize surface energy The strong interaction between outer crystal planes of MNPs is replaced by weak Van Der Waals interaction of PVP covered on their surface They would not able to be aggregated since this Van Der Waals interaction is much more weaker than their Brownian motion in solution

b Thermal decomposition of iron organic precursor method

MNPs with high uniformity in size also can be synthesized through method called thermal decomposition This method uses organometallic precursors such as hydroxylamineferron [Fe(Cup)3], iron pentacarbonyl [Fe(CO)5], ferric

acetylacetonate [Fe(acac)3], iron oleate [Fe(oleate)3] [29] In thermal

decomposition method, these precursors are heated up to their boiling point in a non - polar solvent and decomposed to form MNPs with controllable morphology and narrow size distribution Capping agent, such as fatty acids and hexadecylamine is used in this method for size adjustment Morphology of the nanoparticles is affected by ratio of precursor/non-polar solvent and the heating rate

as co-precipitation method In addition, the environmental problem of this method should be considered since most of used precursors are highly toxic[10].

1.2.2 Core-shell structure magnetite-silica nanoparticles

1.1.2.1 Roles of silica shell

MNPs still exist difficulties to be applicable in biomedicine MNPs was reported about their possibility to be oxidized and perform free radicals that have toxic effect for human body [3] In addition, MNPs synthesized using co-precipitation method are easy to be aggregated due to their high surface energy, whereas MNPs via thermal decomposition method can be monodispersed but hydrophobic and therefore not suitable for biomedicine application [38, 30, 52] Moreover, it is difficult to attach the other crystal materials to MNPs to form multifunctional nanoparticles due to their inconsistency in crystal parameters [46].

Coating surface of MNPs by silica (SiO2) shell can overcomes all of these

problems Silica was reported as non-toxic material [31], therefore coating silica shell can reduces toxicity of magnetite core and improves their biocompatibility The aggregation of MNPs can be prevented by the amorphous structure of silica that results in the decrease of surface energy of MNPs Moreover, the amorphous surface of silica shows high potential for functionalizing with organic groups such as amine, cacboxyl or the other metal nanoparticles[44]

1.1.2.2 Coating silica shell on magnetite nanoparticles a Stöber method

Stöber method, which was discovered the first time by Werner Stöber in 1968 [51], is a kind of sol-gel process to synthesize silica or coated - silica nanoparticles In this method, silica is formed by the hydrolysis of silica -containing precursor The most common silica precursors was used is tetraethyl orthosilicate (TEOS, figure 1.6) This reaction can takes place in both acidic and basic medium For synthesizing free silica nanoparticles, the size distribution by this method is from 0.05 to µm, while it depends on the initial size of nanoparticles precursor when silica is coated The possible process of silica coating reaction can be summarized in figure 1.7

Figure 1.7 Possible processes in silica coating reaction

The first step in silica coating reaction is the hydrolysis of TEOS Using labeling method [6], the mechanism of this reaction was found that the ethoxyl (-OC2H5) groups in TEOS is replaced by hydroxyl (-OH) groups This -OH

group comes from water molecules as reactant, or from the -OH groups on the surface of MNPs

Si(OOC2H5)4+H2O → Si(OC2H5)3OH + C2H5OH

Si(OOC2H5)3OH + H2O → Si(OC2H5)2(OH)2 + C2H5OH

Si(OOC2H5)2(OH)2+ H2O → Si(OC2H5)(OH)3+ C2H5OH

Si(OC2H5)(OH)3+ H2O → Si(OH)4+ C2H5OH

The hydrolysis of TEOS can be proceeded in both acidic or basic catalyst, though the mechanism of hydrolysis step are quite different [11, 36] The intermediates after this hydrolysis step are Si(OC2H5)3OH, Si(OC2H5)2(OH)2,

Si(OC2H5)(OH)3, and Si(OH)4 pH, the initial concentration of TEOS and water

and temperature are main factors that affect to the rate of hydrolysis reaction[11]. Increasing these factors (acidity or basicity, increase temperature or concentration of precursors) will increase the rate of hydrolysis step

Figure 1.8 Competitive reactions between silica growing on silica nanoparticles and silica seeds

Stöber method is one of simplest way to synthesize silica-shell nanoparticles since it requires simple reacting conditions and equipment However, the formation of silica layer is very complicated, that leads to side - reactions such as aggregation of nanoparticles, broad size distribution, or formation of free silica nanoparticles Hence, the condition of silica coating reaction through Stöber method need to be controlled and optimized to produce expected MSNPs

b Inverse microemulsion

Figure 1.9 Synthesis of MNPs by inverse microemulsion method Micro - water droplet (inverse micelles) is formed when the mixture of aqueous phase and organic phase are mixed stably The size of this droplet depends mainly on the ratio of aqueous phase and organic phase and the mixing condition, and that also affect to the size distribution of product Changing amount of magnetite or silica precursors also varies the morphology of MSNPs Increasing concentration of MNPs leads to the formation of multi-core MSNPs, while increasing concentration of silica precursor (usually TEOS) tend to form free silica nanoparticles which does not contain magnetite core[21].

Inverse microemulsion shows its advantage in performing single-core magnetite-silica nanoparticles effectively, whereas the aggregation of magnetite nanoparticles during silica coating reaction in Stöber method is more difficult to be controlled [50] However, coating silica in single MNPs would increases the silica/magnetite ratio, therefore the magnetic property of magnetite-silica nanoparticles would be significantly reduced Moreover, this method has low yield and requires complex conditions such as very high stirring speed and centrifuging

1.2.3 Multifunctional magnetite-silica nanoparticles

Figure 1.10 Some branch of functionalizing silica layer on magnetite - silica nanoparticles

Figure 1.10 illustrates the diversity of silica shell in attaching with various materials to become multifunctional nanoparticles Since silica layer owns amorphous structure and is ease to be modifiable, MSNPs is usually used to attach with other agents, such as metal nanoparticles, drug, quantum dot and enzyme The product is called multifunctional nanoparticles because their property is a combination between the magnetic properties of magnetite core and property of attaching agent

1.1.3.2 Application of multifunctional magnetite - silica nanoparticles a Drug delivery system

Drug delivery using nanomaterials as carrier is currently promising research field To be applied in this drug delivery system, this nanomaterials must satisfy mains standards: to be highly biocompatible, selective transporting to disease cells and effectively release drug Some studies proved that the ideal hydrodynamic diameter of nanoparticles for this application is less than 200 nm [13] The mechanisms of drug release are mainly based on diffusion, magnetic field, temperature, pH, electric field dependent, ultrasonic sound and electromagnetic radiation[61].

amorphous structure can be functionalized to improve the efficiency of drug delivery One of the most promising development of this field is thermosensitive PNIPAAm (poly (Nisopropylacrylamide)) drug loading/release system Due to its phase transition from hydrophylic to hydrophobic state at the critical solubility temperature of 32˚-33˚C – this mechanism can be used as a carrying ligand for hydrophylic drugs

b Hyperthermia

Hyperthermia is considered as a simple and reckless method for direct treatment of disease cells Principles of this method, as shown in figure 1.11, bases on the convertible ability from magnetic to thermal energy of magnetite core First, magnetite - carried agent was attached to selective tumor cells After applying external magnetic field, MNPs absorb magnetic energy and convert it to thermal energy As a results, the temperature of tumor cells increases, and they would be eliminated if their temperature excesses 43ºC[8]

Figure 1.11 Principle of hyperthermia method using MNPs

toxicity of magnetite and modifiable material for functionalization [49] This biomedical application needs magnetite - silica nanoparticles of the highest quality and many clinical tests due to risks of harming normal cells, especially in treatments of the brain tumors

c MRI imaging

One of a most powerful tool to visualize information of body internal structure is magnetic resonance imaging (MRI) This method can observes soft tissues, detect physiological and chemical changes in organism The principles of MRI bases on the change of spin momentum of protons in human body under a strong external magnetic field This instrument work well especially in human body since water takes 70 percent of weight MNPs can improves contrast of MRI image, as shown in figure 1.12, due to their magnetic property Since they have their own magnetization, the relaxation time of proton in water can be modified[47] The criteria of contrast agent should be stability, safety, biodistribution, tolerance [34], but efficiency and quality are poor so actual researches in this field are promising the new generation of high efficiency contrast agents

Figure 1.12 MRI images of human brain without using (left) and using (right) MNPs

Figure 1.13 Structure of magnetite - silica - amine - gold nanoparticles Figure 1.13 illustrates the structure of magnetite-silica-gold nanoparticles, which is the target for synthesizing, investigating and optimizing of this research This multifunctional nanoparticles follows the core - shell structure, which contain MNPs as the core and coated by silica shell This silica layer was modified by attaching with some functional groups and metal nanoparticles This research focuses on attaching amine (-NH2) groups and gold (Au) nanoparticles

Figure 1.14 Procedure of synthesizing MSAANPs

modified MNPs are difficult to be applicable when injecting directly to human body due to its toxicity, while covering silica reduces this harmful effect Amine groups was attached on the silica layer through the hydrolysis of (3-aminopropyl) triethoxysilane (APTES), which also form a new amine - contained silica layer, to connect this nanoparticles with biological molecules such as enzym or anti -gen This nanoparticles was also modified by attaching gold nanoparticles through reduction method for the purpose of detecting by using photo luminescence and local surface plasmon resonance (LSPR)

1.4 Investigation and optimization of experimental procedure 1.4.1 Synthesis of MNPs

MNPs can be synthesized by both co-precipitation and thermal decomposition method To be able to synthesize multifunctional nanoparticles, MNPs should be like - sphere shape and hydrophilic Although MNPs can owns narrower size distribution in thermo-decomposition method, the morphology of products are possibly rods or tube [56] Moreover, these MNPs is hydrophobic due to the addition of fatty acids as surfactant That would be disadvantages when coating MNPs with silica since silica is more hydrophilic material The final products of coating silica reaction could be free silica nanoparticles instead of MNPs

MNPs synthesized by co-precipitation method are hydrophilic due to formation of FeOOH and Fe(OH)2as intermediates [4] These molecules provide

large amount of -OH group on surface of MNPs, that make them become more hydrophilic and suitable for silica coating reaction The problem in this method that need to be controlled is the aggregation of MNPs If the obtained product is big cluster of nanoparticles, it would forms big MNPs that cannot be applied

showed that although PVP cannot prevent aggregation of MNPs entirely, the synthesized cluster of MNPs is stable at the range of diameter from 100 to 200 nm [18] That would be a great convenience since this range of size is still applicable for detection by biosensor In addition, by coating silica on a cluster of MNPs instead of single nanoparticles, the ratio of silica/magnetite would be reduced while the thickness of silica layer can be still the same, that can ensure the magnetic property of magnetite core

1.4.2 Synthesis of MSNPs

Investigation the mechanism and optimization of silica coating reaction is one of the most important part in this research The synthesis of MNPs, functionalization by amine groups and attaching gold nanoparticles are also essential parts for synthesizing the final multifunctional nanoparticles, but the most important step needed to be understandable and optimized is coating silica Firsty, the stability of MNPs in coating silica reaction needs to be investigated and optimized If MNPs is not stable in the condition of coating silica reaction, they would aggregate to form big cluster of magnetite and when silica coating reaction occurs, the obtained product would be big particles coated by silica and can not be applicable Due to the complication in the mechanism of the silica coating reaction, it could leads to by-products such as free silica nanoparticles and silica nanoparticles attached on MNPs The morphology and thickness of silica layer on MNPs also should be optimized to obtain products that still express competent magnetic properties The coverage of silica on surface of MNPs should be completely to stabilize magnetite core and be modifiable to functionalize and attach gold nanoparticles

CHAPTER 2: PRINCIPLES OF MEASUREMENT METHODS 2.1 Dynamic Light Scattering (DLS) measurement

Dynamic light scattering (DLS) is a kind of measurement for determination of size distribution of particles and polymers dispersed in solution DLS can also be called quasi-elastic light scattering or photon correlation spectroscopy The particles size distribution (PSD) in DLS measurement is derived from the variation of the intensity of scattering laser light

Figure 2.1 Working principles of DLS measurement

by a correlator, which is kind of digital processing board (5) The auto correlation function (ACF) and the hydrodynamic size of particles are derived through the analysis of the scattering intensity at different time intervals by the correlator These information is then sent to a computer (6) with a corresponding software to analyse and measure the particles size distribution

2.2 Zeta Potential measurement

Zeta potential (also denoted as ζ - potential) is a term that describes electric potential in a interfacial double layer (DL), as described in figure 2.2 Zeta potential indicates important physical properties of nanoparticles such as absorption rate, aggregating tendency or the interaction with biological system

Figure 2.2 Description of zeta potential

ions that move freely in medium The zeta potential is measured as the electric potential on this slipping plane

In zeta potential measurement, nanoparticles dispersed in liquid medium is applied by a external electric field That lead to the movement of charged nanoparticles, in which their electrophoretic mobility is measured and converted to zeta potential using the Henry equation:

  

3 ) ( 2 F a Ue 

where ε illustrates the dielectric constant of the solvent, η represents the viscosity of solvent and F(κa) is the Henry function.

2.3 Transmission Electron Microscope (TEM) measurement

Transmission electron microscope (TEM) is a kind of microscope that is used to observe samples with internal structure in nanoscale and atomic scale Since TEM use electron beam with very high intensity and low wavelength, it provides much higher resolution than the other common microscopes that use visible light to observe sample TEMs finds application in disease research, chemical identity, semiconductor and nanotechnology research

Figure 2.3 shows the components of TEM machine TEM can be divided into three main components: the illumination system, the objective lens/stages and the imaging system The illumination system consists an electron gun and a set of condenser lenses The electron gun provides electron beam and space for its acceleration to 20 - 1000 keV, while the lenses transfer electron to specimen The objective lens is the most important part of TEM measurement, where electron beam is focused and go through the sample The specimen stage is where all the interaction between sample and electron beam occur The imaging system uses other lens to magnify the intensity of outcome signal TEM images are recorded on a conventional film positioned below the fluorescent screen 2.4 Ultraviolet - visible spectroscopy (UV-VIS)

UV-VIS spectroscopy is considered as one of the most common and important measurement in determining concentration of colored material in analytical chemistry and determining band gap of semiconductors The instrumental components of this equipment is illustrated in figure 2.4 The principle of this measurement bases on the electron transition of material excited by source of ultraviolet (200-380 nm) and visible (380-760 nm) light That leads to the absorbance of this material at some specific wavelengths of incident radiation By measuring the ratio of intensity between income and outcome light at different wavelength, we can plot the UV-VIS spectrum of sample The concentration of sample can be determined using Beer Law:

A = ɛbC = log ( I0/I)bC

Where A is the absorbance of sample, ɛ is the molar absorptivity of sample, I0and I are the intensity of income and out come light respectively, b is the length

Figure 2.4 Instrumental components of UV-VIS measurement 2.5 X-ray Diffraction (XRD)

XRD is an useful tools for determining crystal structure of material In XRD spectrometer, a cathode ray tube is used and filtered to perform monochromatic X-ray radiation (0.7 - Ǻ in wavelength), which is then projected to the sample Due to very short wavelength, X-ray can penetrate the lattice net of sample and this results the constructive interference at some specific angles of incident ray

The principle of this measurement bases on the constructive interference of incident X-rays radiation due to constant distance between two parallel planes in sample At some specific angels θ that satisfy Bragg’s Law (nλ=2d sin θ, where is the wavelength of incident X-ray radiation, d is the interplane distance and n is an integer number), the intensity of constructive interference is highest and then a XRD spectrum of sample is plotted to determine the diffraction angles and their corresponding hkl planes.

2.6 Vibrating sample Magnetometer (VSM)

field and perform an electrical current in a coil that obeys the Faraday’s Law of Induction

Figure 2.5 Working components of VSM measurement

Since the electromagnet is activated before starting the test, the magnetic sample becomes stronger than field that is produced As a result, a magnetic field is formed around the sample and can be analyzed when the vibration begins by calculating the changes occur in relation to the timing of movement The changes of signals are recorded and the hysteresis loop of sample is graphed

2.7 Fourier Transform - Infrared Spectroscopy (FT-IR)

FTIR spectrometers (Fourier Transform Infrared Spectrometer) is an useful tool which mainly applied in organic and analytical chemistry In addition, since FTIR system is able to combine to chromatography, the detection of unstable molecules and the mechanism of chemical reactions and can be investigated

change of dipole moment and the possibility of the energy levels transition Since most organic compounds shows vibrational peaks within 4000 and 400 cm-1, this

CHAPTER 3: EXPERIMENTAL PROCEDURE

3.1 Synthesis and characterization of MNPs, MSNPs, MSANPs and MSAANPs

3.1.1 Magnetite nanoparticles (MNPs)

MNPs were synthesized by co-precipitation method using polyvinyl pyrrolidone (PVP) as surfactant and stabilizer In detail, a solution of 100 mL of distilled water and 5g of PVP (its formula is shown in figure 3.1) was prepared and heated to 70℃ Then, 2.703 g of FeCl3.6H2O and 0.994 g of FeCl2.4H2O

(correspond to 0.01 and 0.005 mole respectively) were dissolved in 30 mL of distilled water and this solution was added to the PVP solution under mechanical stirring Then 30 mL of heated 15% NH4OH solution was added This reaction

underwent mechanical stirring with speed of 600 round per minute (rpm) at 70℃ After 30 minutes reacting, the product was separated using permanent magnet and washed for several times by absolute ethanol The final MNPs was dispersed in 50 mL of absolute ethanol and labeled as MNPs Characterization using TEM, UV-VIS, XRD, DLS, FT-IR and VSM measurements were applied to this MNPs sample for determining their morphology, crystal structure, PSD and magnetic property

Figure 3.1 Chemical formula of PVP 3.1.2 Magnetite/silica nanoparticles (MSNPs)

absolute ethanol After adding 0.5g of PVP, mL of 25% NH4OH solution and

10 mL of distilled water, the solution was sonicated in 30 minutes to ensure the dispersion and stability of magnetite nanoparticles Then various amounts of TEOS was added slowly under mechanical stirring at various temperatures The volume of TEOS and reaction temperature were listed at this following table 3.1

Table 3.1 Reacting condition from sample MS1 to MS7 Sample Volume of TEOS/100 mg

MNPs (mL)

Temperature MS1 0.02 Room temperature (25℃)

MS2 0.1 Room temperature

MS3 0.2 Room temperature

MS4 0.5 Room temperature

MS5 Room temperature

MS5.1 40℃

MS6 Room temperature

MS7 Room temperature

Ammonia solution was added after each 30 minutes of reaction for the first hour for the compensation of ethanol vaporized The products were separated using permanent magnet and washed for times, then was dispersed in 20 mL of absolute ethanol The TEM, UV-VIS, FT-IR, DLS and VSM measurement were applied for some of those MS samples for characterization, investigation and optimization parts

3.1.3 Synthesis of magnetite-silica nanoparticles functionalized by amine groups (MSANPs)

absolute ethanol The final product was re- dispersed in 20 mL of absolute ethanol, labeled as MSANPs and characterized by DLS, FT-IR measurements

Figure 3.2 Chemical formula of APTES 3.1.4 Magnetite/silica/amine/gold nanoparticles (MSAANPs)

The first step of synthesizing MSAANPs is adsorbing gold cation on the silica surface of MSANPs Firstly, 20 mg of MSANPs was dispersed in 30 mL of ethanol After adding mL of 20mM HAuCl4solution, this solution was adjust to

basic medium by adding 0.5 mL of 25% ammonia solution This mixture was sonicated for 45 minutes and left at room temperature overnight (12-16 hours)

After stabilizing process, the gold (III) cation was reduced to Au0by adding

10 mL of 20 mM NaBH4aqueous solution dropwisely under sonicating in hour

After that, this solution was stabilized by ageing process in hours The product was separated using permanent magnet and washed several times by absolute ethanol and was labeled as MSAANPs TEM, UV-VIS, XRD and DLS measurement were applied for characterizing this final magnetite-silica-gold nanoparticles

3.2 Investigation and optimization of synthesis procedure

3.2.1 Investigation of effect of pH on PSD and zeta potential of MNPs

potential of MNPs was similar with the DLS measurement, except that the dispersant in this experiment is distilled water

To investigate effect of pH on sedimentation of MNPs, ten 15 mL-centrifuge tubes was prepared and labeled from to 12, which correspond to their pH A solution of 50 mg MNPs dispersed in 100 mL ethanol was prepared, and its pH was also adjusted as 3.2.1 After each pH adjusting, mL of solution was taken and added to its pH-corresponding centrifuge tubes The sedimentation of these tubes was observed intuitively by taking their photos after 2h, 4h and 24h

3.2.2 Investigation of effect of surfactant on stability of MNPs

A solution of 50 mg of MNPs dispersed in 100 mL of absolute ethanol was prepared PVP was added at concentration of 0.05, 0.1, 0.2, 0.5 and percent step -by-step For the initial solution of MNPs and after each adding step, 10 mL of solution was taken and added to a each 15 mL centrifuge tube These tubes were labeled with its corresponding concentration Then mL of water and 0.2 mL of 25% ammonia solution was added to each tube The sedimentation of these tubes can be observed by taking photos after 2h, 4h and 24h

For experiment investigating PSD and zeta potential, 50 mg of MNPs was dispersed in 100 mL of water PVP was also added with the same steps as above After each adding step, mL of solution was taken for DLS and zeta potential measurement

Sodium citrate also was used in investigation for sedimentation of MNPs In detail, 50 mg MNPs was disperse in solution of 50 mL distilled water and 50 mL absolute ethanol Sodium citrate was added to increase its concentration to 0.2, 0.5, 1, and percent 10 mL of initial solution and solution after each adding step was taken and added to each 15 mL centrifuge tube The sedimentation of these tubes was observed by taking photos at t = 0, 1h and 2h

3.2.3 Investigation of effect of temperature on silica coating reaction

3.2.4 Investigation of effect of TEOS on magnetic properties of MNPs in silica coating reaction

VSM measurement was applied for sample MNPs, MS1, MS2, MS3, MS4 and MS5 to deduce influence of TEOS in silica coating reaction to their magnetic properties

3.2.5 Investigation of mechanism of silica coating reaction

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Characterization of MNPs, MSNPs, MSANPs and MSAANPs 4.1.1 TEM and DLS results

Figure 4.1 (left) TEM image of magnetite nanoparticles

Figure 4.2 (right) Particles size distribution of magnetite nanoparticles calculated from TEM measurement

The core-shell structure of MSNPs can be observed clearly through TEM measurement, as shown in figure 4.3 The darker site represents the magnetite core, due to the bigger atomic number of iron compared with silicon and oxygen The average diameter of single magnetite core is around 20 nm The silica layer, which is the brighter site, cover the magnetite core with the thickness around nm

Gold nanoparticles, represented by black-dots was attached on the surface of silica layer in MSAANPs, which is showed in TEM result is figure 4.4 The size distribution of gold nanoparticles is quite uniform, which is about 10 nm Base on TEM result, it can be concluded that the final nanoparticles has core-shell structure of MSNPs with gold nanoparticles attaching on its surface

Figure 4.4 TEM image of MNAANPs

Figure 4.5 Particles size distribution (PSD) of MNPs, MSNPs, MSANPs and MSAANPs

MNPs exists as a cluster as result of synthesis by co-precipitation method Although PVP cannot separate these clusters completely, their hydrodynamic diameter reduced remarkably from 1000 nm, in which MNPs was synthesized without PVP to about 150 nm This range of size is suitable for biosensors application The hydrodynamic diameter of MSNPs tends to increase about 40 nm after silica coating reaction due to the formation of silica layer and the aggregation of magnetite cluster This aggregation seems continue to occur when MSNPs was functionalized by APTES and attached with gold NPs The hydrodynamic diameter of final MSAANPs is about 450 nm, which is suitable for biosensor application

4.1.2 UV-VIS results

range of wavelength from 300 to 700 nm, since silica is colorless material and magnetite core does not respond with electron transition in range of UV-VIS light[9] It can be seen clearly the peak of gold at wavelength of 530 nm [57, 22] in spectrum of sample MSAANPs, which is not available in UV - VIS spectra of MNPs and MSNPs Therefore, it can be concluded that gold nanoparticles exist in sample MSAANPs The estimated size of gold nanoparticles which can be calculated from its UV-VIS spectrum is about 11.4 nm[41].

Figure 4.6 UV-VIS spectra of MNPs, MSNPs (sample MS5) and MSAANPs 4.1.3 FT-IR results

Figure 4.7 provided proofs about the silica coating and amine -functionalization of MNPs through FT-IR measurement All specific vibration peaks of all these spectra were listed in table 3.1 All spectra of samples MNPs, MSNPs and MSANPs show two strong peaks at 587 and 622 cm-1, which is due

to stretching vibration of Fe-O bonding in magnetite There is no considerable peak at 570 cm-1, which represents the Fe-O vibration in bulk material in all these

MNPs and MSNPs show weak peaks at 1289 and 1432-1453 cm-1, which is due

to the presence of residual PVP in their synthesis processes

Figure 4.7 FT-IR spectra of MNPs, MSNPs and MSANPs

The presence of silica can be confirmed by the peaks at 1066 and 1149 cm-1,

which exist in FT-IR spectra of MSNPs and MSANPs, whereas the spectrum of MNPs did not show them The broad peak around 3400 cm-1 in MNPs and

MSNPs is due to the presence of O-H vibration Since these nanopartices own very large surface area/volume ratio, the intensity of this peak is much stronger compared with corresponding bulk materials Although the strong and broad peak at 3426 cm-1, due to N-H stretching in primary amine group in MSANPs

was overlapped with O-H band and becomes more difficult to realize, the presence of this peak still can be confirmed by the absence of peak at 972 cm-1,

presence of two peaks at 1343 and 1410 cm-1 in FT-IR spectrum of MSANPs,

which are due to C-H and C-N stretching in APTES, also confirmed the coverage of amine groups on the surface of silica layer in MSANPs

Table 4.1 Positions and corresponding type of vibration of MNPs, MSNPs and MSANPs

Position of

peaks (cm-1) Type of vibration MNPs MSNPs MSANPs

587, 622 Fe-O stretching[48] strong strong strong 972 Si-OH stretching[14] no medium no 1066 Si-O vibration combined

with Fe ion[14] no strong strong 1149 O-Si-O stretching[14] no strong strong 1289 C-N stretching in residual

PVP weak weak no

1343 t(CH2)[39] no no weak

1410 C-N stretching in APTES no no weak 1432 - 1453 C-H stretching in residual

PVP weak weak no

1628 N-H symmetric stretching

[60] no no medium

1642 C=O stretching in residual

PVP medium medium no 2921 C-H stretching weak weak weak 3279 N-H symmetric stretching

overtoned[39] no no broad 3398 O-H stretching broad broad broad 3426 N-H stretching in primary

amine no no

Strong (partly overlapped with

4.1.4 VSM results

Figure 4.8 VSM spectra of MNPs and MSNPs (sample MS4)

Figure 4.9 VSM spectra of samples MNPs, MS6 and MSAANPs (1000/H versus Ms)

Table 3.2 shows the value of coercivity (Hc) and saturation remanence (Mr) of these samples From these value, we can deduce the size of single MNPs using Langevin function[32]:

3

) 3

18 (

H M M

kT D

s i sb



 

Where k is the Boltzmann’s constant, T is the temperature (Kelvin scale), Msb represents the saturation magnetization of the corresponding bulk material,

Ms is the saturation magnetization of the nanoparticles; χi is the initial

susceptibility (the slope in its VSM spectrum around origin), and 1/H0 is the

Table 4.2 Magnetic parameters of samples MNP, MS6 and MSAANPs calculated from their VSM spectra

Samples χi Ms(emu/g) 1/H0(Oe-1) H0 (Oe) D (nm)

MNPs 0.0822 58.1 0.003222 310.366 6.81 MS6 0.0558 38.6 0.01383 72.307 8.14

MSAANPs 0.0506 27.2 0.01383 72.307 7.94

Figure 4.10 shows the crystal structure of sample MNPs and MSAANPs through XRD measurement For sample MNPs, main peaks which represent for diffractive planes of magnetite, were observed at positions of 30.68º, 35.92º, 43.44º, 57.16º and 62.18º These 2θ values correspond to (220), (331), (400), (511) and (440) plane respectively [15] It can be concluded that MNPs have face - centered cubic (FCC) reverse spine structure No considerable other peaks was observed, especially diffraction peaks of Fe2O3 confirms the purity of MNPs

These diffractive peaks are similar to XRD spectrum of sample MSAANPs proved that there is no considerable change in crystal structure of MNPs during attaching gold nanoparticles reaction

Three additional peaks that show the presence of gold nanoparticles can be observed in this sample The value of these peaks are 38.23º, 43.25º and 64.66º, which correspond to (111), (200) and (220) planes respectively Base on these values, we can conclude that gold nanoparticles that attach on the surface of MNPs have the faced - centered cubic lattice

The lacttice constants of sample MNPs were calculated and shown in table 4.3 The distance between two consecutive lattice planes that correspond to diffraction angle can be calculated using Brag’s law:

) sin(

2 

 dhkl

n 

where λ is wavelength of X-ray radiation (1.54 Ǻ), n is an integral number, dhklis

diffractive planes and θ is corresponding diffractive angles By using this formula, the average lattice constant of MNPs and MSAANPs were calculated as about 8.31 and 8.314Ao, which is very close to the reference JCPDS No 82-1533, is

8.39Ao.

Table 4.3 Position of diffraction peaks of magnetite in sample MNPs and their crystal parameters

Sample No of

peak 2θ (º) hkl (plane) d (Ao) a (Ao)

MNPs

1 30.68 220 2.911 8.23

2 35.92 311 2.497 8.28

3 43.44 400 2.081 8.32

4 57.16 511 1.61 8.37

5 62.78 440 1.478 8.36

Average crystal constant (Ao) 8.31

MSAANPs

1 31.04 220 2.878 8.14

2 35.46 311 2.528 8.384

3 44.64 400 2.081 8.324

4 57.16 511 1.61 8.366

5 62.84 440 1.477 8.355

Average crystal constant (Ao) 8.314 The crystal parameters of gold nanoparticles in sample MSAANPS were listed and calculated in table 4.4 The average crystal constant that was deduced from this table is 4.108Ao,which is close to referred value of 4.185Ao.

Table 4.4 Position of diffraction peaks of gold nanoparticles in sample MSAANPs and their crystal parameters

Sample No of

peak 2θ (º) hkl (plane) d (Ao) a (Ao) MSAANPs

1 38.23 111 2.351 4.072

2 43.25 200 2.089 4.178

4.2 Investigation and optimization of experimental procedure 4.2.1 Effect of pH on stability of MNPs

Figure 4.11 PSD and zeta potential of MNPs under different pH

Figure 4.11 demonstrates the influence of pH to the PSD and zeta potential of MNPs The insignificant change of average hydrodynamic diameter, which is around 160 nm in range of pH from to 11 indicates that pH did not have considerable influence to the PSD of MNPs at this range of pH At pH 12, the hydrodynamic diameter of MNPs increase considerably to 370 nm and continue increasing after each measurement Therefore, it can be concluded that MNPs tend to aggregate strongly and quickly at this pH

acidic medium, we can conclude MNPs are more ionically repulsive when dispersed in basic medium

Figure 4.12 Sedimentation of MNPs under different pH

However, if we deduce the tendency of aggregation of MNPs from the ionic repulsion, it would contrasts with the results of sedimentation experiment, as shown in figure 4.12 After 24h, MNPs at tube 10, 11, 12, which correspond to pH 10, 11 and 12 aggregated and MNPs can be observed settling down on the bottom of centrifuge tube, while the tube (pH = 9) was aggregating All the other tubes, contain tube (pH = 8), in which its pH that was not adjusted, and tube to (pH from to 7) did not show any slight aggregation or sedimentation

To overcome this problem, three solutions were suggested The first solution is replacing ethanol by water as the medium of silica coating reaction Some experiments about the sedimentation of MNPs in water were proceeded, and the results showed that MNPs were more stable in water and its stability seems not depend on the pH [42] However, using water as medium may increases the rate of hydrolysis of TEOS dramatically and it should be questioned whether this situation could lead to some unexpected reactions

The second solution is about using surfactant to stabilize MNPs before coating silica This is the method that this report focused on to solve the problem of aggregation There would be some question that must be investigated about this method: the kind of surfactant (base on ionic repulsion of steric repulsion), effect of them to the stability of MNPs, their appropriate concentration and mechanism of silica coating reaction when these surfactant were added

Surfactant that base on steric repulsion should be suggested to overcome the aggregation problem Covering surface of MNPs reduces the ionic interaction among MNPs The weak Van der Waals interaction between surfactant layers could not dominate the diffusive movement of MNPs Therefore, theoretically, MNPs can be stabilized using steric surfactant

Figure 4.14 Description of PVP playing a role on the stabilization of MNPs Polyvinylpyrrolidone (PVP) is one of most common surfactant as matrix materials or stabilizer for synthesis of nanoparticles Also due to its negligible toxicity, PVP would be a surfactant that this research focused on to investigate its effect on the stabilization of MNPs The role of PVP was described in figure 4.16

Using acidic medium can be also a solution for the aggregation of MNPs It not only plays a role in catalyzing hydrolysis of TEOS, but also prevents MNPs aggregated However, MNPs can be partly dissolved in low pH [28] Hence, there must be suspicion about coating silica on MNPs in acidic medium

4.2.2 Effect of surfactant on preventing aggregation of MNPs during silica coating reaction

show any role for splitting up magnetite cluster to form smaller clusters or single MNPs

Figure 4.15 PSD and zeta - potential of MNPs under different concentration of PVP

The cover of PVP on the surface of MNPs can be investigated by measuring their zeta potential When the concentration of PVP is from to 0.2 percent, MNPs show similar absolute values of zeta potential, this indicate that the cover of PVP at these concentrations is not considerably When the concentration of PVP increases to 0.5 and percent, the absolute value of zeta potential become smaller dramatically to 4.24 and 2.5 mV respectively This can be explained that the complete coverage of PVP on the surface of MNPs at this concentration of PVP prevents the adsorption of hydroxide ion on the surface of MNPs At this concentration, the kind of dispersion between MNPs was changed, from ionic to steric repulsion

were much more stable in other tubes Therefore, if PVP was used as surfactant to prevent aggregation, its concentration should be bigger than 0.5 percent per 50 mg of MNPs

Figure 4.16 Sedimentation experiment of MNPs under different concentration of PVP

4.2.3 Effect of temperature on silica coating reaction

(a) (b)

A thin silica layer can be observed in TEM result of sample MS5 This silica layer is much thicker in sample MS5.1 due to increasing of reaction temperature This result is suitable with the kinetic of silica coating reaction that increasing temperature make the rate of this reaction increase

However, side-reaction can be considered as serious problem in high-temperature silica coating reaction Increase temperature can make the rate of silica coating reaction increase, but it also proceeds the aggregation of MNPs by the rapid formation of silica layer [2] This problem can be observed by the diameter of sample MS5 and MS5.1 The diameter of MSNPs in sample MS5.1, through TEM measurement, is about 200 nm, whereas it is about μm in sample MS5.1 These results of diameter are quite suitable with the DLS results of these two samples, which are shown in figure 4.18 Therefore, it can be concluded that the silica coating reaction should be proceeded at room temperature

(a) (b)

Figure 4.19 VSM results of sample MNPs, MS1, MS2, MS3, MS4 and MS5 Figure 4.19 shows VSM magnetic properties of sample MNPs, MS1, MS2, MS3, MS4 and MS5 through VSM measurement The value of Ms, Mr and Hc of these samples are also shown in table 4.4

Table 4.5 Comparison between some magnetic parameters of sample MNPs, MS1, MS2, MS3, MS4 and MS5

Sample

Amount of TEOS/100 mg

MNPs (μL)

Ms (emu/g) Mr (emu/g) Hc (Oe)

MNPs 58.1 12.21

MS1 100 63.1 1.72 25.73

MS2 200 55.9 12.21

MS3 500 51.5 0.83 8.82

MS4 1000 47.7 0.42 6.19

MS5 2000 38.6 0.21 4.18

4.2.5 Effect of silica precursor on the mechanism of silica coating reaction

Figure 4.21 The change (Δd) of hydrodynamic diameter of sample MNPs, MS4, MS5, MS6, MS7 during silica coating reaction

The effect and mechanism of TEOS on silica coating reaction was investigated by measuring the change of hydrodynamic diameter of MNPs during coating silica reaction under different concentration of TEOS (figure 4.21) It should be noted that the initial diameters of MNPs cluster were different for each experiment, so that could lead to wrong conclusion when comparing directly their values of diameter Hence, in this case, calculating the change in diameter (∆d) would be better idea

core, had occurred in higher concentration of TEOS The most possible reaction in this case can be the formation of free silica nanoparticles This reaction can be observed more clearly in the samples MS6 and MS7, which used and mL of TEOS per 100 mg of MNPs respectively The average hydrodynamic diameter of of sample MS7 in the first hours was even still smaller than its initial diameter and is the smallest compared with other samples This indicated the formation of silica seed or small silica nanoparticles that reduce the hydrodynamic diameter of sample

The formation of silica nanoparticles in high concentration of TEOS can be proved by the DLS results of these samples after 24 hours reacting, as shown in figure 4.22 DLS spectrum of samples MS6 and MS7 showed the presence of separated peaks The bigger peak represented the MNPs coated by silica, while the peak at 58.8 nm is stand for the silica nanoparticles This silica nanoparticles - peak was not shown in the DLS spectra of sample MS4 and MS5, this indicated that at this concentration of TEOS, the formation of silica nanoparticles did not occur or can be negligible

The yield of coating silica reaction can be calculated simply by converting the thickness of silica layer to the volume of TEOS that underwent hydrolysis and coated on surface of MNPs The results of silica coating efficiency of samples MS4, MS5, MS6 and MS7 are shown in table 4.5 Surprisingly, when the initial amount of TEOS doubled from 0.5 to mL, the actual TEOS coated on the MNPs even decrease 1.8 times, led to the 3.6 times reduction of yield from 80 to 22.04 percent The yield of silica coating continue decreasing to 14.32 and 9.08 percent in sample MS6 and MS7 respectively The actual volume of coated TEOS in these sample are respectively calculated as 0.4, 0.22, 0.286 and 0.454 mL A simple comparison between results of sample MS4 and MS7 shows a surprising conclusion that increasing 10 times amount of TEOS just improves 13.5 percent amount of TEOS that coated on MNPs

Base on these results, we can conclude that the formation of silica nanoparticles at high concentration of TEOS reduces the efficiency of silica coating to MNPs due to the competitive reaction of silica growth on silica nanoparticles The ideal amount of TEOS should be used to prevent formation of silica nanoparticles and increasing yield of silica coating reaction is about mL per 100 mg of MNPs

Table 4.6 Efficiency of silica coating of sample MS4, MS5 and MS7 Sample Diameter of

MNPs (nm)

Amount of TEOS/100 mg

MNPs (mL)

Diameter after 24 silica

coating

Yield of silica coating reaction

(%) MS4

148.1

0.5 191.1 80.0

MS5 174.4 22.04

MS6 180.9 14.32

CONCLUSION

Magnetite nanoparticles (MNPs), magnetite coated by silica nanoparticles (MSNPs), magnetite-silica functionalized by amine (MSANPs) and magnetite-silica-amine-gold nanoparticles (MSAANPs) were synthesized and characterized by various measurement The TEM, XRD and DLS results ensure the morphology, crystal structure and diameter of these nanoparticles Superparamagnetic properties of all samples were confirmed by VSM measurement The cover of silica layer of MNPs and attachment of amine groups and gold nanoparticles can be proved by results from UV-VIS and FT-IR measurement

Some experiments have been performed to investigate and optimize the best condition of these reaction Since MNPs is not stable in basic medium, a steric surfactant such as PVP must be used to prevent its aggregation The ideal concentration of PVP to make MNPs most stable is percent for 100 g of MNPs The silica coating reaction should be proceeded at room temperature The amount of TEOS should be about 0.5 - mL per 100 mg of MNPs to ensure their superparamagnetic property and avoid the formation of free silica nanoparticles during silica coating reaction

FUTURE PLAN

In this research, MNPs, MSNPs, MSANPs and MSAANPs were synthesized successfully and the experimental procedure of MNPs and MSNPs were optimized MSANPs and MSAANPs also should be optimized in their process to obtain product with suitable size, appreciate amount of amine group and gold nanoparticles Some experiment should be continue proceeding to find out the optimized condition for these syntheses, such as effect of amount of APTES on amount of amine group and ability attaching gold nanoparticles, amount of gold-containing precursor (HAuCl4) to the morphology, size distribution and

percentage of gold nanoparticles Some experiment about using other functionalized group and metal nanoparticles, such as carboxylic acid (-COOH) and silver nanoparticles should be considered to be synthesize to compare with the original nanoparticles

REFERENCES

[1] A Donatti, Dario & Ibañez Ruiz, Alberto & Vollet, Dimas (2002) A dissolution and reaction modeling for hydrolysis of TEOS in heterogeneous TEOS-water-HCl mixtures under ultrasound stimulation Ultrasonics sonochemistry 133-8 10.1016/S1350-4177(01)00120-1

[2] Abd Shukor, Syamsul Rizal & Zainal, Nor Ain & Azwana Ab Wab, Hajaratul & Abdul Razak, Khairunisak (2013) Study on the Effect of Synthesis Parameters of Silica Nanoparticles Entrapped with Rifampicin CHEMICAL ENGINEERING TRANSACTIONS 32 2245-2250 10.3303/CET1332375

[3] Barbara A Maher; Imad A M Ahmed; Vassil Karloukovski; Donald A. MacLaren; Penelope G Foulds; David Allsop; David M A Mann; Ricardo Torres-Jardón; Lilian Calderon-Garciduenas (2016) "Magnetite pollution nanoparticles in the human brain" (PDF) PNAS 113 (39): 10797–10801

[4] Blaney, Lee (2007) Magnetite (Fe3O4)Properties, synthesis and applications The Lehigh Review 15 33-81

[5] Boistelle, R & Astier, J.P (1988) Crystallization Mechanisms in Solution Journal of Crystal Growth 90 14-30 10.1016/0022-0248(88)90294-1

[6] Brinker, Charles (1988) Hydrolysis and condensation of silicates: Effects on structure Journal of Non-Crystalline Solids 100 31-50 10.1016/0022-3093(88)90005-1

[7] Bui, Thanh & Nu-Cam Ton, Suong & Duong, Anh & Thai Hoa, Tran. (2017) Dependence of magnetic responsiveness on particle size of magnetite nanoparticles synthesised by co-precipitation method and solvothermal method Journal of Science: Advanced Materials and Devices 10.1016/j.jsamd.2017.11.002

[9] Chaki, Sunil & Malek, Tasmira & Chaudhary, Mahesh & Tailor, Jiten & Deshpande, M (2015) Magnetite Fe3O4 nanoparticles synthesis by wet chemical reduction and their characterization Advances in Natural Sciences: Nanoscience and Nanotechnology 10.1088/2043-6262/6/3/035009

[10] Chin, Suk & Pang, Suh & Tan, Ching-Hong (2011) Green Synthesis of Magnetite Nanoparticles (via Thermal Decomposition Method) with Controllable Size and Shape Journal of Materials and Environmental Science

[11] Chou, Kan-Sen & Chen, Chen-Chih (2008) The critical conditions for secondary nucleation of silica colloids in a batch Stöber growth process Ceramics International - CERAM INT 34 1623-1627 10.1016/j.ceramint.2007.07.009

[12] D Bobo, K J Robinson, J Islam, K J Thurecht, S R Corrie (2016), "Nanoparticle - based medicines: a review of FDA-approved materials and clinical trials to date", Pharmaceutical research Vol 33, Iss 10, pp 2373- 2387

[13] Delgado, Angel & González-Caballero, Fernando & Hunter, R.J & Koopal, Luuk & Lyklema, J (2005) Measurement and Interpretation of Electrokinetic Phenomena (IUPAC Technical Report) Pure and Applied Chemistry - PURE APPL CHEM 77 1753-1805 10.1351/pac200577101753

[14] Ding, H & Zhang, Y.X & Wang, Shihua & Xu, J & Xu, Shicai & Li, G. H (2012) Fe3O4@SiO2 Core/Shell Nanoparticles: The Silica Coating Regulations with a Single Core for Different Core Sizes and Shell Thicknesses Chemistry of Materials 24 4572–4580 10.1021/cm302828d

[15] Dung, Chu (2018) Synthesis of Bifunctional Fe3O4@SiO2-Ag Magnetic–Plasmonic Nanoparticles by an Ultrasound Assisted Chemical Method

[16] Friák, Martin & Schindlmayr, Arno & Scheffler, Matthias (2007) Ab initio study of the half-metal to metal transition in strained magnetite New J Phys 10.1088/1367-2630/9/1/005

cancer nanotheranostics" International journal of pharmaceutics Vol 496, Iss 2, pp 191-218

[18] Gao, Yu & Liu, Yi & Xu, Chenjie (2014) Magnetic Nanoparticles for Biomedical Applications: From Diagnosis to Treatment to Regeneration 10.1007/978-1-4471-4372-7_21

[19] Gasparov, L V.; et al (2000) "Infrared and Raman studies of the Verwey transition in magnetite" Physical Review B.62 (12): 7939

[20] Glynn, M.T., Kinahan, D.J., Ducree, J (2013), CD4 counting technologies for HIV therapy monitoring in resource-poor settings – state-of-the-art and emerging microtechnologies, Lab Chip 13, 2731

[21] Gupta, Ajay & Gupta, Mona (2005) Synthesis and Surface Engineering or Iron Oxide Nanoparticles for Biomedical Applications Biomaterials 26 3995-4021 10.1016/j.biomaterials.2004.10.012

[22] Haiss, Wolfgang & Thanh, Nguyen & Aveyard, Jenny & G Fernig, David (2007) Determination of Size and Concentration of Gold Nanoparticles from UV−Vis Spectra Analytical chemistry 79 4215-21 10.1021/ac0702084

[23] Haque, Navedul & R Khalel, Rafallah & Parvez, N & Yadav, Sanjay & Hwisa, Nagiat & Al-sharif, Shukri & Awen, Bahlul & Molvi, Khurshid (2010) Nanotechnology in Cancer Therapy: A Review Journal of Chemical and Pharmaceutical Research, 2010, 2(5): 161-168 161-168

[24] Hurlbut, Cornelius Searle; W Edwin Sharp; Edward Salisbury Dana (1998) Dana's minerals and how to study them John Wiley and Sons

[25] Jahan, Sheikh & Sadat, Sams & Walliser, Matthew & Haddadi, Azita. (2017) Targeted Therapeutic Nanoparticles: An Immense Promise to Fight against Cancer J Drug Deliv 2017; 2017: 9090325 Volume 2017

[27] K M Taylor-Pashow, J Della Rocca, R C Huxford, W Lin (2010). "Hybrid nanomaterials for biomedical applications", Chemical Communications Vol 46, Iss 32, pp 5832-5849

[28] Kalska-Szostko, Beata & Klekotka, Urszula & Piekut, K & Satula, Dariusz (2014) Stability of Fe3O4 nanoparticles in various model solutions Colloids and Surfaces A: Physicochemical and Engineering Aspects 450 15–24 10.1016/j.colsurfa.2014.03.002

[29] Li, Yang & Afzaal, Mohammad & O'Brien, Paul (2006) The synthesis of amine-capped magnetic (Fe, Mn, Co, Ni) oxide nanocrystals and their surface modification for aqueous dispersibility Journal of Materials Chemistry - J MATER CHEM 16 10.1039/b517351e

[30] Liu, Bin & Zhang, Wei & Yang, Fengkai & Feng, Hailiang & Yang, Xinlin (2011) Facile Method for Synthesis of Fe3O4@Polymer Microspheres and Their Application As Magnetic Support for Loading Metal Nanoparticles The Journal of Physical Chemistry C 115 10.1021/jp204976y

[31] Lu, An-Hui & Salabas, Elena Lorena & Schüth, Ferdi (2007) Lu A.-H , Salabas EL , Schuth F : ‘Magnetic nanoparticles: synthesis, protection, functionalization, and application’, Angew Chem Int Ed Angewandte Chemie (International ed in English) 46 1222-44 10.1002/anie.200602866

[32] Marimón Bolivar, Wilfredo & E González, Edgar (2017) Green synthesis with enhanced magnetization and life cycle assessment of Fe3O4 nanoparticles Environmental Nanotechnology, Monitoring & Management 10.1016/j.enmm.2017.12.003

[33] Massart, René (1981) Preparation of Aqueous Magnetic Liquids In Alkaline and Acidic Media IEEE Transactions On Magnetics 17 1247-1248 10.1109/TMAG.1981.1061188 Cornell, R.M.;

[34] Mihaela Vogt, Carmen (2010) Engineered core-shell nanoparticles for biomedical applications

using a portable microchip count platform in tanzanian HIV-infected patients, PLoS One 6(7), e21409

[36] Nagao, Daisuke & Osuzu, Hideyuki & Yamada, Akira & Mine, Eiichi & Kobayashi, Yoshio & Konno, Mikio (2004) Particle Formation in the Hydrolysis of Tetraethyl Orthosilicate in pH Buffer Solution Journal of colloid and interface science 279 143-9 10.1016/j.jcis.2004.06.041

[37] Nayek, Chiranjib & Manna, Kaustuv & Bhattacharjee, Gourab & Murugavel, Pattukkannu & Obaidat, Ihab (2017) Investigating Size- and Temperature-Dependent Coercivity and Saturation Magnetization in PEG Coated Fe3O4 Nanoparticles Magnetochemistry 19 10.3390/magnetochemistry3020019

[38] O'Brien, Stephen & Brus, Louis & Murray, Christopher (2010). ChemInform Abstract: Synthesis of Monodisperse Nanoparticles of Barium Titanate: Toward a Generalized Strategy of Oxide Nanoparticle Synthesis Cheminform 33 10.1002/chin.200208284

[39] Peña-Alonso, R & Rubio, Fausto & Rubio, Juan & L Oteo, J (2007). Study of the hydrolysis and condensation of Aminopropyltriethoxysilane by FT-IR spectroscopy Journal of Materials Science - J MATER SCI 42 595-603 10.1007/s10853-006-1138-9

[40] R M Cornell, U Schwertmann (2003) The iron oxides : structure, properties, reactions, occurrences, and uses Wiley-VCH, Weinheim,

[41] Rahman, Sanim (2016) "Size and Concentration Analysis of Gold Nanoparticles With Ultraviolet-Visible Spectroscopy," Undergraduate Journal of Mathematical Modeling: One + Two: Vol 7: Iss 1, Article

[44] Rangelov, Stanislav & Pispas, Stergios (2013) Polymer and polymer-hybrid nanoparticles: From synthesis to biomedical applications 10.1201/b15390

[45] Rodriguez, W R Christodoulides, N., Floriano, P.N,, Graham, S., Mohanty, S,, Dixon, M., Hsiang, M., Peter, T., Zavahir, S., Thior, I., Romanovicz, D., Bernard, B., Goodey, A.P., Walker, B.D, McDevitt, J.T (2005), A Microchip CD4 Counting Method for HIV Monitoring in Resource-Poor Settings, PLoS Med 2(7), e182

[46] Rudakovskaya, Polina & Beloglazkina, Elena & Majouga, A.G & Klyachko, Natalia & Kabanov, A.V & Zyk, N.V (2015) Synthesis of magnetite-gold nanoparticles with core-shell structure Moscow University Chemistry Bulletin 70 149-156 10.3103/S0027131415030104

[47] Rümenapp, Christine & Gleich, Bernhard & Haase, Axel (2012). Magnetic Nanoparticles in Magnetic Resonance Imaging and Diagnostics Pharmaceutical research 29 1165-79 10.1007/s11095-012-0711-y

Schwertmann, U., Iron Oxides in the Laboratory : Preparation and Characterization, Willey-Woch, ISBN 3-527-29669-7, Weinheim, Germany, 2000, 55-60

[48] Seyed Sadjadi, Mirabdullah & Fereshteh, Fathi & Farhadyar, Nazanin & Zare, Karim (2012) Synthesize and Characterization of Multifunctional Silica Coated Magnetic Nanoparticles Using Polyvinylpyrrolidone (PVP) as a Mediator Journal of Nano Research 16 43-48 10.4028/www.scientific.net/JNanoR.16.43

[49] Sharma, Hari & Hoopes, P.J (2009) Hyperthermia induced pathophysiology of the central nervous system International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group 19 325-54 10.1080/0265673021000054621

[51] Stöber, Werner; Fink, Arthur; Bohn, Ernst (January 1968) "Controlled growth of monodisperse silica spheres in the micron size range" Journal of

Colloid and Interface Science.26 (1): 62–69.

[52] Sun, Shouheng & Zeng, Hao (2002) Size-Controlled Synthesis of Magnetite Nanoparticles Journal of the American Chemical Society 124 8204-5 10.1021/ja026501x

[53] Sun, T., Morgan, H (2010), Single-cell microfluidic impedance cytometry: a review, Microfluid Nanofluid 8, 423–443

[54] Sundar, Sasikala & Mariappan, Ramalakshmi & Piraman, Shakkthivel. (2014) Synthesis and characterization of amine modified magnetite nanoparticles as carriers of curcumin-anticancer drug Powder Technology 266 321–328 10.1016/j.powtec.2014.06.033

[55] Tartaj, P.; Morales, M.P.; Veintemillas-Verdaguer, S.; Gonzalez-Carreno, T.; Serna, C.J Synthesis, properties and biomedical applications of magnetic nanoparticles, Handbook of Magnetic Materials, 2006, 9, 403

[56] Unni, Mythreyi & M Uhl, Amanda & Savliwala, Shehaab & H Savitzky, Benjamin & Dhavalikar, Rohan & Garraud, Nicolas & Arnold, David & F Kourkoutis, Lena & S Andrew, Jennifer & Rinaldi, Carlos (2017) Thermal Decomposition Synthesis of Iron Oxide Nanoparticles with Diminished Magnetic Dead Layer by Controlled Addition of Oxygen ACS Nano 11 10.1021/acsnano.7b00609

[57] Vetten, Melissa & Tlotleng, Nonhlanhla & Rascher, Delia & Skepu, Amanda & Keter, Frankline & Boodhia, Kailen & Koekemoer, Leigh-Anne & Andraos, Charlene & Tshikhudo, Robert & Mary, Gulumian (2013) Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines Particle and fibre toxicology 10 50 10.1186/1743-8977-10-50

[59] Wu, Y., Tapia, P H., Fisher, G W., Waggoner, A S., Jarvik, J., & Sklar, L A (2013) High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology Cytometry Part A : the journal of the

International Society for Analytical Cytology, 83(2), 220–226 doi:10.1002/cyto.a.22242

[60] Yamaura, Mitiko & Camilo, R.L & Sampaio, L.C & Macedo, M.A & Nakamura, Marcelo & Toma, Henrique (2004) Preparation and Characterization of (3-Aminopropyl)triethoxysilane-Coated Magnetite Nanoparticles Journal of Magnetism and Magnetic Materials 279 210-217 10.1016/j.jmmm.2004.01.094

[61] Zheng, Xin & Xu, Victor & Tan, Yen Nee (2017) Bioinspired Design and Engineering of Functional Nanostructured Materials for Biomedical

iron (II,III) oxide disease research, Barbara A Maher; Imad A M Ahmed; Vassil Karloukovski; Donald A. Hurlbut, Cornelius Searle; W Edwin Sharp; Edward Salisbury Dana Wasilewski, Peter; Günther Kletetschka (1999) "Lodestone: Nature's