Optimization of multifunctional nanoparticles for biosensor application

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VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

LO TUAN SON

OPTIMIZATION OF MULTIFUNCTIONAL

NANOPARTICLES FOR BIOSENSOR

APPLICATION

MASTER'S THESIS

……….

MASTER OF NANOTECHNOLOGY

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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

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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.

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TABLE OF CONTENT
LIST OF FIGURES
LIST OF TABLES

LIST OF ABBREVIATION

CHAPTER 1: GENERAL INTRODUCTION...1

1.1. Targeted nanoparticles and biosensors for disease therapy in biomedicine1
1.2. Multi - functional magnetite-silica-amine-gold nanoparticles (MSAANPs)
... 3

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

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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

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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

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LIST OF FIGURES

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

diagnosis...1

Figure 1.2. Working principles of biosensor using combined CCD camera
and fluorescence...2

Figure 1.3. Working principle of biosensor measuring the change in electrical
impedance... 3

Figure 1.4. Crystal structure of magnetite...4

Figure 1.5. Vibrating sample Magnetometer (VSM) spectrum of MNPs
proves their superparamagnetic property... 4

Figure 1.6. Chemical formula of tetraethyl orthosilicate... 7

Figure 1.7. Possible processes in silica coating reaction... 8

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

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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

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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,

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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

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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

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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].

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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

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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]

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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
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obtained single MNPs can be smaller and narrower when some salt is added to
reacting solution. For examples, addition of 1 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.

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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

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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 2 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

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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.

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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

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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

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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
3 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].

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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

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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

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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

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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.

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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.

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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.

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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

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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.

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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

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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 - 2 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)

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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.

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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

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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)

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absolute ethanol. After adding 0.5g of PVP, 2 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 1 Room temperature

MS5.1 1 40℃

MS6 2 Room temperature

MS7 5 Room temperature

Ammonia solution was added after each 30 minutes of reaction for the first 2
hour for the compensation of ethanol vaporized. The products were separated
using permanent magnet and washed for 3 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)

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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 3 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 1 hour.

After that, this solution was stabilized by ageing process in 2 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

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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 3 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, 5 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 1 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 1 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, 2 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, 2 and 5 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

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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

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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.

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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 5
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

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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

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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

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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,

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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

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4.1.4. VSM results

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

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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
1
0

)

3

18 (

H

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

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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

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Figure 4.10 shows the crystal structure of sample MNPs and MSAANPs
through XRD measurement. For sample MNPs, 5 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 



d

hkl

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.

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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

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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 3 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.

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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 9 (pH = 9) was aggregating. All the
other tubes, contain tube 8 (pH = 8), in which its pH that was not adjusted, and
tube 3 to 7 (pH from 3 to 7) did not show any slight aggregation or
sedimentation.

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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.

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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

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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 0 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 1 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.

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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)

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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 1 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)

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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.

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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 0 58.1 1 12.21

MS1 100 63.1 1.72 25.73

MS2 200 55.9 1 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

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

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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.

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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 2 and 5 mL of
TEOS per 100 mg of MNPs respectively. The average hydrodynamic diameter of
of sample MS7 in the first 2 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 2
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.

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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 1 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 1 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 1 174.4 22.04

MS6 2 180.9 14.32

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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 1 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 - 1 mL per 100 mg of MNPs to ensure their
superparamagnetic property and avoid the formation of free silica nanoparticles
during silica coating reaction.

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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.

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</div>

Optimization of multifunctional nanoparticles for biosensor application

<b>VIETNAM NATIONAL UNIVERSITY, HANOI</b>

<b>VIETNAM JAPAN UNIVERSITY</b>

<b>LO TUAN SON</b>

<b>OPTIMIZATION OF MULTIFUNCTIONAL</b>

<b>NANOPARTICLES FOR BIOSENSOR</b>

<b>APPLICATION</b>

<b>MASTER'S THESIS</b>

<b>……….</b>

<b>MASTER OF NANOTECHNOLOGY</b>

<b>VIETNAM NATIONAL UNIVERSITY, HANOI</b>

<b>VIETNAM JAPAN UNIVERSITY</b>

<b>LO TUAN SON</b>

<b>OPTIMIZATION OF MULTIFUNCTIONAL</b>

<b>NANOPARTICLES FOR BIOSENSOR</b>

<b>APPLICATION</b>

<b>MAJOR: Nanotechnology</b>

<b>CODE: Pilot</b>

<b>RESEARCH SUPERVISOR:</b>

<b>Associate Prof. Dr. NGUYEN HOANG NAM</b>

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