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

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

LO TUAN SON

OPTIMIZATION OF MULTIFUNCTIONAL NANOPARTICLES FOR BIOSENSOR

At first, I would like to express my acknowledgement to my supervisor,Associate Prof Dr Nguyen Hoang Nam, for his advice, instructions, forsupplying researching environment in laboratory and for giving motivationduring 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 groupmeeting, seminars, discussion and suggestion for my research and my futureplans

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

Hanoi, 10th, June, 2019

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

1.4 Investigation and optimization of experimental procedure 16

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

4.1.3 FT-IR results 34

4.1.4 VSM results 37

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

REFERENCES 57

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

Figure 3.1 Chemical formula of PVP 26

Figure 3.2 Chemical formula of APTES 28

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

Figure 4.22 DLS spectra of sample MS4, MS5 MS6 and MS7 after 24h during silica coating reaction 53

LIST OF TABLES

Table 3.1 Reacting condition from sample MS1 to MS7 27Table 4.1 Positions and corresponding type of vibration of MNPs, MSNPsand MSANPs 36Table 4.2 Magnetic parameters of samples MNP, MS6 and MSAANPs

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

MSAANPs and their crystal parameters 41Table 4.5 Comparison between some magnetic parameters of sample MNPs,MS1, MS2, MS3, MS4 and MS5 50Table 4.6 Efficiency of silica coating of sample MS4, MS5 and MS7 54

MSNPs Magnetite - silica nanoparticles

MSANPs Magnetite - silica - amine nanoparticlesMSAANPs Magnetite - silica - amine - gold nanoparticlesPSD Particles size distribution

PVP Poly vinylpyrrolidone

TEM Transmission electron microscopeTEOS Tetraethyl orthosilicate

UV -VIS Ultra violet - visible light

VSM Vibrating sample magnetization

XRD X-ray diffraction

CHAPTER 1: GENERAL INTRODUCTION

1.1 Targeted nanoparticles and biosensors for disease therapy in biomedicine

The current development of Nanotechnology is promising for application inbiomedicine Nanoparticles are kind of material that owns many specicalproperties 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, detectionand therapeutics with various products being approved in clinical[12].

Figure 1.1 Some application of nanoparticles as targeted agent in medical

diagnosisThere are several approach to design biosensors for disease diagnosis.Biosensor that using flow cytometry technique shows its advantage in the rateand accuracy of counting disease cells [59] However, this device is usually

expensive and not be able to be applied in hospitals that have limited budget Forthat reason, many research groups are interested in minimizing and simplizingequipment to reduce costs and improve its portability Microcontroller chips weredesigned and developed by groups such as Massachusetts General Hospital,Harvard Medical School to measure and image size, proportion and uniformity oftargeted cells using digital camera technology (CCD camera) and self -

developed algorithm to count the number of targeted cells in a chambermicroflora[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 fluorescenceFluorescent technology have been developed and combined with CCDcamera in order to increase the accuracy of measurement and simultaneousdetection of targeted cells, as illustrated in figure 1.2 The principle of thismethod is similar to the biosensors that just use single CCD camera, except thatthe system uses two color LEDs and a color image sensor to identifyfluorescently marked cells However, the type of device is still quite bulky and itsaccuracy depends on the specificity of antibodies against used in the device[20].

To overcome this drawback, another method that can be applied is thecounting cells system by measuring electrical impedance [53] As shown in

figure 1.3, targeted cells will be captured by antibodies that are functionalized onthe electrode surface, that leads to the change of electrode impedance Thevariable impedance was measured to estimate the number of targeted cells in thespecimen Although this method own high precision in measuring impedance, theaccuracy of calculated number of targeted cells still depends on the ability to

Figure 1.3 Working principle of biosensor measuring the change in electrical

impedanceDeveloping targeted nanoparticles is a currently promising branch for thedisease diagnosis using biosensor Some measurement can be applied fordetecting disease such as measuring concentration of cancer cell orsimultaneously 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 bemodified by attaching with functional groups such as amine, carboxylic acid toconnect with biological molecules [25] Moreover, some metal nanoparticles

such as gold, silver or zinc also can be attached for detection by photoluminescence or localized surface plasmon resonance (LSPR)

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

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

Figure 1.5 Vibrating sample Magnetometer (VSM) spectrum of MNPs

proves their superparamagnetic property

When the diameter of MNPs reduces to below 30 nm, the number ofexchange - 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 hysteresisloop in its magnetization spectrum, also the value of coercivity and saturationremanence (can be determined by taking intercept of VSM spectrum to the Oxand Oy axes respectively) are approximately zero, as shown in figure 1.5

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

1.2.1.3 Synthesis of magnetite nanoparticles

(Fe(H2O)6)3+ → FeOOH + 3H++4H2O

Fe2++2OH-→ Fe(OH)2

2FeOOH + Fe(OH)2→ Fe3O4+ 2H2OCo-precipitation method shows its advantage in requiring simple reactingcondition and equipment In addition, this method is applicable for producing alarge amount of MNPs However, adjustments should be applied to obtain MNPswith narrow size distribution, suitable size of single nanoparticles and gooddispersion In details, narrow size distribution of MNPs can be obtained if thenucleation and growth process can be proceeded separately [47] Hence, high

temperature should be required to obtain unique MNPs The size distribution of

(1.1)

(1.2)(1.3)(1.4)

obtained single MNPs can be smaller and narrower when some salt is added toreacting solution For examples, addition of 1 M NaCl can minimizes the averagediameter of the nanoparticles for 1.5 nm[5].

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

b Thermal decomposition of iron organic precursor method

MNPs with high uniformity in size also can be synthesized through methodcalled thermal decomposition This method uses organometallic precursors such

as hydroxylamineferron [Fe(Cup)3], iron pentacarbonyl [Fe(CO)5], ferricacetylacetonate [Fe(acac)3], iron oleate [Fe(oleate)3] [29] In thermal

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

This method can produces MNPs with narrow size distribution [55] and also

various morphology that can be controlled by changing reaction parameters.However, thermal decomposition cannot produce large amount of nanoparticles

as co-precipitation method In addition, the environmental problem of thismethod 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 wasreported about their possibility to be oxidized and perform free radicals that havetoxic effect for human body [3] In addition, MNPs synthesized using

co-precipitation method are easy to be aggregated due to their high surfaceenergy, whereas MNPs via thermal decomposition method can be monodispersedbut 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

1.1.2.2 Coating silica shell on magnetite nanoparticles

a Stöber method

Figure 1.6 Chemical formula of tetraethyl orthosilicate

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 tetraethylorthosilicate (TEOS, figure 1.6) This reaction can takes place in both acidic andbasic medium For synthesizing free silica nanoparticles, the size distribution bythis method is from 0.05 to 2 µm, while it depends on the initial size ofnanoparticles precursor when silica is coated The possible process of silicacoating reaction can be summarized in figure 1.7

-Figure 1.7 Possible processes in silica coating reactionThe first step in silica coating reaction is the hydrolysis of TEOS Usinglabeling method [6], the mechanism of this reaction was found that the ethoxyl

(-OC2H5) groups in TEOS is replaced by hydroxyl (-OH) groups This -OHgroup comes from water molecules as reactant, or from the -OH groups on thesurface of MNPs

Si(OOC2H5)4+H2O → Si(OC2H5)3OH + C2H5OHSi(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

(1.5)(1.6)(1.7)(1.8)

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 waterand temperature are main factors that affect to the rate of hydrolysis reaction[11].

Increasing these factors (acidity or basicity, increase temperature orconcentration of precursors) will increase the rate of hydrolysis step

Bases on many factors, the next process can be deposition of intermediates

on the surface of MNPs or nucleation and self-growing processes to perform freesilica nanoparticles [11] Since the deposition is more thermodynamically stable

than the nucleation process, this second step prefers to the silica growing on thesurface of MNPs in low reaction rate If some factors are changed to promote thisprocess, such as increasing temperature or concentration of TEOS, the nucleation

of silica seed would becomes dominant That leads to a competitive reactions ofsilica growing between on the surface of MNPs and surface of silica seeds[1], as

shown in figure 1.8 As a consequence, the formation of free silica nanoparticlesreduces the efficiency of silica coating reaction and also reduces the quality ofMSNPs

Figure 1.8 Competitive reactions between silica growing on silica

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

b Inverse microemulsion

Microemulsion is a method for synthesizing MSNPs which have thick silicalayer and narrow size distribution Since MNPs synthesized by co-precipitationmethod are hydrophilic, the water - in - oil system, or inverse microemulsion isused to coat silica layer on MNPs

Figure 1.9 Synthesis of MNPs by inverse microemulsion methodMicro - water droplet (inverse micelles) is formed when the mixture ofaqueous phase and organic phase are mixed stably The size of this dropletdepends mainly on the ratio of aqueous phase and organic phase and the mixingcondition, and that also affect to the size distribution of product Changingamount 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 formfree silica nanoparticles which does not contain magnetite core[21].

Inverse microemulsion shows its advantage in performing single-coremagnetite-silica nanoparticles effectively, whereas the aggregation of magnetitenanoparticles 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-silicananoparticles would be significantly reduced Moreover, this method has lowyield and requires complex conditions such as very high stirring speed andcentrifuging

1.2.3 Multifunctional magnetite-silica nanoparticles

1.2.3.1 Introduction

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 variousmaterials to become multifunctional nanoparticles Since silica layer ownsamorphous structure and is ease to be modifiable, MSNPs is usually used toattach with other agents, such as metal nanoparticles, drug, quantum dot andenzyme The product is called multifunctional nanoparticles because theirproperty is a combination between the magnetic properties of magnetite core andproperty 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 researchfield To be applied in this drug delivery system, this nanomaterials must satisfy

3 mains standards: to be highly biocompatible, selective transporting to diseasecells and effectively release drug Some studies proved that the idealhydrodynamic 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 andelectromagnetic radiation[61].

Multifunctional silica coated - magnetite nanoparticles are potential choice in

amorphous structure can be functionalized to improve the efficiency of drugdelivery One of the most promising development of this field is thermosensitivePNIPAAm (poly (Nisopropylacrylamide)) drug loading/release system Due toits phase transition from hydrophylic to hydrophobic state at the critical solubilitytemperature of 32˚-33˚C – this mechanism can be used as a carrying ligand forhydrophylic drugs.

b Hyperthermia

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

Figure 1.11 Principle of hyperthermia method using MNPs

In this hyperthermia application, multifunctional MSNPs can be used as atargeting agent While magnetite core kills tumor tissues by overheating method,silica shell plays a role as a stabilizer for magnetite core, protective material for

toxicity of magnetite and modifiable material for functionalization [49] This

biomedical application needs magnetite - silica nanoparticles of the highestquality and many clinical tests due to risks of harming normal cells, especially intreatments of the brain tumors

c MRI imaging

One of a most powerful tool to visualize information of body internalstructure is magnetic resonance imaging (MRI) This method can observes softtissues, detect physiological and chemical changes in organism The principles ofMRI bases on the change of spin momentum of protons in human body under astrong external magnetic field This instrument work well especially in humanbody since water takes 70 percent of weight MNPs can improves contrast ofMRI image, as shown in figure 1.12, due to their magnetic property Since theyhave their own magnetization, the relaxation time of proton in water can bemodified[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 efficiencycontrast agents

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

MNPs

1.3 Multi - functional MSAANPs applied for biosensor

Figure 1.13 Structure of magnetite - silica - amine - gold nanoparticlesFigure 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, whichcontain MNPs as the core and coated by silica shell This silica layer wasmodified by attaching with some functional groups and metal nanoparticles Thisresearch focuses on attaching amine (-NH2) groups and gold (Au) nanoparticles.

Figure 1.14 Procedure of synthesizing MSAANPsThe synthesis of this nanoparticles was followed by procedure that is shown

in figure 1.14 MNPs was synthesized using co-precipitation method of ferrousand ferric salt solutions with PVP as a stabilizer The MNPs then was coated bysilica layer through Stöber method This silica layer plays a role as an amorphoussurface to be easier in modifying than the crystal surface of MNPs Moreover, the

modified MNPs are difficult to be applicable when injecting directly to humanbody due to its toxicity, while covering silica reduces this harmful effect Aminegroups 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 nanoparticlesthrough reduction method for the purpose of detecting by using photoluminescence 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 thermaldecomposition method To be able to synthesize multifunctional nanoparticles,MNPs should be like - sphere shape and hydrophilic Although MNPs can ownsnarrower size distribution in thermo-decomposition method, the morphology ofproducts are possibly rods or tube [56] Moreover, these MNPs is hydrophobic

due to the addition of fatty acids as surfactant That would be disadvantageswhen coating MNPs with silica since silica is more hydrophilic material Thefinal products of coating silica reaction could be free silica nanoparticles instead

showed that although PVP cannot prevent aggregation of MNPs entirely, thesynthesized 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 ofMNPs instead of single nanoparticles, the ratio of silica/magnetite would bereduced while the thickness of silica layer can be still the same, that can ensurethe magnetic property of magnetite core

1.4.2 Synthesis of MSNPs

Investigation the mechanism and optimization of silica coating reaction isone of the most important part in this research The synthesis of MNPs,functionalization by amine groups and attaching gold nanoparticles are alsoessential parts for synthesizing the final multifunctional nanoparticles, but themost important step needed to be understandable and optimized is coating silica.Firsty, the stability of MNPs in coating silica reaction needs to be investigatedand 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 coatingreaction occurs, the obtained product would be big particles coated by silica andcan not be applicable Due to the complication in the mechanism of the silicacoating reaction, it could leads to by-products such as free silica nanoparticlesand silica nanoparticles attached on MNPs The morphology and thickness ofsilica layer on MNPs also should be optimized to obtain products that stillexpress competent magnetic properties The coverage of silica on surface ofMNPs should be completely to stabilize magnetite core and be modifiable tofunctionalize and attach gold nanoparticles

Therefore, understanding and optimizing silica coating reaction need to beproceeded to obtained multifunctional nanoparticles with suitable size, gooddispersion, narrow size distribution, suitable thickness of silica layer and nopresence of by-product These criteria that were investigated and optimized areshown in figure 1.15

Figure 1.15 Criteria of MSAANPs needed to be optimized in this research

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 Theparticles size distribution (PSD) in DLS measurement is derived from thevariation of the intensity of scattering laser light

Figure 2.1 Working principles of DLS measurement

The typical components of DLS system are illustrated in figure 2.1 Amonochromatic light source, usually a laser beam (1) was shot through samplewhich is contained in a cell or cuvette (2) If the size of nanoparticles are biggerthan one tenth of the wavelength of laser light, the Mie scattering would happenand its intensity is measured by a detector The position of detector are usually173˚ (A) or 90˚ (B) depends on the model of equipment The attenuator (4) plays

a role in adjusting the intensity of the scattering laser The upper and lower limit

of transmission that the attenuator can adjust is 100% and 0.0003% respectively

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

2.2 Zeta Potential measurement

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

Figure 2.2 Description of zeta potentialWhen a charged nanoparticles are dispersed in a liquid medium, it attractsions with opposite charge strongly on its surface to form a charged thin layercalled “Stern layer” This Stern layer induces “diffusive ion layer” that is thickerand more loosely attracted and these two surfaces are called “electric doublelayer” (EDL) When nanoparticles move in liquid medium, there is a “slippingplane” between the ions that their movement depend on the nanoparticles and the

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

In zeta potential measurement, nanoparticles dispersed in liquid medium isapplied by a external electric field That lead to the movement of chargednanoparticles, in which their electrophoretic mobility is measured and converted

to zeta potential using the Henry equation:

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 SinceTEM use electron beam with very high intensity and low wavelength, it providesmuch 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 Instrumental components of TEM

(2.1)

Figure 2.3 shows the components of TEM machine TEM can be dividedinto three main components: the illumination system, the objective lens/stagesand the imaging system The illumination system consists an electron gun and aset of condenser lenses The electron gun provides electron beam and space forits acceleration to 20 - 1000 keV, while the lenses transfer electron to specimen.The objective lens is the most important part of TEM measurement, whereelectron beam is focused and go through the sample The specimen stage iswhere all the interaction between sample and electron beam occur The imagingsystem uses other lens to magnify the intensity of outcome signal TEM imagesare 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 andimportant measurement in determining concentration of colored material inanalytical chemistry and determining band gap of semiconductors Theinstrumental components of this equipment is illustrated in figure 2.4 Theprinciple 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 incidentradiation By measuring the ratio of intensity between income and outcome light

at different wavelength, we can plot the UV-VIS spectrum of sample Theconcentration of sample can be determined using Beer Law:

A = ɛbC = log ( I0/I)bCWhere 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

of cell containing sample (usually 1 cm) and C is the concentration of sample

(2.2)

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 XRDspectrometer, a cathode ray tube is used and filtered to perform monochromaticX-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 andthis results the constructive interference at some specific angles of incident rayThe principle of this measurement bases on the constructive interference ofincident X-rays radiation due to constant distance between two parallel planes insample 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 aXRD spectrum of sample is plotted to determine the diffraction angles and their

corresponding hkl planes.

2.6 Vibrating sample Magnetometer (VSM)

VSM is a useful tool to measure the magnetic properties of material In VSMequipment, the sample is contained in a vibrating system that oscillate duringmeasurement, as shown in figure 2.5 This vibration varies the external magnetic

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

Figure 2.5 Working components of VSM measurementSince the electromagnet is activated before starting the test, the magneticsample 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 bycalculating 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 usefultool which mainly applied in organic and analytical chemistry In addition, sinceFTIR system is able to combine to chromatography, the detection of unstablemolecules and the mechanism of chemical reactions and can be investigated.When sample is exposed to infrared radiation, it will be excited to higherlevel of vibrational energy The absorbance of each bonding in moleculescorresponds to one or some specific peak in FT-IR spectrum, in which based onthem we can deduce which bonding are available in sample Each peakcorresponds to one kind of vibration of bonding (overtoned vibration is notcounted) The wavenumber (or frequency) is determined by the gaps between

change of dipole moment and the possibility of the energy levels transition Sincemost organic compounds shows vibrational peaks within 4000 and 400 cm-1, thisregion is commonly used for FI-IR measurement.

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 polyvinylpyrrolidone (PVP) as surfactant and stabilizer In detail, a solution of 100 mL ofdistilled water and 5g of PVP (its formula is shown in figure 3.1) was preparedand 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 ofdistilled water and this solution was added to the PVP solution under mechanicalstirring Then 30 mL of heated 15% NH4OH solution was added This reactionunderwent mechanical stirring with speed of 600 round per minute (rpm) at 70℃.After 30 minutes reacting, the product was separated using permanent magnetand 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 MNPssample for determining their morphology, crystal structure, PSD and magneticproperty

Figure 3.1 Chemical formula of PVP

3.1.2 Magnetite/silica nanoparticles (MSNPs)

Eight different samples of MNPs coated by silica, which were denoted fromMS1 to MS7 (including sample MS5.1), were synthesized with different amounts

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 thedispersion and stability of magnetite nanoparticles Then various amounts ofTEOS was added slowly under mechanical stirring at various temperatures Thevolume 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

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

A solution of 40 mg of sample MS5 in 30 mL of absolute ethanol and 10 mL

of water was prepared After 15 minutes sonicating, 2 mL of (3-amino propyl)triothoxysilane (APTES, its chemical formula was illustrated in figure 3.2) wasadded The reaction was under sonicating for 45 minutes at 40℃ The product