development of encapsulated antimicrobial additives …
TRANSCRIPT
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
Ref. code: 25605722040507ZOJ
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
Ref. code: 25605722040507ZOJ
ii
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
Ref. code: 25605722040507ZOJ
iii
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
Ref. code: 25605722040507ZOJ
iv
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.
Ref. code: 25605722040507ZOJ
v
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
Ref. code: 25605722040507ZOJ
vi
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
Ref. code: 25605722040507ZOJ
vii
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
Ref. code: 25605722040507ZOJ
viii
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
Ref. code: 25605722040507ZOJ
ix
4.11 EDS data comparison between APTES-modified Cu/SiO2 microspheres and 51
GLYMO-modified Cu/SiO2 microspheres
Ref. code: 25605722040507ZOJ
x
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
5000 magnification at 2 kV
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
Ref. code: 25605722040507ZOJ
xi
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
TEOs to water,right) with 1000 magnification at 20 kV
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)
4.11 The percent inhibition of Sample 28 plotted as a function of Cu/SiO2 microsphere 44
content in comparison with the commercial antifungal paint(TOA213) after 6 months
4.12 The percent inhibition of Sample 28 plotted as a function of Cu/SiO2 microsphere 45
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.
Ref. code: 25605722040507ZOJ
xii
A2 Antimicrobial activities effect of Sample 5 on P.funiculosum by varies amount of 62
Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213
and only acrylic paint(control) in week 2.
A3 Antimicrobial activities effect of Sample 5 on P.funiculosum by varies amount of 63
Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213
and only acrylic paint(control) in week 3
A4 Antimicrobial activities effect of Sample 5 on P.funiculosum by varies amount of 64
Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213
and only acrylic paint(control) in week 4
B1 Antimicrobial activities effect of Sample 8 on P.funiculosum by varies amount of 65
Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213
and only acrylic paint(control) in week 1.
B2 Antimicrobial activities effect of Sample 8 on P.funiculosum by varies amount of 66
Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213
and only acrylic paint(control) in week 2.
B3 Antimicrobial activities effect of Sample 8 on P.funiculosum by varies amount of 67
Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213
and only acrylic paint(control) in week 3.
B4: Antimicrobial activities effect of Sample 8 on P.funiculosum by varies amount of 68
Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213
and only acrylic paint(control) in week 4.
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.
Ref. code: 25605722040507ZOJ
xiii
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.
Ref. code: 25605722040507ZOJ
1
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
Ref. code: 25605722040507ZOJ
2
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-
Ref. code: 25605722040507ZOJ
3
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).
Ref. code: 25605722040507ZOJ
4
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 nano- particle, 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
Ref. code: 25605722040507ZOJ
5
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)
Ref. code: 25605722040507ZOJ
6
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].
Ref. code: 25605722040507ZOJ
7
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 nano- particles, 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
Ref. code: 25605722040507ZOJ
8
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
Ref. code: 25605722040507ZOJ
9
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
Ref. code: 25605722040507ZOJ
10
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].
Ref. code: 25605722040507ZOJ
11
Figure 2.4 : Microstructures of six different aerogels prepared using different catalysts[45].
2.6 Modification of silica
Modification of silica using co- polymers 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
Ref. code: 25605722040507ZOJ
12
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.
Ref. code: 25605722040507ZOJ
13
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.
Ref. code: 25605722040507ZOJ
14
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.
Ref. code: 25605722040507ZOJ
15
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.
Ref. code: 25605722040507ZOJ
16
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
Ref. code: 25605722040507ZOJ
17
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
Ref. code: 25605722040507ZOJ
18
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.
Ref. code: 25605722040507ZOJ
19
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
Ref. code: 25605722040507ZOJ
20
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
Ref. code: 25605722040507ZOJ
21
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 for APTES grafted SiO2 particles
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 for APTES grafted SiO2 particles
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.
Ref. code: 25605722040507ZOJ
22
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 for APTES grafted SiO2 particles
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
Ref. code: 25605722040507ZOJ
23
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.
Ref. code: 25605722040507ZOJ
24
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
Ref. code: 25605722040507ZOJ
25
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.
Ref. code: 25605722040507ZOJ
26
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.
Ref. code: 25605722040507ZOJ
27
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
Ref. code: 25605722040507ZOJ
28
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
5000 magnification at 2 kV
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
5000 magnification at 2 kV
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
TEOs to water,right) with 1000 magnification at 20 kV
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
TEOs to water,right) with 1000 magnification at 20 kV
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
34
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
35
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
36
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
37
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
38
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
Figure 4.11 : The percent inhibition of Sample 28 plotted as a function of Cu/SiO2
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
1% 5% 10% 20% commercial
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.
Figure 4.12 : The percent inhibition of Sample 28 plotted as a function of Cu/SiO2
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
1% 5% 10% 20% commercial
antifungal
% I
nh
ibit
ion
Week 1
Week 2
Week 3
Week 4
Ref. code: 25605722040507ZOJ
46
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
48
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,
Ref. code: 25605722040507ZOJ
49
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.
Ref. code: 25605722040507ZOJ
50
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
Ref. code: 25605722040507ZOJ
51
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
GLYMO-modified Cu/SiO2 microspheres
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
Ref. code: 25605722040507ZOJ
52
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.
Ref. code: 25605722040507ZOJ
53
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-
Ref. code: 25605722040507ZOJ
54
modified Cu/SiO2 microspheres showed the better performance on both antifungal and
antibacterial than commercial antimicrobial paint (TOA213).
Ref. code: 25605722040507ZOJ
55
Reference
1. Hamouda, I.M.,(2012). Current perspectives of nanoparticles in medical and dental
biomaterials. Journal of biomedical research,26(3): p. 143-151.
2. Santo, C.E., et al.,(2011). Bacterial killing by dry metallic copper surfaces. Applied and
environmental microbiology,77(3): p. 794-802.
3. Konieczny, J. and Z. Rdzawski,(2012). Antibacterial properties of copper and its alloys.
Archives of Materials Science and Engineering,. 56(2): p. 53-60.
4. Palza, H.,(2015). Antimicrobial polymers with metal nanoparticles. International
journal of molecular sciences, 16(1): p. 2099-2116.
5. Mahltig, B., et al.,(2010) Antimicrobial coatings on textiles–modification of sol–gel
layers with organic and inorganic biocides. Journal of sol-gel science and technology, 55(3):
p. 269-277.
6. Lemire, J.A., J.J. Harrison, and R.J. Turner,(2013). Antimicrobial activity of metals:
mechanisms, molecular targets and applications. Nature Reviews Microbiology, 11(6): p. 371.
7. Chen, S., et al.,(2014). Synergistic antibacterial mechanism and coating application of
copper/titanium dioxide nanoparticles. Chemical Engineering Journal, 256: p. 238-246.
8. Hajipour, M.J., et al.,(2012). Antibacterial properties of nanoparticles. Trends in
Biotechnology, 30(10): p. 499-511.
9. Kim, Y.-H., et al.,(2012). Evaluation of copper ion of antibacterial effect on
Pseudomonas aeruginosa, Salmonella typhimurium and Helicobacter pylori and optical,
mechanical properties. Applied Surface Science, 258(8): p. 3823-3828.
10. Valodkar, M., et al.,(2011). Synthesis and anti-bacterial activity of Cu, Ag and Cu–Ag
alloy nanoparticles: a green approach. Materials Research Bulletin, 46(3): p. 384-389.
11. Zielecka, M., et al.,(2011) Antimicrobial additives for architectural paints and
impregnates. Progress in Organic Coatings. 72(1): p. 193-201.
12. Quang, D.V., et al.,(2012). Preparation of amino-functionalized silica for copper
removal from an aqueous solution. Journal of Industrial and Engineering Chemistry, 18(1): p.
83-87.
13. Witucki, G.L.,(1993). A silane primer: chemistry and applications of alkoxy silanes.
Journal of coatings technology, 65: p. 57-57.
14. Zheng, S. and J. Li,(2010). Inorganic–organic sol gel hybrid coatings for corrosion
protection of metals. Journal of Sol-Gel Science and Technology, 54(2): p. 174-187.
15. Deflorian, F., et al.,(2008). Integrated electrochemical approach for the investigation of
silane pre-treatments for painting copper. Progress in Organic Coatings, 63(3): p. 338-344.
Ref. code: 25605722040507ZOJ
56
16. Mahltig, B. and H. Böttcher,(2003). Modified silica sol coatings for water-repellent
textiles. Journal of sol-gel science and technology, 27(1): p. 43-52.
17. Mahltig, B. and A.(2010). Fischer, Inorganic/organic polymer coatings for textiles to
realize water repellent and antimicrobial properties—A study with respect to textile comfort.
Journal of Polymer Science Part B: Polymer Physics, 48(14): p. 1562-1568.
18. Kim, M., et al.,(2003). Encapsulation of water-soluble dye in spherical sol-gel silica
matrices. Journal of sol-gel science and technology, 27(3): p. 355-361.
19. Tamon, H., T. Kitamura, and M. Okazaki,(1998). Preparation of silica aerogel from
TEOS. Journal of colloid and interface science, 197(2): p. 353-359.
20. Barberena-Fernández, A., P. Carmona-Quiroga, and M. Blanco-Varela,(2015).
Interaction of TEOS with cementitious materials: Chemical and physical effects. Cement and
Concrete Composites, 55: p. 145-152.
21. Mahltig, B., et al.,(2010). Silver Nanoparticles in SiO. Acta Chim. Slov, 57: p. 451-457.
22. Kumar, R., S. Howdle, and H. Münstedt,(2005). Polyamide/silver antimicrobials: effect
of filler types on the silver ion release. Journal of Biomedical Materials Research Part B:
Applied Biomaterials, 75(2): p. 311-319.
23. Díaz-Visurraga, J., et al.,(2011). Metal nanostructures as antibacterial agents. Science
And Technology Against Microbial Pathogens: Research, Development and Evaluation.
Badajoz: Formatex, : p. 210-218.
24. Ruparelia, J.P., et al.,(2008). Strain specificity in antimicrobial activity of silver and
copper nanoparticles. Acta Biomaterialia, 4(3): p. 707-716.
25. Palza, H., R. Quijada, and K. Delgado,(2015). Antimicrobial polymer composites with
copper micro-and nanoparticles: Effect of particle size and polymer matrix. Journal of
Bioactive and Compatible Polymers: Biomedical Applications, p. 0883911515578870.
26. Dizaj, S.M., et al.,(2014). Antimicrobial activity of the metals and metal oxide
nanoparticles. Materials Science and Engineering: C, 44(0): p. 278-284.
27. Rai, M., A. Yadav, and A. Gade,(2009). Silver nanoparticles as a new generation of
antimicrobials. Biotechnology advances, 27(1): p. 76-83.
28. Anyaogu, K.C., A.V. Fedorov, and D.C. Neckers,(2008) Synthesis, characterization,
and antifouling potential of functionalized copper nanoparticles. Langmuir, 24(8): p. 4340-
4346.
29. Ren, G., et al.,(2009). Characterisation of copper oxide nanoparticles for antimicrobial
applications. International journal of antimicrobial agents, 33(6): p. 587-590.
Ref. code: 25605722040507ZOJ
57
30. Damm, C., H. Münstedt, and A. Rösch,(2008). The antimicrobial efficacy of polyamide
6/silver-nano-and microcomposites. Materials Chemistry and Physics, 108(1): p. 61-66.
31. Borkow, G. and J. Gabbay,(2004). Putting copper into action: copper-impregnated
products with potent biocidal activities. The FASEB journal, 18(14): p. 1728-1730.
32. Grass, G., C. Rensing, and M. Solioz,(2011). Metallic Copper as an Antimicrobial
Surface. Applied and Environmental Microbiology, 77(5): p. 1541-1547.
33. Delgado, K., et al.,(2011). Polypropylene with embedded copper metal or copper oxide
nanoparticles as a novel plastic antimicrobial agent. Letters in applied microbiology, 53(1): p.
50-54.
34. Stohs, S. and D. Bagchi,(1995). Oxidative mechanisms in the toxicity of metal ions.
Free Radical Biology and Medicine, 18(2): p. 321-336.
35. Rahman, I.A. and V. Padavettan,(2012). Synthesis of Silica Nanoparticles by Sol-Gel:
Size-Dependent Properties, Surface Modification, and Applications in Silica-Polymer
Nanocomposites—A Review. Journal of Nanomaterials, 2012: p. 15.
36. Schmidt, H.K., et al., Aqueous sol-gel derived nanocomposite coating materials.
Materials Research Society,159.
37. Dorcheh, A.S. and M. Abbasi,(2008). Silica aerogel; synthesis, properties and
characterization. Journal of materials processing technology, 199(1): p. 10-26.
38. Rubio, F., J. Rubio, and J. Oteo,(1998). A FT-IR study of the hydrolysis of
tetraethylorthosilicate (TEOS). Spectroscopy Letters, 31(1): p. 199-219.
39. Donatti, D. and D. Vollet,(1996). Study of the hydrolysis of TEOS-TMOS mixtures
under ultrasound stimulation. Journal of non-crystalline solids, 204(3): p. 301-304.
40. Fardad, M.,(2000). Catalysts and the structure of SiO2 sol-gel films. Journal of
Materials science, 35(7): p. 1835-1841.
41. Nawaz, K., S.J. Schmidt, and A.M. Jacobi,(2014). Effect of catalyst used in the sol–gel
process on the microstructure and adsorption/desorption performance of silica aerogels.
International Journal of Heat and Mass Transfer, 74: p. 25-34.
42. Kesmez, Ö., et al.,(2010). Effect of amine catalysts on preparation of nanometric SiO2
particles and antireflective films via sol–gel method. Journal of sol-gel science and technology,
56(2): p. 167-176.
43. Shan, J. and H. Tenhu,(2007) Recent advances in polymer protected gold nanoparticles:
synthesis, properties and applications. Chemical Communications, (44): p. 4580-4598.
44. Yilgör, E. and I. Yilgör,(2014). Silicone containing copolymers: Synthesis, properties
and applications. Progress in Polymer Science, 39(6): p. 1165-1195.
Ref. code: 25605722040507ZOJ
58
45. Oktay, B. and N. Kayaman-Apohan,(2013). Polydimethylsiloxane (PDMS)-based
antibacterial organic–inorganic hybrid coatings. Journal of Coatings Technology and
Research, 10(6): p. 785-798.
46. Jung, H.-S., D.-S. Moon, and J.-K. Lee,(2012). Quantitative analysis and efficient
surface modification of silica nanoparticles. Journal of nanomaterials, 2012 p. 48.
47. Lee, C.H., et al.,(2011). Preparation and characterization of surface modified silica
nanoparticles with organo-silane compounds. Colloids and Surfaces A: Physicochemical and
Engineering Aspects, 384(1): p. 318-322.
48. Dai, M., et al.(2009). FTIR study of copper agglomeration during atomic layer
deposition of copper. in MRS Proceedings. Cambridge Univ Press.
49. Cuoq, F., et al.,(2013). Preparation of amino-functionalized silica in aqueous
conditions. Applied Surface Science, 266: p. 155-160.
50. Lin, J., J.A. Siddiqui, and R.M. Ottenbrite,(2001). Surface modification of inorganic
oxide particles with silane coupling agent and organic dyes. Polymers for Advanced
Technologies, 12(5): p. 285-292.
51. Dobry, D.E., et al.,(2009). A model-based methodology for spray-drying process
development. Journal of pharmaceutical innovation, 4(3): p. 133-142.
52. Mohammadkhani, A., et al.,(2011). Characterization of surface properties of ordered
nanostructures using SEM stereoscopic technique. Microelectronic Engineering, 88(8): p.
2687-2690.
53. De Muynck, W., N. De Belie, and W. Verstraete,(2010). Antimicrobial mortar surfaces
for the improvement of hygienic conditions. Journal of Applied Microbiology, 108(1): p. 62-
72.
54. Reithmeier, E., T. Vynnyk, and T. Schultheis,(2010). 3D-measurement using a
scanning electron microscope. Applied Mathematics and Computation, 217(3): p. 1193-1201.
55. Yang, Z., et al.,(2004). Novel Inorganic/Organic Hybrid Electrolyte Membranes.
Prepr. Pap.-Am. Chem. Soc., Div. Fuel Chem, 49(2): p. 599.
56. Macan, J., et al.(2003). Preparation of Epoxy-Based Organic-Inorganic Hybrids by Sol-
Gel Process. in International Conference MATRIB 2003, Materials, Tribology, Processing.
57. Macan, J., et al.(2004). Thermal degradation kinetics of epoxy-silica organic-inorganic
hybrid materials. in Third International Conference on Polymer Modification, Degradation
and Stabilisation MoDeSt 3.
58. Godnjavec, J., et al.,(2012). Stabilization of rutile TiO2 nanoparticles with glymo in
polyacrylic clear coating. Materiali in tehnologije, 46(1): p. 19-24.
Ref. code: 25605722040507ZOJ
59
59. González, M.G., J. Baselga, and J.C. Cabanelas,(2012). Applications of FTIR on epoxy
resins-identification, monitoring the curing process, phase separation and water uptake,
INTECH Open Access Publisher.
60. Nikolic, G., et al.,(2010). Fast fourier transform IR characterization of epoxy GY
systems crosslinked with aliphatic and cycloaliphatic EH polyamine adducts. Sensors, 10(1):
p. 684-696.
61. Macan, J. and H. Ivanković.(2006). Influence of Hydrolysis Conditions on Curing and
Properties of an Epoxy-Silane Based Hybrid Material. in MATRIB 2006-Materials, Tribology,
Processing.
62. Lan, L., G. Gnappi, and A. Montenero,(1993). Infrared study of EPOXS-TEOS-TPOT
gels. Journal of materials science, 28(8): p. 2119-2123.
63. Ebrahiminezhad, A., et al., (2013). Preparation of novel magnetic fluorescent
nanoparticles using amino acids. Colloids and Surfaces B: Biointerfaces, 102: p. 534-539.
64. Hui, L., et al.,(2013). Adsorption behavior and adsorption mechanism of Cu (II) ions
on amino-functionalized magnetic nanoparticles. Transactions of Nonferrous Metals Society
of China, 23(9): p. 2657-2665.
65. Zorainy, M.Y., et al.(2017). Preparation and Characterization of Modified Silica-Epoxy
Hybrid Ceramic Coatings. in MATEC Web of Conferences. EDP Sciences.
66. Sinkó, K., (2010). Influence of chemical conditions on the nanoporous structure of
silicate aerogels. Materials, 3(1): p. 704-740.
67. Launer, P.J., Infrared analysis of organosilicon compounds: spectra-structure
correlations. Silicone compounds register and review, 1987. 100. from
https://www.gelest.com/wp-content/uploads/5000A_Section1_InfraredAnalysis.pdf
68. Al-Oweini, R. and H. El-Rassy,(2009). Synthesis and characterization by FTIR
spectroscopy of silica aerogels prepared using several Si (OR) 4 and R′′ Si (OR′) 3 precursors.
Journal of Molecular Structure, 919(1): p. 140-145.
69. Lai, S.M., et al.,(2013). Preparation and properties of melt‐blended polylactic
acid/polyethylene glycol‐modified silica nanocomposites. Journal of Applied Polymer Science,
130(1): p. 496-503.
Ref. code: 25605722040507ZOJ
60
Appendices
Ref. code: 25605722040507ZOJ
61
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.
Ref. code: 25605722040507ZOJ
62
Week 2
Figure A2 : 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 2.
Ref. code: 25605722040507ZOJ
63
Week3
Figure A3 : 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 3
Ref. code: 25605722040507ZOJ
64
Week4
Figure A4 : 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 4
Ref. code: 25605722040507ZOJ
65
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
Cu/SiO2 microspheres in acrylic paint at 10% 20% 30%wt compared with TOA213 and only
acrylic paint(control) in week 1.
Ref. code: 25605722040507ZOJ
66
Week 2
Figure B2 : Antimicrobial activities effect of Sample 8 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 2.
Ref. code: 25605722040507ZOJ
67
Week 3
Figure B3 : Antimicrobial activities effect of Sample 8 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 3.
Ref. code: 25605722040507ZOJ
68
Week 4
Figure B4 : Antimicrobial activities effect of Sample 8 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 4.
Ref. code: 25605722040507ZOJ
69
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.
Ref. code: 25605722040507ZOJ
70
Week2
Figure C2 : 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 2.
Ref. code: 25605722040507ZOJ
71
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.
Ref. code: 25605722040507ZOJ
72
Week 4
Figure C4 : 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 4.
Ref. code: 25605722040507ZOJ
73
Appendix D
Result of antimicrobial activities of Sample 28 ( TEOS to GLYMO 1:0.17
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.
Ref. code: 25605722040507ZOJ
74
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.
Ref. code: 25605722040507ZOJ
75
Appendix F
Result of antimicrobial activities of Sample 28 ( TEOS to GLYMO 1:0.17
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.
Ref. code: 25605722040507ZOJ
76
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.
Ref. code: 25605722040507ZOJ