DEVELOPMENT OF ENCAPSULATED ANTIMICROBIAL ADDITIVES …

DEVELOPMENT OF ENCAPSULATED ANTIMICROBIAL

ADDITIVES TO EXTEND BIOACTIVITY

BY

KANITA BOONRUANG

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

(ENGINEERING AND TECHNOLOGY)

SIRINDHORN INTERNATIONAL INSTITUTE OF TECHNOLOGY

THAMMASAT UNIVERSITY

ACADEMIC YEAR 2017

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Abstract

DEVELOPMENT OF ENCAPSULATED ANTIMICROBIAL ADDITIVES TO EXTEND

BIOACTIVITY

by

KANITA BOONRUANG

Bachelor: Chemical Engineering, Sirindhorn International Institute of Technology,

Thammasat University, 2014

Master of Science: Engineering and Technology, Sirindhorn International Institute of

Technology, Thammasat University, 2018

The development of silica microspheres that contain immobilized copper has led to the

application of antibacterial or antifungal paint additive that is effective, yet safe to human

beings. However, the fast release of copper when the microspheres suspended in water-based

solvent remains problematic as it reduces the useful lifetime of their applications, such as paint

additives. A slow and controlled release is often preferred to achieve efficient and prolonged

antimicrobial effect. In this study, the release of copper from acrylic-coating can be controlled

by varying the silica to copper ratio and by forming the microspheres with complex pore

properties. The goal of the research was to develop antimicrobial additives for paint. The

additives were synthesis by modified silica-copper microparticles with 3-

glycidyloxypropyltrimethoxysilane (GLYMO) and 3-Aminopropyl)triethoxysilane (APTES).

This work involvs the incorporation of copper (II) in form of copper acetate (Cu(CO2CH3)2)

to silica microparticles by using tetraethoxysilane (TEOS) as a precursor. Copper loading

capacity and copper adherence on silica surface were enhanced by addition of GLYMO and

APTES. The objective is to produce Cu/SiO2 microspheres to be mixed in antimicrobial paint

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that can inhibit growth of Escherichia coli (E.coli) and Penicillium funiculosum (P.

funiculosum). The effects in the addition of GLYMO/APTES to TEOS solution were studied.

The sol-gel formation with acid catalysts was used in combination with a spray dryer to obtain

Cu/SiO2 microspheres. The morphology of Cu/SiO2 microspheres was observed using Field

Emissive Scanning Electron Microscope (FE-SEM) to determine the particle size and particles

size distributions. Energy-dispersive spectrometry (EDS) and X-ray Fluorescence (XRF) used

an elemental analysis method to identify and quantify all compounds. The specific functional

groups of modified surfaces was confirmed by Fourier Transform Infrared Spectroscopy

(FTIR). Thermogravimetric Analysis (TGA) was used to measure percent weight loss to

determine the organosilane content in Cu/SiO2 microspheres. ImageJ program was used to

determine the area of microorganism growth on the paints surface, which was used to calculate

the inhibition rate. The higher proportion of additives present in paints, the higher the

antimicrobial activities.

Keyword: Copper, Sol-Gel Process, Antimicrobial, Paint, Cu/SiO2 microspheres

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Acknowledgements

First of all, I am deeply grateful to my advisor, Assistant Professor Dr. Wanwipa

Siriwatwechakul for the continuous support of my master study and research, who gave me a

great experience and always encouraged me. The door to Assistant Professor Dr. Wanwipa

Siriwatwechakul office was always open whenever I ran into a trouble spot or had a question

about my research or writing. Her guidance helped me in all the time of research and writing

of this thesis. I could not have imagined having a better advisor and mentor for my master

study.

Besides my advisor, I would like to thank the experts who were involved in my thesis:

Assistant Professor Dr. Paiboon Sreearunothai, Dr. Pimpa Limthongkul and Dr. Passarin

Jongvisuttisun, for their insightful comments and encouragement, but also for the hard question

which incented me to widen my research from various perspectives. Special thanks to Mr.Panus

Sundarapura, graduated student at Sirindhorn International Institute of Technology,

Thammasat University for his participations and contributions in performing the experiments.

I would also like to express my appreciation to Ms. Aye yu yu Swe and Ms. Thanh P Vu,

graduated student at Sirindhorn International Institute of Technology, Thammasat University

for their help and suggestions along this thesis work.

I am grateful for full scholarship and sponsor project of the thesis work provided by

Sirindhorn International Institute of Technology and Siam Research and Innovation ( SRI by

SCG cement).

Finally, I would like to express my gratitude to my family, who always gave a support

and continuous encouragement throughout my years of study. This accomplishment would not

have been possible without them.

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Table of Contents

Chapter Title Page

Signature page i

Abstract ii

Acknowledgements iv

Table of Contents v

List of Figures viii

List of Tables ix

1 Introduction 1

1.1 Introduction 1

1.2 Research Objective 2

1.3 Scope of research 2

2 Literature review 4

2.1 Antimicrobial Properties of Metal 4

2.2 Metal toxicity mechanism 4

2.3 Metal Nanoparticles 7

2.4 Approach for making copper nanoparticles 7

2.5 Sol-Gel Process 8

2.6 Modification of silica 11

2.7 Spray Drying Process 13

2.8 Characterization methods 14

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3 Methodology 16

3.1 Materials and equipment 16

3.2 Experiment and procedure 17

3.2.1 Synthesis of Cu/SiO2 microspheres 17

3.2.2 Modification of Cu/SiO2 microspheres 18

3.3 Characterizations methods 25

3.4 Antimicrobial activities test 25

3.4.1 Sample preparation 26

3.4.2 Microbial preparation and growth media 26

3.4.3 Contact killing on paint surface 27

4 Result and Discussion 28

4.1 SEM analysis for Acid/Base catalyst selection 28

4.2 SEM and gelation time test analysis for amount of copper acetate 28

selection

4.3 SEM -EDS analysis for ratio of TEOS to water selection 30

4.4 Antimicrobial activities of Cu/SiO2 microspheres 32

4.5 Study GLYMO-modified Cu/SiO2 microspheres 34

4.5.1 Gelation time test of GLYMO-modified Cu/SiO2 36

4.5.2 SEM EDS and XRF of GLYMO-modified Cu/SiO2 36

4.5.3 Fourier Transform Infrared Spectroscopy (FTIR) of 38

GLYMO-modified Cu/SiO2 microspheres

4.5.4 Thermogravimetric analysis (TGA) of GLYMO-modified 40

Cu/SiO2microspheres

4.5.6 Antimicrobial activities of GLYMO modified-Cu/SiO2 microspheres 42

and long term study

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4.6 Study APTES-modified Cu/SiO2 microspheres 46

4.6.1 Synthesis APTES-modified Cu/SiO2 microspheres via two-pot 46

synthesis

4.6.2 Characterization of APTES-modified Cu/SiO2 microspheres via 48

one pot synthesis

4.6.3 Characterization of APTES-modified Cu/SiO2 microspheres via 51

one pot synthesis

5 Conclusion and Recommendations 53

Reference 55

Appendices 60

Appendix A 61

Appendix B 65

Appendix C 69

Appendix D 73

Appendix E 74

Appendix F 75

Appendix G 76

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List of Tables

Tables Page

3.1 Different amount of copper acetate in solution for study copper effect 17

3.2 Different ratio of TEOS to DI water in solution to study effect of water 18

3.3 Conditions for a gelation time study at different ratios of TEOS to GLYMO in 19

sol-gel solution

3.4 Operating conditions of two-pot systhesis (a) 22

3.5 Operating conditions of two-pot systhesis (b) 23

3.6 Conditions for gelation time study at different amount of APTES and copper 24

acetate in sol-gel process

3.7 Conditions for spray dryer 24

4.1 Gelation time test of different amount of copper acetate in solution 29

4.2 EDS data of Cu/SiO2 microspheres for samples prepared with different ratios of 32

TEOS to water

4.3 Average percent inhibition on fungal growth of Sample 5 33

4.4 Average percent inhibition on fungal growth of Sample 8 33

4.5 Gelation time of different ration TEOS to GLYMO in sol-gel solution 34

4.6 EDS data of GLYMO-modified Cu/SiO2 microspheres 37

4.7 XRF data of GLYMO-modified Cu/SiO2 microspheres compare to non-modified 38

Cu/SiO2 microspheres

4.8 Summary of Relevant Peaks of GLYMO-modified Cu/SiO2 microspheres 39

4.9 Organic Content of GLYMO-modifiedCu/SiO2 microspheres determined from 41

TGA 41

4.10 Gelation time of different amount of APTES and copper acetate in sol-gel 49

process

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4.11 EDS data comparison between APTES-modified Cu/SiO2 microspheres and 51


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List of Figures

Tables Page

2.1 A summary of the main mechanisms behind the antimicrobial behavior [4] 4

2.2 Antibacterial mechanisms of metal toxicity[6]. 6

2.3 Metallic nanomaterial in various shapes and sizes [6]. 7

2.4 Anitibacterial mechanism of metal ion toxicity 8

2.5 Schematical drawing of the equations of hydrolysis and condensation in sol-gel 9

process[15].

2.6 Schematical drawing of the sol gel process in acid catalyst and base catalyst. 10

2.7 Microstructures of six different aerogels prepared using different catalysts[45]. 11

2.8 Homocondensation of hydrolyzed APTES molecule for anhydrous grafting [52]. 12

2.9 (3-Glycidyloxypropyl) trimethoxysilane (GLYMO) 13

3.1 Counting Chamber for prepare microbial suspension 27

4.1 SEM images of SiO2 microspheres produced by base catalyst (left) and acid 28

catalyst (right) and dried by spray drier with 10000 magnification at 2 kV.

4.2 SEM images of Cu/SiO2 microspheres produced by spray drier at different amount 29

of copper acetate to tetraethoxysilane (TEOS): 0.1%w/v (left), 0.2%w/v(right) with

5000 magnification at 2 kV

4.3 SEM images of Cu/SiO2 microspheres produced by spray drier at different amouth 30

of copper acetate to tetraethoxysilane (TEOS) 0.5%w/v (left), 2.4%w/v (right) with


4.4 SEM images of Cu/SiO2 microspheres produced by spray drier at different ratio of 31

tetraethoxysilane (TEOS): DI water ; Sample 5 (1:4 TEOs to water, left), Sample 6 (1:6

TEOs to water,right) with 1000 magnification at 20 kV

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4.5 SEM images of Cu/SiO2 microspheres produced by spray drier at different ratio of 31

tetraethoxysilane (TEOS): DI water ; Sample 7 (1:8 TEOs to water, left), Sample 7 (1:9


4.6 SEM images of GLYMO-modified Cu/SiO2 microspheres produced by spray drier : 36

Sample 24 (left), Sample 28 (right)

4.7 FTIR Absorbance graph of Sample 5(blue), Sample 24(green), Sample 28(red) and 40

pure GLYMO(pink).

4.8 dTGA graph comparison between Sample 5, Sample 24, Sample 28 and Pure 41

GLYMO

4.9 Particle size distribution of GLYMO modified Cu/SiO2 microspheres: Sample 28 42

(top) and Sample 24 (bottom)

4.10 The percent inhibition of Sample 28 plotted as a function of Cu/SiO2 microsphere 43

content in comparison with the commercial antifungal paint(TOA213)


content in comparison with the commercial antifungal paint(TOA213) after 6 months


content in comparison with the commercial antifungal paint(TOA213) after 1 year

4.13 : Paper coated with GLYMO-modified Cu/SiO2 microspheres shows 100% bacterial 46

inhibition as no metallic-grey color is on the surface (left). Uncoated filter paper covered

with E. coli, which appears as metallic grey on EMB agar (right).

4.14 FTIR analysis of APTES grafted silica particles (Sample32) before and after wash. 47

4.15 FTIR graph of APTES-modified Cu/SiO2 microspheres (Sample 47, red) 50

compared with Pure SiO2 (blue)

4.16 SEM-EDS images of 0.1% v/v APTES-modified Cu/SiO2 microspheres produced 51

by spray drier

A1 Antimicrobial activities effect of Sample 5 on P.funiculosum by varies amount of 61

Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213

and only acrylic paint(control) in week 1.

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and only acrylic paint(control) in week 3



and only acrylic paint(control) in week 4

B1 Antimicrobial activities effect of Sample 8 on P.funiculosum by varies amount of 65









B4: Antimicrobial activities effect of Sample 8 on P.funiculosum by varies amount of 68



C1 Antimicrobial activities effect of Sample 28 on P.funiculosum by varies amount of 69

Cu/SiO2 microspheres in acrylic paint at 1% 5% 10%and 30%wt compared with

TOA213 in week 1.

C2 Antimicrobial activities effect of Sample 28 on P.funiculosum by varies amount of 70

Cu/SiO2 microspheres in acrylic paint at 1% 5% 10%and 30%wt compared with

TOA213 in week 2.

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C3 Antimicrobial activities effect of Sample 28 on P.funiculosum by varies amount 71

of Cu/SiO2 microspheres in acrylic paint at 1% 5% 10%and 30%wt compared with

TOA213 in week 3.

C4 Antimicrobial activities effect of Sample 28 on P.funiculosum by varies amount 72

of Cu/SiO2 microspheres in acrylic paint at 1% 5% 10%and 30%wt compared with

TOA213 in week 4.

D1 Antimicrobial activities effect of Sample 28 with 5%wt of Cu/SiO2 microspheres 73

in acrylic paint on P.funiculosum by perform at 6 months ; week1 , week2 week3

and week 4 respectively.

E1 Antimicrobial activities effect of TOA213 on P.funiculosum by perform at 6 74

months ; week1 , week2 week3 and week 4 respectively.

F1 Antimicrobial activities effect of Sample 28 with 5%wt of Cu/SiO2 microspheres 75

in acrylic paint on P.funiculosum by perform at 1 year; week1 , week2 week3 and

week 4 respectively. 75

G1 Antimicrobial activities effect of TOA213 on P.funiculosum by perform at 76

1 year ; week 1 , week 2 week 3 and week 4 respectively.

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Chapter 1

Introduction

1.1 Introduction

Within construction industry, contamination of paint and cement products by biofilm

is a serious problem because it can affect human health and aesthetic. Therefore, antimicrobial

substances may be added to inhibit the growth of biofilm on the paint and cement products.

Metal nanoparticles such as silver and copper are wildly used as one of the most effective

antimicrobial agents due to their large surface area to volume ratios [ 1] . Copper and its

derivatives received attention as a preferred material for door knobs, railings, and grab bars in

public facilities. Certain studies have revealed that they were able to kill bacteria on the

contaminated surface in just under two hours [2] . Moreover copper is cheaper than silver and

gold and can also be easily mixed with polymers. It is relatively stable both in terms of chemical

and physical properties [ 1, 3, 4] . Generally, it is believed that not the metal itself acts as

biocides but the release of its ions that is responsible for the antimicrobial properties [ 4- 6] .

Metal ions can directly damage microbial cell walls, and disrupt the cells’ structural integrity.

Other researchers also suggested that the copper ions have direct oxidative damage on the DNA

structure of microbes and result in deadly mutations [3, 4, 6].

The antimicrobial properties of silica microspheres with immobilized metallic biocides

such as copper and silver are well known [7-11]. Immobilized metallic biocides with silica

particles were found by many studies because silica has a high superficial area, high pore

volume and durability in many kinds of solvents, and it is a good absorbent substrate [7-13].

Silica is used to synthesize corrosion protection coating [14, 15] and water repellent coating

[16, 17], encapsulate water- soluble dye [18], produce aerogel [19], and strengthen cement

mortar by lowering its porosity and permeability [20].

Sol- gel method has been one of the leading process to create silica particles. The

process is the formation of an oxide network through the hydrolysis and condensation reactions

of a precursor in a liquid [5, 21]. Tetraethoxysilane (TEOS) is one of the most commonly used

silica alkoxide that has been used to produce the silica sol because it is cheap, environmental

friendly, and harmless to human [14, 15, 18]. However, copper particles adsorbed onto the

porous structure of the silica can be released too quickly; hence it limits the long term

effectiveness of the antimicrobial activity [11]. A controlled release of the metal ions is often

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preferred to achieve prolonged antimicrobial effect[22]. The release of copper ion can be

controlled by modification of silica support.

This study aims to apply copper’ s antimicrobial properties to paints, by incorporating

copper immobilized silica (Cu/ SiO2) microparticles as a paint additive. We proposed a

technique to improve the stability of the copper loading in silica microparticles by modifying

the structures with 3-Glycidyloxypropyl trimethoxysilane (GLYMO) and 3-

Aminopropyltriethoxysilane ( APTES) . X-Ray Fluorescence (XRF), Scanning electron

microscopy (SEM), Energy Dispersive Spectroscopy (EDS), and Particle Size Distribution

(PSD) are used to observe the morphology, particle size, properties, and copper content. The

Fourier Transform Infrared Spectroscopy (FTIR) and Thermogravimetric Analysis (TGA) are

applied for identify specific functional group of GLYMO and APTES and their content. The

modified Cu/SiO2 microspheres are mixed with acrylic-based paint for antimicrobial test using

fungus (Penicillium funiculosum) and bacteria gram- negative (Escherichia coli). Long term

antimicrobials properties of the modified Cu/ SiO2 microspheres was studied by repeated the

test after 6 months and one year.

1.2 Research Objective

The objective of this study is to develop antimicrobial additives in paints with the following

objectives:

1. Optimize ratio of GLYMO and APTES added in Cu/SiO2 microspheres and propose

the optimum ratio of modified Cu/SiO2 microspheres in cooperated with paints.

2. Study antimicrobial effects of the modified Cu/SiO2 microspheres in comparison with

commercial antimicrobial agent (TOA213).

3. Study long term effect on antimicrobial activities of modified Cu/SiO2 microspheres in

comparison with commercial antimicrobial agent (TOA213).

1.3 Scope of research

Silica surface a great number of silanol group ( ≡ S − OH) , which can be

modified to attach a variety of functional groups for specific applications. This research will

focus on applying 3- Glycidyloxypropyl trimethoxysilane (GLYMO) and 3-

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Aminopropyltriethoxysilane (APTES) in silica gel solution at various ratios and investigate its

antimicrobial activities by using fungus (P. funiculosum) and bacteria gram-negative (E. coli).

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Chapter2

Literature review

2.1 Antimicrobial Properties of Metal

Practical antimicrobial substances must be harmful to microbes but have least effects

on human. Metals have been used prevalently as antimicrobial materials for decades even

earlier than the discovery of antibiotic. Antimicrobial activity of metal can be used in many

form such as nanoparticle, metallic compound and coating [ 6] . It can be either in form of

element or compound. Metal ions inhibit the growth of microbes by disturbing the duplication

system leading to cell dead [6, 23].

Figure 2.1 : A summary of the main mechanisms behind the antimicrobial behavior [4]

2.2 Metal toxicity mechanism

Some of metal ions in generals can be co- transported or bind to some atoms of donor

ligands, such as O, N and S, through strong and selective interactions. It has been postulated

that, external metal ions or their complexes can replace original metals present in biomolecules

leading to cellular dysfunction . This phenomenon is called ionic mimicry or molecular

mimicry, depending on whether metal ions or metal complexes are involved[6, 24]. In this

way, some metals can promote the destruction of Fe– S clusters, for instance from bacterial

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dehydratases that is particularly vulnerable to site-specific inactivation by toxic metals. Metals

can also replace non-catalytic metal-binding sites inhibiting enzyme activity [1].

For example, cupric ions (Cu2+) in particular are able to form organic complexes with

sulfur- , nitrogen- or oxygen- containing functional group present in the microorganism. This

may result in defects in the conformational structure of nucleicacids and proteins, besides

changes in oxidative phosphorylation and osmotic balance. Finally, microbe exposed to toxic

doses of some metals upregulate genes involved in the elimination of ROS generating oxidative

stress[2, 4].

The previous studies suggest that only one amino acid residue in any given protein is

susceptible to metal catalyzed oxidation and that such residues are adjacent to metal- binding

sites. Carbonyls are product that formed by the metal catalyzed oxidation of several amino acid

side chains, and level of carbonyl groups is used as a marker of oxidative protein damage.

Oxidation of the amino acid side chains in proteins may cause loss of catalytic activity,

therefore, trigger protein degradation in vivo. Therefore, in principle, metals could catalyzed

site-specific damage might be responsible for metal toxicity. In addition to destruction of active

site, metal substitutions at non- catalytic metal- binding sites can inhibit enzyme activity and

another mechanism of site-specific enzyme inhibition that can result in metal poisoning[6].

After metal ions enter into the cell, the Fenton reaction will generate free radicals,

which later react and damage protein, membrane, DNA and etc. The damage causes by

oxidation of free radical were called “oxidative stress”. Oxidative stress is key determinant of

cell damage by metal ions. There are several reactions that emit free radical into cells. These

reactions are shown below from equation (1) to (7). The damage that cause by oxidative stress

can be in form of reactive oxygen species, antioxidant depletion, protein dysfunction, loss of

enzyme activity, impaired membrane function, interference with nutrient assimilation and

genotoxicity[2, 6].

Fe2+ + H2O2 → Fe3+ + OH- + OH∙ Fenton Reaction (1)

Fe3+ + O2∙- → Fe2+ + O2 (2)

O2∙- + H2O2 → O2 + OH- + OH∙ Haber-Weiss Reaction (3)

Fe3+ + reduced antioxidant → Fe2+ + oxidized antioxidant (4)

OH∙ + RSH → H2O + RS∙ Thiyl Radical (5)

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OH∙ + (R)3CH → H2O + (R)3C∙ Carbon-Centered Radical (6)

(R)3C∙ + O2 → (R)3COO∙ Peroxyl Radical (7)

Figure 2.2 : Antibacterial mechanisms of metal toxicity[6].

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2.3 Metal Nanoparticles

Metals which are used as microbial inhibitors can be applied in several forms depending

on the applications. Three common methods are metallic nanoparticles, metallic compounds

and metallic coatings [ 6] . Nanoparticles have been fabricated using various metals combined

with organic and inorganic moieties. Metallic nanomaterial have strong antibacterial properties

especially those made of gold and copper[4, 25]. Many nanoparticles are reported for their

ability to physically interact with the cell surfaces of some bacteria [6, 26]. Nanoparticle

toxicity could be due to several attributes, including traits that are particle specific ( such as

size, shape or surface charge) and traits that control the release of metal ions. The toxic mode

of action of nanoparticles has also been associated with ROS generation and membrane

disruption [8, 23, 26]. Commercially, antimicrobial activities coming from inorganic biocides

and directly applies to applications, but due to the environmental and safety aspects the

antimicrobial activity has to be reached by the modification of nanoparticles. Since the high

surface to volume ratio of nanoparticles strongly relates to the antimicrobial properties because

the large surface area of the particles is in contact with bacteria effluent [1, 23]. The

nanoparticles containing metal compound interact with the elements of bacterial membranes

resulting in changing of structure leading to the cell death [8, 23, 27].

Figure 2.3 : Metallic nanomaterial in various shapes and sizes [6].

2.4 Approach for making copper nanoparticles

One especially successful approach incorporates Ag, Cu and Zn ions into a zeolite

carrier; metal ions embedded in the zeolite matrix exchange with other positive ions from

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moisture in the environment, leading to the release of the toxic metal ions. This technology has

been used to make antimicrobial textiles, house wares, medical equipment and devices [27].

Another important development has been the use of Cu nanoparticle as an antimicrobial

surface. In fact, most of studies regarding antimicrobial metal nanoparticles focused on the

metal ion release instead of the particle absorbed by the bacteria [24, 28, 29]. When water

containing dissolved oxygen reaches the metal particles, the standard corrosion process occurs

[30, 31]. Then, ions resulting from corrosion or dissolution process can finally be released[4].

The loss of bacterial cell viability has been correlated with the uptake of Cu ions and

increase in production of ROS [6, 32-34]. It has been hypothesized that this leads to lipid

peroxidation, loss of membrane integrity, loss of cytoplasmic content. Cu generates ROS and

further damage the cell and finally leads to cell death[23]. However, degraded at a rate that

increases with respect to the Cu content of the particles [6, 32].

Figure 2.1 : Anitibacterial mechanism of metal ion toxicity

2.5 Sol-Gel Process

Advantages of the sol-gel process are that it is a cheap and operate at low- temperature

that allows for control of the product composition. The process is the formation of an oxide

network through condensation reactions of a precursor in a liquid. Hydrolysis of alkoxyilanes

precursors in either acid or alkaline medium results in the formation of silica gel[5, 21]. There

are several factors that can affect sol- gel process for example, the pH of the mixture, the ratio

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of alkoxyilanes precursors to water, the speed of mixing and mixing time. Characteristics of

the product depend on specific combinations of these parameters [11]. Drying process and

additional substrates added to the mixture during sol- gel process also play important roles in

the formation of porous structure of the material. Materials with different physical

characteristics that can be obtained by this method ranges from amorphous xerogel, also known

as aerogel, to advanced coating materials and fibers, as well as nanospheres [11].

Figure 2.2 : Schematical drawing of the equations of hydrolysis and condensation in sol-gel

process[15].

The hydrolysis of alkoxyilanes molecule forms reactive silanol groups. The

condensation between silanol groups produces water while the condensation between silanol

groups and ethoxy groups produces ethanol. Both condensation reactions result in siloxane

bridges (Si-O-Si) that form the entire silica structure. The formation of silica particles involves

nucleation followed by particle growth. Depending on the reaction conditions, the resulting

silica forms spherical or gel network [35].

The sol- gel process mechanism is composed of two steps which are hydrolysis and

condensation [13, 14, 19, 20, 36-38]. It is prepared by the combination of metal organic as a

precursor, solvent, and catalyst to promote the reaction. TEOS is a common silica alkoxide that

has been used to produce the silica sol because it is cheap, environmental friendly, and harmless

to human [14, 15, 18]. Silica alkoxide is reported to be slow in hydrolysis when compared with

other alkoxide [39], thus suitable for the process that does not need the short-timed gelation. In

addition, the tetramethoxysilane (TMOS) can also be used to synthesize the silica gel [19], but

Eq.1

Eq.2

Eq.3

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the study of Donatti and Vollet shows that it causes rapid hydrolysis in sol- gel process and

results in quick gelation [39].

2.5.1 The Selection of Acid/Base Catalyst

Silicon alkoxides generally react slowly with water, but the reaction process, hydrolysis

and condensation, can be sped up by the use of acid and base catalysts. Acid catalysts can be

any monoprotic acid ( example, HCL or HF) and a basic catalyst usually uses ammonia,

ammonium hydroxide or ammonium fluoride [40]. Different catalysts result in different

characteristics of silica gel[11]. In acidic conditions, reaction rate decrease as more alkoxy

group is hydrolyzed. Reaction occurred at terminal Si favored as linear polymer product. While

basic condition reaction rate increase as more alkoxy group are hydrolyzed. Reaction occurred

at central Si favored result in branched polymer product chain [41, 42].

Figure 2.3 : Schematical drawing of the sol gel process in acid catalyst and base catalyst.

Nawaz and Schmidt have experimented with six different catalysts in order to evaluate

the effect of the catalyst on the microstructure of the gel. It can be observed from the SEM

images that the pore size depends on the catalysts used in preparation. Samples using acid-

based catalysts resulted in gels with higher pore density with smaller pore size, while basic

catalysts resulted in larger pore size and smaller pore density [41].

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Figure 2.4 : Microstructures of six different aerogels prepared using different catalysts[45].

2.6 Modification of silica

Modification of silica using copolymers attracts interest during the past decades due

to its application which produce multifunctional particles with a variety of ordered structures

[43]. In particular, silica- modified networks can lead to desirable combinations of properties

such as high backbone flexibility, low glass transition temperatures and good thermal and

oxidative stability [44]. In 2013, Oktay and Kayaman-Apohan prepared antibacterial organic-

inorganic hybrid coatings using polydimethysiloxane ( PDMS) as the copolymer to be

integrated into the silica matrix. It was found that the hybrid coating has a higher decomposition

temperature compared to the control sample, showing superior thermal property. This could be

due to the well-dispersed silica network formed from the inorganic precursor [45].

Modification of the silica surface with amino or thiol functionalized

organoalkoxysilane can be used to stabilize the metal particles. 3-Aminopropyltriethoxysilane

(3-APTES) is organoalkoxysilane which easily hydrolyzes in water. It was suggested that the

reaction occurs through the interaction between the amino group of APTES and surface Si–OH

group in anhydrous condition, forming a stable surface attachment [12, 46]. In general,

functionalization of these materials can be carried out by two methods. The first method is

simultaneous co- condensation between a silica precursor and organoalkoxysilane to form

functionalized silica after in a single-step process. In the second method, a functional group is

attached to the silica substrate by means of a coupling reaction between the silanol groups on

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the silica surface and a selected organosilane [12, 47]. The experiment by Dang Viet Quang

also shows that 3-APTES grafted on silica beads are able to adsorbed silver or copper particles

due to complexes formation and electrostatic interaction [48]. The grafted layer is

hydrolytically stable in water at room temperature. Moreover APTES may polymerize in water

at the silica surface and thus lead to a large grafting yield [49]. Bing et al., have measured the

property of grafting density by using cupric ion adsorption between amino-modified silica

particles and unmodified silica particles, which result shows that APTES modification

significantly changed the cupric ion adsorption capacity of the silica particles; while the

unmodified silica particles had little adsorption capacity. High grafting density on the modified

silica surface brings a high functionality. The result shows that the addition of APTES toTEOS

can enhance the silica structure to anchor the cupric ion than unmodified amino surface of silica

structure.

Figure 2.5 : Homocondensation of hydrolyzed APTES molecule for anhydrous grafting

[52].

3-Glycidyloxypropyl trimethoxysilane (GLYMO) is cross-link structure precursor that

generally used as adhesion promoter in coating industry [15, 50]. GLYMO bonding in form of

van der Waals bond during the liquid stage and later covalent bonds, which has strong adhesive

force. In the study of dye encapsulation by Kim et al., the GLYMO-doped silica can retain the

dye particle in the matrices due to its ability to lower the pore size of the silica gel structure

and thus increase the pore density. Due to its ability which can lower the leaching rate of

entrapped molecule and become hydrophobic at certain ratio of liquid to precursor [16]. The

study proposed that the larger pore silica gel is unable to trap the dye molecule long enough

and also allows the water molecule to penetrate the pore to resolve the dye molecule, and thus

leads to the higher leaching rate and dye loss. They also propose that the effect of GLYMO to

silica gel is that it acts as both physical entrapment to lower pore size and covalent entrapment.

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Chu et al. reported that addition of GLYMO to silica matrix can act as the binder, lead to

increase the thickness of the silica structures, increase density, and enhance the adhesion to

polymer substrate. In addition, the usage of GLYMO surface- modification is also used as a

copper pretreatment process [14].

Figure 2.9 : (3-Glycidyloxypropyl) trimethoxysilane (GLYMO)

2.7 Spray Drying Process

Spray drying technology is widely used to convert liquids ( solutions, emulsions,

suspension or slurries) to solid powders with an acceptable level of degradation and oxidation

of volatile compounds. This is done by removing the moisture component from the liquid

solution[21]. Common applications of spray drying are found in food, chemical and materials

industries. Spray drying process depends greatly on the preparation, homogenization,

atomization, dispersion and subsequently dehydration of the feed solution. All spray dryers use

some type of atomizer or spray nozzle to sprayed solution into a chamber, a droplets of the

solution are releasing through the nozzle and in contact with the hot air thus turning it from

liquid to powder form. The hot drying air can be passed as a co-current or counter-current flow

to the atomizer direction. Final products will collected via cyclone[51]. The advantages of

using spray drying include the ability to produce powders of desired particle size and moisture

content by manipulating operating conditions. It is a continuous and easy operation with a

quick response time and also applicable to both heat sensitive and heat resistant materials .This

makes spray drying a suitable method of drying for the production of silica powder with

uniform composition in large scale.

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2.8 Characterization methods

2.8.1 Scanning Electron Microscope (SEM)

Scanning electron microscope is equipment used to investigate samples less than

micron length small [52, 53]. Inside the column of the microscope, the cathode heated up by

electric current emits electrons into the vacuum. Below the cathode, a metallic disk with a

central hole is installed, also known as the anode. The anode is connected with a positive pole

of a high voltage source and the cathode with a negative pole. The electric field between the

cathode and anode accelerates the electrons downwards. These electrons are called the primary

electrons which form a broad diverging beam which is directed onto the specimen. An

electromagnetic lens focuses this beam finely on the specimen surface to form secondary

electrons. In conclusion, the scanning electron microscope detects these secondary electrons

and uses them to build up an image which will be shown on the monitor[54].

2.8.2 Energy Dispersive X-Ray Spectroscopy (EDS)

The Energy Dispersive X- Ray Spectroscopy ( EDS) was used in conjunction to the

SEM, and was used for the elemental analysis of the specimen’ s surface topology. Because

each element has a unique atomic structure, therefore, having a specific peak of its x- ray

emission. The sample was bombarded with electron beam and turn the sample into excited

state. Then, the excited state caused the x-ray emission from the sample and was detected by

the detector. Eventually, the element or chemical analysis will be done. It can be used to

determine the percentage of each element was contained in each sample. In this study EDS was

used to see how much copper was present, and this amount can be compared to how much

copper was incorporated in microspheres.

2.8.3 X-ray fluorescence spectrometer (XRF)

X-ray Fluorescence (XRF) is a technique to determine the elemental content in samples.

The primary x-ray beam collides the atoms in the samples, they become ionized. If the radiation

energy is high enough, it can displace an inner electron, and the atom becomes unstable and an

outer electron will replace the inner electron. When this happens, energy is released in form

of secondary x-ray and is termed fluorescent radiation. The secondary x-ray is a characteristic

of a transition between specific electron orbitals in a particular element, the resulting

fluorescent X-rays then can be used to detect the abundances of elements that are present in the

sample.

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2.8.4 Thermogravimetric analysis (TGA)

The TGA is a thermal analysis, and operates by measuring the amount of weight loss

as a function of increasing temperature and time [55-57]. The TGA data and its differential

(DTGA) can be used to analyze the amount of organic or inorganic components by comparing

with known curves of each component, determined by experimentation. The result showed the

peak of the amount of organic loss in weight percent.

2.8.5 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR measurement is used to confirm the functional groups that are responsible for the

absorption property. Each specific peak indicates the functional groups. By analyzing how

well the sample absorbs light at each wavelength, and producing a graph of a wavelength range,

this data can be compared to a reference of how functional groups behave under the same

conditions. The expected structure and the functional groups for identify APTES are aliphatic

amine group (N-H) at ~1650 cm−1, ~1550 cm−1 and ~3400cm−1. The observed peak at CH2

group is at ~2921cm−1. The presence of GLYMO can identify by glycidyl group and methoxy

group (-O-CH3). Glycidyl group can be identified by epoxy band at 3050 cm-1, 1250 cm-1, and

915 cm-1 and methoxy peaks are located ~2800cm−1 [58-62].

2.8.6 Particle Size Distribution (PSD)

PSD is a technique for characterizing the relative amount of the particles present

according to the size. The distribution of the particles also analyzed by PSD.

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Chapter 3

Methodology

3.1 Materials and equipment

3.1.1 Material for the Synthesis of Cu/SiO2 Powder and Modified Cu/SiO2 Powder

3-Aminopropyltriethoxysilane (Sigma Aldrich, Singapore)

3-glycidyloxypropyltrimethoxysilane (Merck, Germany)

Nitric acid (Merck, Germany)

Tetraethoxysilane (Merck, Germany)

Copper (II)acetate monohydrate (Sigma Aldrich, Singapore)

Deionized Water

Glassware

Orbital Shaker

Parafilm M®

Spray Dryer (SDE-5 Euro, Euro best, Thailand)

3.1.2 Material for Testing the Antimicrobial Properties

Penicillium funiculosum stock

Escherichia coli stock

Potato Dextrose Agar (PDA), (HiMedia, India)

Eosin Methylene Blue Agar, (HiMedia, India)

Sodium Chloride (Qrec, New Zeland)

Tryptic Soy Agar, (HiMedia, India)

Tryptic Soy Broth , (HiMedia, India)

Autoclave machine

Incubator

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3.2 Experiment and procedure

3.2.1 Synthesis of Cu/SiO2 microspheres

3.2.1.1 Sol-Gel preparation with catalyst selection

Base Catalyst

Preparation of Cu/SiO2 microspheres were based on a method which described by

Zielecka et al [11]. Hydrolysis of TEOS, which resulted in silica formation was catalyzed by

ammonia solution at pH 11. Ethanol, DI water, and ammonia were mixed with a

EtOH:NH3:H2O ratio of 200:1:55 (v/v). This mixture was added with TEOS (TEOS: H2O =

23:55 v/v) and stirred for 3 hours.

Acid Catalyst

Acid based process was done, according to B Mahltig [5]. TEOS was mixed with water

at the ratio TEOS to DI water of 1:9 (v:v). The hydrolysis of TEOS was performed by adding

0.15% v/v of nitric acid (anhydrous HNO3) to adjust the pH of the mixture to be below 3 and

continued stirring for 3 hours.

3.2.1.2 Effects of the amount of copper acetate on gelation.

From the previous studies, the amount of TEOS was added to DI water was fixed at a

ratio of 1:9 (v:v). HNO3 was added with the concentration 0.15% (by volume). Continue

stirring for 3 hours, and then copper acetate was added. The amount of copper ( II) acetate,

according to Table 1, varied and continued stirring for another 1 hours before feeding the

solution into the spray dryer.

Table 3.1 : Different amount of copper acetate in solution for study copper effect

Sample Copper acetate,

w/v

TEOS

(mL)

DI Water

(mL)

HNO3

(mL)

Copper (II) acetate

(g)

1 0.1% 20 180 0.3 0.188

2 0.2% 20 180 0.3 0.376

3 0.5% 20 180 0.3 0.940

4 2.4% 20 180 0.3 4.700

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3.2.1.3 Study the ratio of TEOS to water

After the ratio of copper acetate to solution was determined, the effects of water was

also studied. The amount of DI water added to TEOS varied as shown in Table 2, because the

ratio of TEOS to water is one of factor that affects the final product properties[19]. HNO3 was

added with the concentration 0.15% (by volume). The ratio copper acetate was kept at 0.2%

(w/v). The mixture was stirred for 3 hours, and the copper acetate was added and continued

stirring for another 1 hours.

Table 3.2 : Different ratio of TEOS to DI water in solution to study effect of water

Sample Ratio of TEOS to Water, (v:v) TEOS (mL) DI Water (mL) HNO3 (mL)

5 1:4 400 1600 3

6 1:6 285.7 1714.3 3

7 1:8 222.2 177.78 3

8 1:9 200 1800 3

3.2.2 Modification of Cu/SiO2 microspheres

3.2.2.1 GLYMO modified-Cu/SiO2 microspheres

Previous research reported that adding GLYMO into sol gel solution will retard the

gelation time, which is impractical for large scale processing. Thus, the amount of copper

acetate and the catalyst, which responsibility for the acceleration of gelation time was studied

to find a suitable condition for spray drying. The ratio of silica precursors (TEOS and

GLYMO) to water was fixed 1:4 (v:v). The solution was stirred for 3.5 h. Copper (II) acetate

was added and stirred for another 30 minutes. the gelation time was observed of each sample.

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Table 3.3 : Conditions for a gelation time study at different ratios of TEOS to GLYMO in sol-gel solution

Sample TEOS:GLYMO

(v:v)

TEOS

(mL)

GLYMO

(mL)

DI Water

(mL)

Copper acetate

(w/v)

Copper (II) acetate

(g)

Nitric acid

(mL)

9 1:0.25 8 2 40 0.2% 0.1 0.06

10 1:0.17 8.57 1.43 40 0.2% 0.1 0.06

11 1:0.125 8.88 1.12 40 0.2% 0.1 0.06

12 1:0.1 9.10 0.90 40 0.2% 0.1 0.06

13 1:0.25 8 2 40 0.2% 0.1 0.2

14 1:0.17 8.57 1.43 40 0.2% 0.1 0.2

15 1:0.125 8.88 1.12 40 0.2% 0.1 0.2

16 1:0.1 9.10 0.90 40 0.2% 0.1 0.2

17 1:0.25 8 2 40 0.6% 0.3 0.06

18 1:0.17 8.57 1.43 40 0.6% 0.3 0.06

19 1:0.125 8.88 1.12 40 0.6% 0.3 0.06

20 1:0.1 9.10 0.90 40 0.6% 0.3 0.06

21 1:0.25 8 2 40 1% 0.5 0.06

22 1:0.17 8.57 1.43 40 1% 0.5 0.06

23 1:0.125 8.88 1.12 40 1% 0.5 0.06

24 1:0.1 9.10 0.90 40 1% 0.5 0.06

25 1:0.25 8 2 40 3% 1.5 0.06

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26 1:0.25 8 2 40 5% 2.5 0.06

27 1:0.25 8 2 40 7% 3.5 0.06

28 1:0.17 8.57 1.43 40 3% 1.5 0.06

29 1:0.125 8.88 1.12 40 3% 1.5 0.06

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3.2.2.2 APTES Modified-Cu/SiO2 microspheres

Synthesis APTES-modified Cu/SiO2 microspheres via two pot synthesis

The two-pot synthesis method requires pure silica particles prior to surface modification

process. Sol-gel method was first used to prepare pure silica particles (SiO2) by follow B

Mahltig [5]. The second step involved the surface modification of SiO2 by APTES. Then,

copper acetate will be applied to the finished APTES grafted-SiO2.

Method 1 for APTES grafted SiO2 particles

Method 1 was modified from the study by A. Ebrahiminezad [63] by varies amount of

APTES and reaction time. Silica particles (SiO2) was pretreated with hydrochloric solution at

pH 3 for 30 min. 4.2 g of pretreated SiO2 particles were suspended in 150 mL of ethanol to

water solution (1:1 by volume) and sonicated to get uniform dispersion. APTES was added to

the suspension while maintaining the temperature at 40 C in a water bath. Continue sonication

for another 10 minutes under N2 atmosphere. The reaction was allowed for 8 and 13 hours at

40 C with continuous stirring. The amount of APTES and the reaction time was detailed in

Table 4 below. Finally the grafted silica powder were filtered and washed with ethanol and DI

water, and dried in an oven overnight at 50 C.


Method 2 was modified from the study by A. Ebrahiminezad [63] by varies operating

temperature and drying temperature. First, pretreated SiO2 particles 5.6 g were suspended in

200 mL of toluene and sonicated to get uniform dispersion. 0.1 mL of APTES was added to

the suspension while maintaining the temperature at 50 C in a water bath. Then, continue

sonication for another 10 minutes under N2 atmosphere. The reaction was carried out for 18

hours at different temperatures. The amount of APTES and the reaction temperature is detailed

in Table 4-5 below. Finally the grafted silica particles were filtered and washed with toluene,

and dried in an oven overnight at 50 C.


Method 3 was modified from the study by F. Cuoq [49]. 4 g of SiO2 particles were

added to 80 mL of water and then 2x10-3mol of APTES was introduced to the suspension. The

reaction was carried out for 18h under stirring at room temperature. Finally the grafted silica

powder were filtrated and rinsed then dried in oven at 40C overnight.

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Rinsing procedure:

Grafted silica was removed by rinsing in 150 mL of water for 7h. at room temperature with

continuous stirring. pH of the suspensions was adjusted to pH 3 to prevent the dissolution of

the silica. The suspensions were then filtrated and rinsed with 300 mL of DI water.


Method 4 was modified from the study by Hui Li [64]. 3g of SiO2 particles was added

to 24 mL of DI water and 120 mL ethanol. Under continuous stirring, 1.5 mL ammonia solution

(30%, w/v) and 2.7 mL APTES were added to the mixture. The reaction was allowed to proceed

at room temperature for 3h. The suspensions were then filtrated and washed with ethanol and

DI water the dried at 50C overnight.

Table 3.4 : Operating conditions of two-pot systhesis (a)

Sample 30

(Method1)

Sample 31

(Method1)

Sample 32

(Method1)

Sample 33

(Method2)

Solution 75 mL of DI

water

75 mL of

ethanol

75 mL of DI

water

75 mL of

ethanol

75 mL of DI

water

75 mL of

ethanol

200 mL of

Toluene

Amount of SiO2 4.2 g. 4.2 g. 4.2 g. 5.6 g.

Amount of

APTES

16.8 mL

(10 v/v%)

8.4 mL

(5 v/v%)

16.8 mL

(10 v/v%)

0.1 mL

(0.49 v/v%)

Operating Temp.

(C)

40 40 40 50

Reaction time (h) 2 18 18 18

Drying temp (C) 50 overnight 50 overnight 50 overnight 40 overnight

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Table 3.5 : Operating conditions of two-pot systhesis (b)

Sample 34

(Method2)

Sample 35

(Method3)

Sample 36

(Method4)

Solution 200 mL of

Toluene

80 mL of water 24 mL of DI water

120 mL of ethanol

1.5 mL ammnoia

solution

Amount of SiO2 5.6 g. 4 g. 3 g.

Amount of APTES 0.1 mL

(0.49 v/v%)

0.93602 mL

(1.15 v/v%)

2.7 mL

(1.82 v/v%)

Operating Temp.

(C)

120 C Room

temperature

Room temperature

Reaction time (h) 18h 18h 3h

Drying temp (C) 55 for 3 hr. 40 overnight 50 overnight

Synthesis APTES-modified Cu/SiO2 microspheres via one pot synthesis

This method is a simultaneous co-condensation between the silica precursor and a

selected organoalkoxysilane to form functionalized silica. Previous studies showed that adding

APTES into the solution of a silica precursor will accelerate the gelation time, which caused

the gelation time to be too fast for processing. Thus, the amount of APTES, ratio of TEOS to

solvent and copper acetate, were varied to find a suitable condition for spray drying while the

ratio of TEOS to solvent (water and ethanol) was fixed at 1:4 (by volume). The mixture was

subjected to stirring, after a few minutes when solution was well mixed, 0.15% (by volume) of

nitric acid was added as the catalyst. The solution was stirred while APTES was added and

continued stirring for 3.5 h. Copper (II) acetate was added and stir for another 30 min. The

gelation time was observed of each sample.

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Table 3.6 : Conditions for gelation time study at different amount of APTES and copper

acetate in sol-gel process

Sample TEOS

(mL)

Ethanol

(mL)

DI Water

(mL)

APTES

(v/v)

APTES

(mL)

Copper

(II)

acetate

(w/v)

Copper

(II)

acetate

(g)

37 10 - 40 0.1% 0.05 0.2% 0.1

38 10 - 40 0.5% 0.25 0.2% 0.1

39 10 - 40 1% 0.5 0.2% 0.1

40 10 40 - 0.5% 0.25 1% 0.5

41 10 40 - 0.5% 0.25 2% 1

42 10 40 - 0.5% 0.25 3% 1.5

43 10 20 20 0.5% 0.25 0.2% 0.1

44 10 10 30 0.5% 0.25 0.2% 0.1

45 10 8 32 0.5% 0.25 0.2% 0.1

46 10 8 32 0.5% 0.25 0.5% 0.25

47 10 8 32 0.5% 0.25 1% 0.5

48 10 8 32 0.5% 0.25 1.5% 0.75

3.2.1.2 Spray Drying Process

The sol gel suspension was supplied to spray dryer to obtain Cu/SiO2 microspheres,

which were used for testing the efficiency of antimicrobial activities. The parameters for

operating the spray dryer are as stated in the Table 7 below:

Table 3.7 : Conditions for spray dryer

Temperature Inlet 200°C

Temperature Outlet 95°C

Air pressure 0.1 bar

Feed rate 7-7.5 mL/min

Blower speed 2,400 rpm

Nozzle AO 140-6-37-70-ss

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Nozzle CAP 40100 DF-SS

3.3 Characterizations methods

Cu/SiO2 microspheres were characterized by using scanning electron microscopy

(SEM) and Energy-dispersive spectrometry (EDS) were used to determine the particle size,

morphology and all element content in microspheres. SEM-EDS data were performed at Do

SEM 24 hr, Thailand. X-ray fluorescence spectrometer (XRF) was used to identify and quantify

all compounds in every sample by obtained from Thailand Institute of Nuclear Technology

(Public Organization) . Particle Size Distribution (PSD) is a technique for characterizing the

relative amount of the particles present according to the size. The GLYMO modified Cu/SiO2

microparticles were dispersed in DI water and characterized by Dynamic Light Scattering

(Zetasizer Nanoseries, model S4700) from National Nanotechnology Center (NANOTEC),

Pathum Thani, Thailand.Fourier transform infrared spectroscopy (FTIR) was also used to

confirm the functional groups that are responsible for the absorption property Cu/SiO2

microspheres were grinded and mixed with Potassium bromide (KBr) and pressed into platelets

prior to being recorded on NicoletiS50 in transmission mode. Then Thermogravimetric

analysis (TGA) can be used to confirm the presence of APTES and GLYMO on the synthetic

microspheres. Yhe significant weight loss of organic group occurs at the temperature range of

350 – 600 °C at the heating rate of 10 °C/min under N2 (g) [56, 57, 65] and performed at Center

of Scientific Equipment for Advanced Research, Thammasat University.

3.4 Antimicrobial activities test

Fungi and gram-negative bacteria are selected to test for the antimicrobials capabilities

of the Cu/SiO2 microspheres. Acrylic-based paint was modified with Cu/SiO2 microspheres

and tested with the cultures of model microbials. Penicillium funiculosum (P. funiculosum)

was chosen as a model for fungus. Escherichia coli (E. coli) was chosen as a model for gram-

negative bacteria. The procedures were performed under aseptic conditions to prevent

contamination. The microbial growth was analyzed by using the image processing program,

ImageJ (US National Institute of Health) to evaluate the area covered by the microbes.

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3.4.1 Sample preparation

This method was modified from M. Zielecka et al. [11] Filter papers number 1 with 90

mm. diameter, Whatman for size 5x5 cm2 were prepared and autoclaved for use as sterile paper

media. Acrylic base paint was pre-prepared by mix acrylic paint with sterilized RO water at

ratio 1:1 (by volume). Then acrylic base paint was mix with selected Cu/SiO2 microspheres at

difference w/v ratio (1 - 30 wt%). The modified-paints were coated onto sterile paper media

with 5 replicate in each condition and air-dried for 24 hours at room temperature. The paper

coated with commercial antimicrobial paint (TOA 213 Water Repellent) was used as a positive

control, and the paper coated with acrylic base paint alone was used as a negative control.

3.4.2 Microbial preparation and growth media

3.4.2.1 Antifungal experiment

The method was modified from ASTM D2574-9700. Sub-culture of P.funiculosum was

spread on Potato dextrose agar (PDA) to prepare stock suspension and incubate at 30±2°C at

least 3 days, but no more than 7 days. Stock suspension is start by dilute cultured P.funiculosum

with 9 mL sterile NaCl solution, such that the resultant spore suspension contains 0.8 to 1.2 by

106 spores/mL as determine with a counting chamber. To evaluate the antibacterial efficiency

of Cu/SiO2 microspheres, Potato dextrose agar (PDA) were used as growth media.

3.4.2.2 Bacteria experiment

First, take 2 mL of bacteria inoculum and incubate in 8 mL of Tryptic Soy Broth (TSB)

with a tube cap loosely closed at 37± 2 °C for 24 hours. Then spread the bacteria from TSB

onto the Tryptic Soy Agar ( TSA) plate, and continue incubating at the same condition for

another 24 hours. After that, use the same method of dilution as the antifungal test so that the

concentration of bacteria is diluted to approximately 106 CFU/mL. To evaluate the antibacterial

efficiency of Cu/SiO2 microspheres, different growth media were used. For gram negative

bacteria (E.Coli), Eosin Methylene Blue agar (EMB) was used because it is a both selective

and differential growth medium. E.Coli growth will turn the media greenish/grey metallic

color.

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For counting the number of microbial, the equation below was applied:

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 1 𝑚𝑙 = 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑥 1𝑚𝑙

4 𝑥 10−6

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 =𝑠𝑢𝑚 𝑜𝑓 𝑠𝑝𝑜𝑟𝑒𝑠 𝑖𝑛 5 𝑐𝑒𝑙𝑙𝑠

5

3.4.3 Contact killing on paint surface

The painted paper as placed onto the growth media surface. Note that all glassware and

solutions used in association with the fungi should be autoclaved prior to use to minimalize the

risk of contamination. An auto- pipette was used to drop 25 microliters of the microbial-

containing-solution on the painted paper media in the prepared petri dishes, and a cell-spreader

was used to spread the solution evenly on the surface of the plate. The surface must be inspected

to confirm that the entire surface was well covered. The procedures involving the application

of microbial cultures to the painted surfaces was done by the same applicator for every sample.

The processes were performed under a Class II Biosafety cabinet, and in aseptic conditions to

prevent contaminant from the environment. The petri dishes were incubated at 30±2°C, over a

period of 4 weeks for the antifungal experiment and 37± 2 °C for a week on the antibacteria

experiment. Finally, the antimicrobial activity was evaluated by the percent coverage of the

microbial growth on the sample using ImageJ program. The percent inhibition is defined in

Equation (1) below.

% 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (1 −𝐴𝑟𝑒𝑎 𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑚𝑖𝑐𝑟𝑜𝑏𝑒𝑠 𝑜𝑛 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒

𝐴𝑟𝑒𝑎 𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑚𝑖𝑐𝑟𝑜𝑏𝑒𝑠 𝑜𝑛 𝑡ℎ𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) 𝑥 100 ---(1)

Figure 3.1 : Counting Chamber for prepare microbial suspension

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Chapter 4

Result and Discussion

4.1 SEM analysis for Acid/Base catalyst selection

The type of catalysts used in a sol-gel process was said to be one the main factor that

shapes the physical properties, e. g. pore size, surface topology, of the silica gel [19]. SEM

micrographs showed that Cu/SiO2 microspheres from both acid- and base-catalyzed processes

have the particle size around 5-10 μm. The acid-catalyzed process presented particles with

more smooth surfaces with spherical shapes than those obtained from the base-catalyzed

process. By compared Cu/SiO2 microspheres yield between acid and base catalyst process, acid

process yields particles with higher level of integrity. Heavier particles which resulted from

acid process can be collected easier then it help to increase the process yield. The limitation of

base catalyst process is that the powder is small and light, so sample collection is challenged.

In order to produce bigger and heavier powder, acid catalyst process was proposed.

Figure 6 : SEM images of SiO2 microspheres produced by base catalyst (left) and acid

catalyst (right) and dried by spray drier with 10000 magnification at 2 kV.

4.2 SEM and gelation time test analysis for amount of copper acetate selection

As seen from Table 8, the increases in copper acetate in the reaction resulted in shorter

gelation time. When the amount of copper acetate to sol-gel solution is 0.5% (w/v), gelation

occurred within 30 min and Sample 4 were gelled within 10 min. It could be said that Sample

3 and Sample 4 cannot be spray-dried because the gelation time was too fast. Sample 1 and

Sample 2 showed a practical time scale by starting to gel at more than 3 and 2 hours

Ref. code: 25605722040507ZOJ

29

respectively. However, the greater copper loading showed better antimicrobial properties so

the amount of copper acetate at 0.2% w/v was selected to use in further experiment.

Table 4.1 : Gelation time test of different amount of copper acetate in solution

Sample Copper acetate, (w/v) Gelation time

1 0.1% ~ 3 hours

2 0.2% ~ 2 hours

3 0.5% ~ 30 minutes

4 2.4% < 10 minutes

Figure 4.2: SEM images of Cu/SiO2 microspheres produced by spray drier at different

amount of copper acetate to tetraethoxysilane (TEOS): 0.1%w/v (left), 0.2%w/v(right) with


Ref. code: 25605722040507ZOJ

30

Figure 4.3 : SEM images of Cu/SiO2 microspheres produced by spray drier at different

amouth of copper acetate to tetraethoxysilane (TEOS) 0.5%w/v (left), 2.4%w/v (right) with


SEM was performed to observ the morphology and size of the final products. As the

copper acetate was increased, the product deformity was observed. Sample 1-3 presented a

spherical shape morphology with the diameter around 5-10 μm. At the highest amount of

copper acetate ,2.4% w/v, parts of resulted products had rod shape instead of sphere (Figure

4.3).

4.3 SEM -EDS analysis for ratio of TEOS to water selection

SEM results showed that all samples have similar morphology with diameters around

10-20 μm with spherical shape and smooth surface. Sample 8, with the ratio 1:9 of TEOS to

water, the final product had the lightest blue in color and light density. For Sample 5 ,1:4 (v:v)

TEOS to water, had the darkest blue color and more dense structure. Due to the physical

difference among the samples, it can be conducted that the ratio between TEOS to water might

affect the formation of the silica particles, which conform to the previous literature review

about the effect of water, at higher volume ratio of alkoxy group to water the hydrolysis is

faster than condensation and result in very loose gel networks[66]. Every samples were sending

to SEM- EDS analysis for morphology and determine copper content to examine these results.

Ref. code: 25605722040507ZOJ

31

Figure 4.4 : SEM images of Cu/SiO2 microspheres produced by spray drier at different ratio

of tetraethoxysilane (TEOS): DI water ; Sample 5 (1:4 TEOs to water, left), Sample 6 (1:6


Figure 4.5 : SEM images of Cu/SiO2 microspheres produced by spray drier at different ratio

of tetraethoxysilane (TEOS): DI water ; Sample 7 (1:8 TEOs to water, left), Sample 7 (1:9


Ref. code: 25605722040507ZOJ

32

Table 4.2 : EDS data of Cu/SiO2 microspheres for samples prepared with different ratios of

TEOS to water

Weight %

Sample 5 Sample 6 Sample 7 Sample 8

Element TEOS:Water,

1:4

TEOS:Water,

1:6

TEOS:Water,

1:8

TEOS:Water,

1:9

C 10.92 17.13 4.91 4.37

O 52.79 49.90 58.85 60.67

Si 31.31 29.49 33.51 32.74

Cu 4.98 3.48 2.73 2.22

Cu : Si 0.159 0.118 0.081 0.067

EDS shows the difference in copper content for each sample. Sample 5 gave the highest

copper content followed by Sample 6, 7 and 8 respectively. This can be concluded that that

Sample 4 with the ratio of TEOS to water equal to 1:4 (by vol) had the highest surface area due

to more surface area for copper to contain in microspheres. This assumption is with the

literature that the effect of water with the highest surface area (~1,000m2g-1) could be achieved

at stoichiometric amount of water (molar ratios~4) [66]. Therefore, Sample 5 should be best

suited for use as antimicrobial additives because the physical characteristics is practical for

spray drying process, and it contained the highest amount of copper.

4.4 Antimicrobial activities of Cu/SiO2 microspheres

In order to confirm the antimicrobial abilities of Cu/SiO2 microspheres, the modified

paint with Cu/SiO2 microspheres was tested with P. funiculosum, and compared to the

commercial antimicrobial (TOA213). In this test, the fungal growth was observed every 1 week

for 1-month duration.

ImageJ program was used to analyze the percent inhibition of fungal growth.

Commercial antimicrobial paint showed the poorest antimicrobial performance compared to

Sample 5 and Sample 8 with the percent inhibiting around 10%. Then the result between

Sample 5 and Sample 8 compared the same concentration of Cu/SiO2 microspheres mixed with

paint. Both samples showed the same tendency of antimicrobial activity. The performance

Ref. code: 25605722040507ZOJ

33

(percent inhibition) increased as the amount Cu/SiO2 microspheres mixed in paint increased. It

could be concluded that the increased amount of Cu/SiO2 microspheres in paint increased

copper content, which resulted in increased antimicrobial performance. More than 50% growth

inhibition can be archived by using at least 20%wt of Cu/SiO2 microspheres while 30%wt of

Cu/SiO2 microspheres gave the best performance. At week 4, the performance of Sample 5 was

reduced to 62% inhibition while Sample 8 showed poorer performance with 40% inhibition. It

showed that sample 5 gave a better performance than Sample 8. Thus we concluded that that

the TEOS to water ratio of 1:4 is the best for produce Cu/SiO2 microspheres.

Table 4.3 : Average percent inhibition on fungal growth of Sample 5

Time

10%wt of

Cu/SiO2

microspheres

20%wt of

Cu/SiO2

microspheres

30%wt of

Cu/SiO2

microspheres

Commercial

antimicrobial

paint

(TOA213)

Acrylic

paint

Week1 32.75%±7.08% 51.01%±6.79% 84.87%±6.32% 4.96%±4.50% 0%

Week2 28.98%±7.84% 49.96%±6.62% 75.24%±7.93% 8.07%±4.69% 0%

Week3 18.50%±5.55% 29.92%±11.58% 69.16%±7.91% 8.11%±4.60% 0%

Week4 15.67%±4.69% 29.29%±11.58% 62.28%±9.11% 7.85%±4.50% 0%

Table 4.4 : Average percent inhibition on fungal growth of Sample 8

Time

10%wt of

Cu/SiO2

microspheres

20%wt of

Cu/SiO2

microspheres

30%wt of

Cu/SiO2

microspheres

Commercial

antimicrobial

paint (TOA213)

Acrylic

paint

Week1 24.50% 61.36% 71.96% 36.12% 0%

Week2 9.73% 40.97% 47.80% 19.84% 0%

Week3 10.36% 33.68% 42.89% 10.01% 0%

Week4 9.99% 30.93% 40.01% 10.84% 0%

Ref. code: 25605722040507ZOJ

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4.5 Study GLYMO-modified Cu/SiO2 microspheres

Table 4.5 : Gelation time of different ration TEOS to GLYMO in sol-gel solution

Sample TEOS:GLYMO

(v:v)

TEOS

(mL)

GLYMO

(mL)

DI Water

(mL)

Copper acetate

(w/v)

Copper (II)

acetate (g)

Nitric acid

(mL)

Gelation

time

9 1:0.25 8 2 40 0.2% 0.1 0.06 > 48 hr.

10 1:0.17 8.57 1.43 40 0.2% 0.1 0.06 > 48 hr.

11 1:0.125 8.88 1.12 40 0.2% 0.1 0.06 > 48 hr.

12 1:0.1 9.10 0.90 40 0.2% 0.1 0.06 > 48 hr.

13 1:0.25 8 2 40 0.2% 0.1 0.2 > 48 hr.

14 1:0.17 8.57 1.43 40 0.2% 0.1 0.2 > 48 hr.

15 1:0.125 8.88 1.12 40 0.2% 0.1 0.2 > 48 hr.

16 1:0.1 9.10 0.90 40 0.2% 0.1 0.2 > 48 hr.

17 1:0.25 8 2 40 0.6% 0.3 0.06 > 48 hr.

18 1:0.17 8.57 1.43 40 0.6% 0.3 0.06 ~12 hr. 30

min.

19 1:0.125 8.88 1.12 40 0.6% 0.3 0.06 ~9 hr.

20 1:0.1 9.10 0.90 40 0.6% 0.3 0.06 ~7 hr.

21 1:0.25 8 2 40 1% 0.5 0.06 > 48 hr.

22 1:0.17 8.57 1.43 40 1% 0.5 0.06 ~8 hr.

Ref. code: 25605722040507ZOJ

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23 1:0.125 8.88 1.12 40 1% 0.5

0.06 ~5 hr. 30

min.

24 1:0.1 9.10 0.90 40 1% 0.5 0.06 ~4 hr.

25 1:0.25 8 2 40 3% 1.5 0.06 > 48 hr.

26 1:0.25 8 2 40 5% 2.5 0.06 > 48 hr.

27 1:0.25 8 2 40 7% 3.5 0.06 > 48 hr.

28 1:0.17 8.57 1.43 40 3% 1.5 0.06 ~12 hr.

29 1:0.125 8.88 1.12 40 3% 1.5 0.06 ~7 hr.

Ref. code: 25605722040507ZOJ

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4.5.1 Gelation time test of GLYMO-modified Cu/SiO2

Compared with Samples 1-4 which gelled within 3 hours, Samples 9-12 took more than

48 hrs to gel. This experiment also showed that the increase copper drives the reaction faster,

resulting in the reduction of gelation time. However, the increased amount of HNO3 does not

gave a major reduction in gelation time. In conclusion, increasing amount of copper acetate

can drive the reaction faster, and the trend also shows that more GLYMO will slow down the

gelation process. From gelation time test, the following samples were selected to proceed to

the spray dry process by increasing the total volume to be 2L.

1) Sample 24 with 1% copper acetate w/v, 0.06 mL of nitric acid

2) Sample 28 with 3% copper acetate w/v, 0.06 mL of nitric acid

Sample 24 was selected due to it has practical gelation time of 4 hours. Sample 28 is chosen as

the candidate for the greater GLYMO content with greater copper acetate loading capacity, and

can gel within 12 hours.

4.5.2 SEM EDS and XRF of GLYMO-modified Cu/SiO2

Figure 4.6 : SEM images of GLYMO-modified Cu/SiO2 microspheres produced by spray

drier : Sample 24 (left), Sample 28 (right)

The scanning electron micrographs of GLYMO-modified Cu/SiO2 microspheres were

shown in Figure 4.6. The particles size is about 10-30 µm in spherical shape with smooth

surfaces. The presence of copper on the surface of GLYMO-modified Cu/SiO2 microspheres

was confirmed by EDS analysis. The content of copper was shown in Table 13.

Ref. code: 25605722040507ZOJ

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According to EDS analysis, the copper content in GLYMO-modified Cu/SiO2

microspheres found in Samples 24 and 28 had the ratio of copper to silica of 0.176 and 0.465

respectively. The amount of copper on Sample 28 was higher than Sample 24 because the

loading copper amount was up to 3%wt compared to Sample 24, where the loading copper

amount was about 1%wt. However, it can be concluded that sample which has greater GLYMO

content will have greater copper loading capacity.

For XRF analysis (Table 14), GLYMO-modified Cu/SiO2 microsphere and non-

modified Cu/SiO2 microsphere were measured from Nuclear Technology Service Center. The

result has shown the same trend with EDS analysis that sample which has greater GLYMO

content will has greater amount of copper.

Table 4.6 : EDS data of GLYMO-modified Cu/SiO2 microspheres

Element Sample 24

TEOS to GLYMO, 1:0.10 (vol)

Sample 28

TEOS to GLYMO, 1:0.17 (vol)

C 14.01 17.11

O 45.89 37.59

Si 34.10 30.93

Cu 6.01 14.38

Cu : Si 0.176:1 0.465:1

Ref. code: 25605722040507ZOJ

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Table 4.7 : XRF data of GLYMO-modified Cu/SiO2 microspheres compare to non-modified

Cu/SiO2 microspheres

Element Compound

Sample 5

TEOS to Water

1:4 (vol)

Sample 24

TEOS to GLYMO

1:0.10 (vol)

Sample 28

TEOS to GLYMO

1:0.17 (vol)

S SO3 - 0.05 -

Zn ZnO 0.02 - -

Fe Fe2O3 0.11 0.06 0.08

Ca CaO 0.39 0.30 0.35

P P2O2 1.08 0.81 1.08

Si SiO2 43.20 41.85 35.22

Cu CuO 3.51 6.37 17.24

Cu : Si 0.081:1 0.152:1 0.490:1

4.5.3 Fourier Transform Infrared Spectroscopy (FTIR) of GLYMO-modified Cu/SiO2

microspheres

KBr-FTIR measurement was used to compare between spectra of non-modified

Cu/SiO2 microspheres (Sample 5, 1:4 v:v TEOS to water), Pure GLYMO (non-reacted with

anything) and GLYMO-modified Cu/SiO2 microspheres (Sample 24, 1:0.1 v:v TEOS to

GLYMO and Sample 28, 1:0.17 v:v TEOS to GLYMO) under the specified laboratory

conditions in order to verify the presence of GLYMO on GLYMO-modified Cu/SiO2

microspheres. GLYMO structure composed of glycidyl group and methoxy group. Glycidyl

group can be identified by epoxy peak of FT-IR, which 3050 cm-1, 1250 cm-1,and 915 cm-1[58-

62]. However the peak 3050 cm- 1 is unable to be used because it is overlapped by the silanol

(Si-OH) and hydroxyl (-OH) peak that is broad at 3200 – 3600 cm-1. Furthermore, the peak

1250 cm-1 can only be seen in Pure GLYMO sample (Figure 4.7); it is not found in both Sample

24 and Sample 28. Likewise, the peak 915 cm- 1 also shows only in Pure GLYMO sample

(shown at 907.8 cm-1). Therefore, the epoxy group cannot be used as an indicator.

The peak that can be used as an identifier comes to the methoxysilane group, which in

GLYMO contains 3 methoxy groups (-O-CH3) [15, 67-69]. In this case, the characteristic peak

assigned to -Si-O-CH3 emerges sharply at 2870 cm-1, and strongly around 1100-1080 cm-1[15,

67, 68]. The peak around 2870 cm-1 can be seen only in the sample that contains GLYMO

Ref. code: 25605722040507ZOJ

39

which are Pure GLYMO, Sample 24, Sample 28 but not in Sample 5 (non-modified).

Therefore, it can qualitatively verify the presence of the GLYMO in Cu/SiO2 microspheres due

to its characteristic peak of –Si-O-CH3. The summary of the major peaks found in the FTIR

analysis is listed in Table 15.

Table 4.8 : Summary of Relevant Peaks of GLYMO-modified Cu/SiO2 microspheres

Structural

Unit

Type of

Vibration

Wavenumber

(cm-1)

Pure

GLYMO

Sample

24

Sample

28

Sample 5

(non-

modified)

-Si-O-H O-H and

-Si-OH 3600-3200 3440.8 3405.9 3421.2 3447.5

-Si-O-CH3 νas C-H 2870 2879.1 2894.8 2890.3 -

H-O-H δ H-O-H 1620 1636.9 1636.5 1623.4 1636.6

-Si-O-Si- νas Si-O-Si 1130-1000 - 1070.7 1070.4 1039.5

Ref. code: 25605722040507ZOJ

40

Figure 4.7 : FTIR Absorbance graph of Sample 5(blue), Sample 24(green), Sample 28(red)

and pure GLYMO(pink).

4.5.4 Thermogravimetric analysis (TGA) of GLYMO-modified Cu/SiO2

microspheres

GLYMO-modified Cu/SiO2 microspheres were analyzed by TGA method to confirm

the organic content (GLYMO) in each ratio quantitatively. From dTGA graph, the comparison

between pure GLYMO(non-reacted with anything), Sample 5, Sample 24 and Sample 28

showed the curve of both modified-GLYMO have the three majors change. Firstly, around 30-

190 oC, due to the loss of humidity, secondly, around 140-350 oC, which corresponds to the

decomposition of the crosslinked organic polymer network in epoxy and lastly at 350-600 oC,

it might be from GLYMO decomposition. As can be seen from the graph, Sample 5 has the

greatest water content, following by Sample 24 and Sample 28. Thus could be concluded that

water molecules cannot retain well in the silica matrix with higher GLYMO content. In

additions, the experiment conducted by Macan et al. stated that the higher GLYMO content

will result in the shift to higher degradation temperature [56]. The result was consistency with

Macan et al. by the significant drop of Pure GLYMO is stop around 725°C while Sample28

and Sample 24 at 600 °C.

* S am _30_4_2016_K B R _P ure G LY MO

* S am _30_4_2016_K B R _TE O S 1-4

* S am _30_4_2016_K B R _1-10_1

* S am _30_4_2016_K B R _1-6_1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8A

bs

orb

an

ce

1000 1500 2000 2500 3000 3500 4000

Wav enumbers (cm-1)

Si-O-Si Si-O-CH3

Si-OH

Ref. code: 25605722040507ZOJ

41

Table 4.9 : Organic Content of GLYMO-modifiedCu/SiO2 microspheres determined from

TGA

Sample

Name

Organic

Content (wt %)

Theoretical Ratio of

TEOS:GLYMO

(w/w)

Experimental Ratio of

TEOS:GLYMO

(w/w)

Sample 24 11.064 1:0.114 1:0.126

Sample 28 14.651 1:0.190 1:0.177

Figure 4.8 : dTGA graph comparison between Sample 5, Sample 24, Sample 28 and Pure

GLYMO

Ref. code: 25605722040507ZOJ

42

4.5.5 Particle Size Distribution of GLYMO-modified Cu/SiO2 microspheres

Figure 4.9 : Particle size distribution of GLYMO modified Cu/SiO2 microspheres: Sample 28

(top) and Sample 24 (bottom)

The particle size distribution result shows that the Sample 28 and Sample 24 silica

powder had the Z-average mean particle size of 1651 and 843 nm, respectively. From Figure

4.9, each sample displays only one particular particle size intensity peak, indicating that the

powder acquired from spray drying process has a low polydispersity, i.e. particle size is

homogeneous. The size of Sample 28, which greater amount of GLYMO was added, is twice

bigger than Sample 24, indicating that GLYMO played a significant role in the formation of

particle structure during sol-gel synthesis.

4.5.6 Antimicrobial activities of GLYMO modified-Cu/SiO2 microspheres and long term

study

The EDS/XRF results in previous section showed that Sample 28 had higher copper

content in microspheres than Sample 24. So Sample 28 was more preferable to use in the test

for antimicrobial activities.

Size Distribution by Intensity

Inte

nsi

ty (

Percen

t)

Size (d. nm)

Size (d. nm)

Inte

nsi

ty (

Percen

t)

Ref. code: 25605722040507ZOJ

43

4.5.6.1 Antifungal activities of GLYMO modified-Cu/SiO2 microspheres

Performed Immediately

Figure 4.10 : The percent inhibition of Sample 28 plotted as a function of Cu/SiO2

microsphere content in comparison with the commercial antifungal paint(TOA213)

Figure 4.10 show that at week 4, the commercial antimicrobial paint (TOA213) had

78.91% inhibition, which was only marginally better than Sample 28 with 1%wt of GLYMO

modified-Cu/SiO2 microspheres. The other samples exhibited an average percent inhibition

rate well above 80% over the course of four weeks. Sample 28 with 10%wt of GLYMO

modified-Cu/SiO2 microspheres, in particular, showed an outstanding ability with more than

99% inhibition rate. The performance of the antifungal paint made from GLYMO modified-

Cu/SiO2 microspheres exhibit a steady trend over time. This is true with the exception of

Sample 28 with 1%wt GLYMO modified-Cu/SiO2 microspheres, which ended the first week

of incubation with 47.46% inhibition. The trend also demonstrated that the higher amount of

GLYMO modified-Cu/SiO2 microspheres added, the higher the inhibition rate. The basic

assumption of this experiment was that GLYMO would help retaining the release of copper

also increase copper loading capacity to microspheres, which would result in a higher

percentage of fungi inhibition.

0

10

20

30

40

50

60

70

80

90

100

1% 5% 10% 20% commercial

antifungal

% I

nh

ibit

ion

Week 1

Week 2

Week 3

Week 4

Ref. code: 25605722040507ZOJ

44

Performed at 6 Months


microsphere content in comparison with the commercial antifungal paint(TOA213) after 6

months

Long-term antifungal property over 6 months was conducted. Figure 4.11 showed that

all contents of Sample 28 resulted in higher fungal inhibition over the TOA213 except 1wt.%,

indicating that Sample 28 has a better antifungal performance than TOA213.

Performed at 1 year

The trend of 1-year experiment still showed better antimicrobial ability of Sample 28

over TOA213. The antifungal ability of TOA213 significantly decreases after one year while

the modified Cu/SiO2 microspheres (Sample 28) can retain most of its antifungal ability.

Sample 28 with 5%wt, 10%wt, and 20%wt of Cu/SiO2 microspheres content were able to

inhibit the fungal growth more than 85% over a month of experiment. Meanwhile, TOA213

hads a lower inhibiting ability after being kept for a year. The explanation for the better

performance of Sample 28 could be the fact that it had a greater amount of copper loaded than

Sample 24, which increases the longevity of the product, as there was more copper to inhibit

the microbial growth. This is also possible because of the higher TEOS:GLYMO ratio of

Sample 28, which allowed the higher copper loading capacity. However, Sample 28 with 10

0

10

20

30

40

50

60

70

80

90

100


antifungal

% I

nh

ibit

ion

Week 1

Week 2

Week 3

Week 4

Ref. code: 25605722040507ZOJ

45

wt. % and 20 wt.% content had strong blue color, which could make a color aberration when

added to painting. Therefore, the Sample 28 with content of 1 wt.% and 5 wt.% were preferred

as an additive to paint due to its antifungal ability, its longer durability, and its lesser effect to

color deviation.


microsphere content in comparison with the commercial antifungal paint(TOA213) after 1

year

4.5.6.2 Antibacterial activities of GLYMO modified-Cu/SiO2 microspheres

For gram- negative bacteria (E. coli), GLYMO modified-Cu/SiO2 microspheres

archived 100% inhibition while the paper coated with TOA213 cannot inhibit bacterial growth

completely. Only 90+% inhibition is seen after one week, as some of the metallic grey colonies

were observed. Also, the sample contains only paints still show some degree of inhibition

around 75% . This means that paint, despite not being modified with the antimicrobial agent,

is still be able to inhibit bacterial growth by itself. However, the sample paper that was not

coated with any surface modifiers could not inhibit the bacterial growth, as the entire surface

was covered with bacterial colonies (Figure 4.13, right).

0

10

20

30

40

50

60

70

80

90

100


antifungal

% I

nh

ibit

ion

Week 1

Week 2

Week 3

Week 4

Ref. code: 25605722040507ZOJ

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Figure 4.13 : Paper coated with GLYMO-modified Cu/SiO2 microspheres shows 100%

bacterial inhibition as no metallic-grey color is on the surface (left). Uncoated filter paper

covered with E. coli, which appears as metallic grey on EMB agar (right).

4.6 Study APTES-modified Cu/SiO2 microspheres

4.6.1 Synthesis APTES-modified Cu/SiO2 microspheres via two-pot synthesis

There were 7 samples (Sample30-36) used two-pot synthesis, where each sample was

washed with solvent before put in oven-dry. The samples were then characterized for amine

group with FTIR to make sure that APTES was grafted of surface of SiO2 before applying

grafted APTES-SiO2 with copper acetate.

Ref. code: 25605722040507ZOJ

47

Figure 4.14 : FTIR analysis of APTES grafted silica particles (Sample32) before and after

wash.

FTIR results showed no characteristic peak in every sample (Sample 30-36), which

meant that no amine group was detected. The assumption was that APTES gelled among itself,

not attached on the SiO2 surface. Thus it could be washed by solvents during washing step. To

confirm this assumption Sample 32 which run the reaction overnight, result in gelled solution,

was characterized. Then the gelled solution was dry at 50 °C overnight in the oven. The

APTES-modified SiO2 was separated into 2 samples. For the first sample, the APTES-modified

SiO2 were examined with FTIR to find amine groups, and the second one was washed by

solvent before tested by FTIR. Figure 4.14 shows the result of Sample 32 that had not been

washed with solvents having symmetrical vibration of CH2 at 1560 cm-1, aliphatic amine peak

at 1560 cm-1 and CH2 bending at 1478 cm-1 and 1314 cm-1 but the amine peak was undetected

for Sample 32 that washed with solvent. The result may come from incorrect way of synthesis

causes the grafted APTES unable to attach on the SiO2 surface. By this one-pot synthesis was

studied to modify Cu/SiO2 microspheres with APTES.

688

.44

780

.78

917

.88

131

4.43

147

6.78

156

0.26

292

8.66

B atch3_B efore w as h

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8A

bs

570

.1790

.7

927

.8

327

3.6

374

4.5

385

4.0

B atch3_A fter w ash

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Ab

s

500 1000 1500 2000 2500 3000 3500 4000

Wav enumbers (cm-1)

-CH2- Amine Group

Si-OH

Si-O-Si

Si-O-Si

Ref. code: 25605722040507ZOJ

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4.6.2 Characterization of APTES-modified Cu/SiO2 microspheres via one pot synthesis

4.6.2.1 Gelation time test of APTES-modified Cu/SiO2 microspheres via one pot

synthesis

Sample 37-48 were tested for gelation time because the reaction time between APTES

and water was short, which caused the solution gel within a short period of time and unable to

use in spray dry process. According to Table 17, the gelation time was too fast if using only

water as a solvent, and process with pure ethanol was too long. Therefore, the solution was

proceeded with both water and ethanol as a solvent. It was found that by mixing APTES 0.5%

(by vol) in a mixture of water and ethanol, the solution has a gelation time about 1.5 hrs. While

the other condition was found that the gelation time was short and unusable for spray dry.

Therefore, Sample 47 was chosen to proceed to spray dry process.

4.6.2.2 Fourier Transform Infrared Spectroscopy (FTIR) of APTES-modified

Cu/SiO2 microspheres via one pot synthesis

Sample 47 was characterize by FTIR (Figure 4.15) to ensure that APTES is present in

APTES- modified Cu/SiO2 microspheres. CH2 bending peaks was observed which located at

1631 cm-1, and 1384 cm-1, and 3400 cm-1, but at 3400 cm-1 band overlapped with stretching

vibration N-H. The peak at 1631 cm-1 was also seen in pure SiO2, this peak could not be used.

Thus, the peaks located at 1384 cm-1 was used to confirm APTES presence in APTES-

modified Cu/SiO2 microspheres,

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Table 4.10 : Gelation time of different amount of APTES and copper acetate in sol-gel process

Sample TEOS (mL) Ethanol

(mL)

DI Water

(mL)

APTES

(v/v)

APTES

(mL)

Copper (II) acetate

(w/v)

Copper (II)

acetate (g)

Gelation time

37 10 - 40 0.1% 0.05 0.2% 0.1 ~ 1 hr.

38 10 - 40 0.5% 0.25 0.2% 0.1 ~30 min

39 10 - 40 1% 0.5 0.2% 0.1 immediately

40 10 40 - 0.5% 0.25 1% 0.5 > 24 hr.

41 10 40 - 0.5% 0.25 2% 1 > 24 hr.

42 10 40 - 0.5% 0.25 3% 1.5 > 24 hr.

43 10 20 20 0.5% 0.25 0.2% 0.1 ~ 2 hr.

44 10 10 30 0.5% 0.25 0.2% 0.1 ~ 1 hr.

45 10 8 32 0.5% 0.25 0.2% 0.1 ~ 3 hr.

46 10 8 32 0.5% 0.25 0.5% 0.25 ~ 2 hr.

47 10 8 32 0.5% 0.25 1% 0.5 > 1.5 hr.

48 10 8 32 0.5% 0.25 1.5% 0.75 < 30 min.

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Figure 4.15 : FTIR graph of APTES-modified Cu/SiO2 microspheres (Sample 47, red) compared with Pure SiO2 (blue)

538.

456

3.0

790.

6

927.

3

1041

.2

1635

.63254

.8

1062

.72

1083

.88

1105

.06

1384

.33

1631

.92

3451

.35

P ureS i

* E xp17_part1_01

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Abso

rba

nce

500 1000 1500 2000 2500 3000 3500 4000

Wav enumbers (cm-1)

Amine Group

Si-OH

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4.6.3 Characterization of APTES-modified Cu/SiO2 microspheres via one pot synthesis

4.6.3.1 SEM-EDS analysis of APTES-modified Cu/SiO2 microspheres via one-

pot synthesis

Figure 4.16 : SEM-EDS images of 0.1% v/v APTES-modified Cu/SiO2 microspheres

produced by spray drier

The scanning electron micrographsof APTES- modified Cu/SiO2 microspheres are

shown in Figure 4.16. The particles size was about 10-30 µm in spherical shape. The surface

of the samples which modified by APTES had rough surface compared to the samples that

modified with GLYMO. The presence of copper on the surface of APTES -modified Cu/SiO2

microspheres was confirmed by EDS analysis. The content of copper is showed in Table 19.

Table 4.11 : EDS data comparison between APTES-modified Cu/SiO2 microspheres and


Weight %

Element Sample 47

(0.5% v/v APTES)

Sample 24

TEOS to GLYMO,

1:0.1 (vol)

Sample 28

TEOS to GLYMO,

1:0.17 (vol)

C 19.23 17.11 14.01

O 47.86 37.59 45.89

Si 20.48 30.93 34.10

Cu 12.43 14.38 6.01

Cu : Si 0.607:1 0.176:1 0.465:1

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EDS analysis for copper content showed that Sample 47, 24 and 28 had the ratio of

copper to silica of 0.607, 0.465 and 0.176 respectively. It cannot determine, which one was

the best sample because during the synthesis process each sample has been loaded with

different amount of copper. The amount of copper on Sample 24 was higher than sample28

because the loading copper amount was up to 3%wt compared to Sample 28, where the l copper

amount was about 1 wt. %. However, it can be concluded that that sample which had greater

GLYMO content had greater copper loading capacity. In the case of APTES- modified Cu/SiO2

microspheres, Sample 47 that had 1 wt % copper, had copper content more than Sample 24. It

can be concluded that modification of Cu/SiO2 microspheres with APTES show a better

efficiency to adsorb copper than modify with GLYMO. Due to very low amount of obtained

powder form APTES-modified Cu/SiO2 microspheres so this sample cannot be characterized

further.

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Chapter 5

Conclusion and Recommendations

Functionalizing silica particles opens the door to countless useful applications in many

areas such as medicine, paint, waste water treatment, etc. This project attempted to apply the

knowledge of copper loaded in modified-organoalkoxysilane (GLYMO and APTES) for

antimicrobial applications, particularly in paint. Using copper loaded silica particles proved to

be effective in inhibiting the growth of fungi and bacteria. However the, useful lifetime of

application is limited due to the copper content and releasing rate of copper from silica

particles. So a slow and increase loading capacity is often preferred to achieve prolonged

antimicrobial effect.

In this study, the Cu/SiO2 microspheres were synthesized by sol-gel and spray drying

process with different starting materials to study the effect of GLYMO and APTES on the

ability to inhibit microbial growth, including long term performance compared to commercial

antimicrobial paint (TOA213). For GLYMO-modified Cu/SiO2 microspheres, Sample 24

(TEOS to GLYMO 1:0.1 v:v)and Sample 28 (TEOS to GLYMO 1:0.17 v:v) were found that

the copper loading capacity increased when adding a proper amount of GLYMO to TEOS sols,

which leaded to the better performance in inhibiting microbial growth. APTES-modified SiO2

microspheres were successfully synthesized with the one-pot method using mixture of ethanol

and water as a solvent. XRF and EDS were used to measure the copper content and show that

the APTES-modified SiO2 microspheres had higher copper content in SiO2 microspheres than

GLYMO-modified SiO2 microspheres(Sample 28). TGA and FTIR were used to confirm the

presence of APTES and GLYMO by detecting amine group and methoxy group in modified

SiO2 microspheres. SEM micrographs showed that the copper stayed in the form of flake

around the microspheres with the size of diameter around 5-10 μm. Moreover, the PSD results

showed that higher GLYMO content also resulted in a larger particle size microspheres.

Sample 28 was twice as large as Sample 24. Finally, the acrylic paint mixed with 5 wt.% of

Sample 28 showed the preferable quality to be an additive to paint, as its ability to inhibit P.

funiculosum growth was over 85% immediately after sample preparation, 6-month, and 1-year

of antifungal experiment. This sample exhibited the best antifungal ability, as well as its

durability, and its ability to maintain copper stability. The antibacterial result was even better

as it could inhibit 100% of gram-negative (E. coli) bacterial growth. Besides, GLYMO-

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modified Cu/SiO2 microspheres showed the better performance on both antifungal and

antibacterial than commercial antimicrobial paint (TOA213).

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Appendices

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Appendix A

Result of antimicrobial activities of Sample 5 (TEOS to Water 1:4 V:)

Week 1

Figure A1: Antimicrobial activities effect of Sample 5 on P.funiculosum by varies amount of

Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213 and only

acrylic paint(control) in week 1.

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Week 2

Figure A2 : Antimicrobial activities effect of Sample 5 on P.funiculosum by varies amount of



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Week3



acrylic paint(control) in week 3

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Week4



acrylic paint(control) in week 4

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Appendix B

Result of antimicrobial activities of Sample 8 (TEOS to Water 1:9 v:v)

Week 1

Figure B1 : Antimicrobial activities effect of Sample 8 on P.funiculosum by varies amount of



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Week 2




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Week 3




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Week 4




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Appendix C

Result of antimicrobial activities of Sample 28 ( TEOS to GLYMO 1:0.17

v:v) by perform immediately

Week1

Figure C1 : Antimicrobial activities effect of Sample 28 on P.funiculosum by varies amount

of Cu/SiO2 microspheres in acrylic paint at 1% 5% 10%and 30%wt compared with TOA213

in week 1.

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Week2



in week 2.

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Week 3

Figure C3: Antimicrobial activities effect of Sample 28 on P.funiculosum by varies amount of

Cu/SiO2 microspheres in acrylic paint at 1% 5% 10%and 30%wt compared with TOA213 in

week 3.

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Week 4



in week 4.

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Appendix D


v:v) by perform at 6 months for week 1-4

Figure D1 : Antimicrobial activities effect of Sample 28 with 5%wt of Cu/SiO2 microspheres

in acrylic paint on P.funiculosum by perform at 6 months ; week1 , week2 week3 and week 4

respectively.

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Appendix E

Result of antimicrobial activities of Commercial antimicrobial ( TOA213)

by perform at 6 months for week 1-4

Figure E1 : Antimicrobial activities effect of TOA213 on P.funiculosum by perform at 6

months ; week1 , week2 week3 and week 4 respectively.

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Appendix F


v:v) by perform at 1 year for week 1-4

Figure F1 : Antimicrobial activities effect of Sample 28 with 5%wt of Cu/SiO2 microspheres

in acrylic paint on P.funiculosum by perform at 1 year; week1 , week2 week3 and week 4

respectively.

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Appendix G

Result of antimicrobial activities of Commercial antimicrobial ( TOA213)

by perform at 1 year for week 1-4

Figure 7 : Antimicrobial activities effect of TOA213 on P.funiculosum by perform at 1 year

; week 1 , week 2 week 3 and week 4 respectively.

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