ii
DEVELOPMENT OF A MICROFLUIDIC DEVICE AND ELECTRONIC
INFUSION SYSTEM TO FABRICATE MICROCAPSULES OF CALCIUM
ALGINATE
YAP HIUNG YIN
A thesis submitted in
fulfilment of the requirements for the award of the
Degree of Master of Electrical Engineering with Honors
Faculty of Electrical and Electronic Engineering
Universiti Tun Hussein Onn Malaysia
JAN 2016
iv
To my beloved father (Yap Kok Min),
mother (Cheah Mei Yoong), sister
and brother
v
ACKNOWLEDGEMENT
I praise to God for giving me the strength to overcome all the difficulties of my
master project. I would like to express my most appreciation and gratitude to my
supervisor, PM. Dr. Soon Chin Fhong for her relentless effort in giving guidance and
encouragement through this project. I believe that this project would never be
produced superbly if not because of her. Although she was busy with her project, she
still sacrifices a lot of precious time for giving help and support to make me not only
success in understanding the project but also to learn how to become a better
researcher.
Besides, special thanks to my parent Mr. Yap Kok Min and Mrs. Yap, my
family, friends and technicians and those involved directly or indirectly in this
project for their support, motivation and sharing their knowledge until this project
completed.
Last but not least, I would also like to thank financial support from Research
Acculturation Grant Scheme (RACE Vot No. 1515) and Post Graduate Incentive
Scheme (GIPS Vot No. 1406) of Universiti Tun Hussein Onn Malaysia.
vi
LIST OF ASSOCIATED PUBLICATIONS
H. Y. Yap, C. F. Soon, N. H. Muhd Nor, M. S. Saripan, M. Z. Sahdan, K. S. Tee,
“Synthesis and Characterization of Polymeric Microspheres by Using Suspension
Polymerization Technique”, International Integrated Engineering Summit (IIES),
2014. (Presented)
K. S. Tee, M. S. Saripan, H. Y. Yap, C. F. Soon, M. Z. Sahdan, “Development of A
Mechatronic Syringe Pump for Microfluidic Device Based on Polyimide Film”,
International Integrated Engineering Summit (IIES), 2014. (Presented)
H. Y. Yap, K. S. Tee, K. Ahmad, N. Zainal, S. C. Wong, W. Y. Leong, C. F. Soon,
“Customizing A High Flow Rate Syringe Pump For Injection Of Fluid To A
Microfluidic Device Based On Polyimide Film”, Malaysian Technical Universities
Conference on Engineering and Technology (MUCET), 2015. (Presented)
vii
ABSTRACT
Microfluidic is used to perform mixing, flowing and dispersing of fluid in micro
volume and they are widely applied in biomedical sciences, bioengineering and food
sciences. Microfabrication technique based on microelectronic technology has the
capability to produce microfluidic devices in micro or nano-metric scale but this
technique involves costly process and toxic chemicals. In this project, we proposed
the use of patterned vinyl adhesive template to produce a microfluidic device on a
work bench that was applied to generate microbeads of Calcium Alginate for
microencapsulation of cells. The design of the microfluidic device was simulated in
the COMSOL Multiphysics software to provide emulsification of two fluids. In this
work, an infusion pump of high flow rate (1000 to 5000 µl/min) was developed to be
used together with a commercial syringe pump that could pump the fluid flow to
emulsify the continuous and disperse phases leading to the production of microbeads.
The human keratinocyte cell lines (HaCaTs) encapsulated in the microbeads were
characterised using Fourier Transform Infrared Spectroscopy (FTIR) and 4',6-
diamidino-2-phenylindole (DAPI) staining. The fabrication process of the
microfluidic device was simple, safe and yet highly reproducible. Nonetheless, the
device developed was able to produce narrow distribution of microbeads with
diameter from 500 to 800 µm. At a volume of 20 µl Sodium Alginate solution, 50 ±
10 pieces of microbeads can be produced in 5 seconds. HaCaTs were successfully
embedded in the microbeads using this simple and efficient technique.
viii
ABSTRAK
Peranti mikrobendalir digunakan untuk melaksanakan percampuran, pengaliran dan
pemisahan cecair dalam isipadu yang sangat sedikit iaitu dalam unit mikro, dan ia
sering digunakan di bidang bioperubatan, biokejuruteraan dan sains makanan. Teknik
mikropembikinan berdasarkan dari teknologi mikroelektronik mempunyai keupayaan
untuk menghasilkan peranti mikrobendalir dalam mikro atau nano metrik tetapi
teknik tersebut memerlukan proses yang mahal dan penggunaan bahan kimia toksik.
Dalam tesis ini, kami mencadangkan peranti mikrobendalir dibuat dengan
menggunakan templat pelekat vinil dan digunakan untuk menghasilkan Kalsium
Alginat mikromanik-manik bagi membalut sel-sel. Reka bentuk bagi peranti
mikrobendalir telah disimulasi dengan menggunakan perisian COMSOL Multiphysics
untuk menyediakan pengemulsian dua cecair. Dalam tesis ini, pam infusi yang
mempunyai kadar aliran yang tinggi (1000 to 5000 µl/min) telah dibina dan diguna
bersama-sama dengan pam picagari komersial yang boleh mengepam aliran cecair
untuk melakukan aliran yang mempunyai dua jenis fasa iaitu pengemulsian
berterusan dan pemecahan sebagai pembawa kepada pengeluaran mikromanik-manik.
Sel keratinocyte manausia yang dibalut dalam mikromanik-manik telah dilakukan
pencirian dengan menggunakan Jelmaan Fourier Spektroskopi Inframerah (FTIR)
dan pewarnaan 4',6-diamidino-2-phenylindole (DAPI). Proses fabrikasi untuk peranti
mikrobendalir adalah mudah dan boleh diulangi. Peranti yang dibina mempunyai
keupayaan untuk menghasilkan pengedaran sempit mikromanik-manik dari 500
kepada 800 µm. Dengan menggunakan 20 µl larutan Natrium Alginat, mikromanik-
manik dapat dihasilkan 50 ± 10 biji dalam 5 saat. Sel-sel telah berjaya dikulturkan
dalam mikromanik-manik dengan menggunakan teknik yang mudah dan cekap.
ix
CONTENTS
TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT v
LIST OF ASSOCIATED PUBLICATIONS vi
ABSTRACT vii
CONTENTS
LIST OF TABLES
ix
xiii
LIST OF FIGURES xiv
LIST OF SYMBOLS AND ABBREVIATIONS xviii
LIST OF APPENDICES xx
CHAPTER 1 INTRODUCTION 1
1.1 Project Background 1
1.2 Problem Statement 2
1.3 Objectives 3
1.4 Scope 4
x
CHAPTER 2 LITERATURE REVIEW 5
2.1 Introduction 5
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
2.13
Background Study of Microfluidic Device
Development
Microfluidic Simulation in COMSOL
Multiphysics and Navier Stoke Equation
Fluid Dynamic
Electronic Infusion System
Techniques to Produce Microbeads
Previous Work of Synthesis Microbeads by
Using Microfluidic Device
Polymers Used For Fabrication of Microbeads
Calcium Alginate
Calcium Alginate Used For Microencapsulation
of Cells
Human Keratinocyte Cell Lines
A Review on Stepper Motor and Motor Driver
Arduino-Uno Microcontroller
5
7
8
11
13
17
18
20
21
22
23
25
2.14 Summary 26
CHAPTER 3 RESEARCH METHOLOGY 28
3.1 Introduction 28
3.2 COMSOL Multiphysics Simulation 29
3.3 Fabrication of the Microfluidic Devices 31
3.4 Development of an Electronic Infusion System
for Purging the Microfluidic Device
33
xi
3.4.1 The Mechatronic Design 33
3.4.2 Programming the Microcontroller to Control the
Motor Speed
36
3.4.3 Characterisation of the Infusion System 41
3.5 Production of Calcium Alginate Microbeads by
Using the Syringe Pump, Electronic Infusion
System and Microfluidic Device
43
3.6 Fourier Transform Infrared Spectroscopy of the
Calcium Alginate Microbeads
45
3.7
3.7.1
3.7.2
Encapsulation of Cells Using Calcium Alginate
Microcapsules
Culture of Human Keratinocyte Cell Lines
Microencapsulation of Cells
45
45
46
3.8 Summary 47
CHAPTER 4 RESULTS AND DISCUSSION 48
4.1 Introduction 48
4.2 Simulation of the Fluid Flow and Pressure of the
Microfluidic Device
48
4.3 Fabrication of Microfluidic Device 51
4.4 The Electronic Infusion System 53
4.4.1 The Characterisation of the Infusion System 53
4.5 Size Distribution of the Microbeads 56
4.6 Chemistry Content of the Microbeads Produced 63
4.7 Cells Encapsulation Using Calcium Alginate
Microcapsules
63
xii
4.8 Summary 65
CHAPTER 5 CONCLUSIONS 66
5.1 Future Works 67
5.2 Contribution 67
REFERENCES 69
APPENDICES 81
xiii
LIST OF TABLES
1.1 Fabrication of the microfluidic device 3
2.1 The summary of three lead screw threads 12
2.2 Comparison of the lead screw with different start 13
2.3 The advantages and disadvantages of the stepper
motor
14
2.4 Three types of polymerisation techniques with
different sequence of stirred and added solution
16
2.5 The different flow rate and dimension of channel
influenced the size of the microbeads
18
2.6 Summary of the polymers used as microbeads 19
2.7 The encapsulation of alginate microbeads 22
3.1 The specification of sodium alginate and paraffin
oil
31
4.1 The simulation results for velocity and pressure 51
4.2 The optimisation of the microfluidic design 51
4.3 The design specification of electronic infusion
system
56
xiv
LIST OF FIGURES
2.1 The liquid flow in a pipe. 8
2.2 The depiction of the liquid sheared by the nitrogen gas.
The parameter wm is the minimal width of the liquid.
9
2.3 Laminar flow in a microfluidic channel. 10
2.4 Turbulent flow in a microfluidic channel. 10
2.5 The principal of continuity applied in a pipe or a
microfluidic channel.
11
2.6 The different between a ball screw and lead screw. 12
2.7 The (a) single, (b) double and (c) triple start were
defined by the axial distance of lead (l), where p is the
pitch.
13
2.8 Flow focusing of microfluidic device to produce PDMS
microbeads.
14
2.9 The precision particle fabrication (PPF) system. 15
2.10 The experimental setup for suspension polymerisation. 16
2.11 The structural characteristics of alginates. 20
2.12 The ionic crosslinking of Na-alginate and Ca2+. 21
2.13 Normal skin of human which consists of epidermis and
dermis.
23
2.14 H-bridge circuit where 1 is gate, 2 is drain and 3 is
source for IRFZ44 MOSFET.
24
2.15 The schematic diagram in L298N. 24
2.16 The fly-back diode applied in motor driver L298N. 25
2.17 The hardware of Arduino-Uno microcontroller
(ATmega238).
26
xv
3.1 The flow chart of project methodology 29
3.2 The pattern of the microfluidic device. 30
3.3 A piece of vinyl adhesive template with microfluidic
pattern.
32
3.4 The process of microfluidic device fabrication. 33
3.5 The conceptual model of the infusion system. 34
3.6 The motor coupler to connect between lead screw and
motor shaft.
34
3.7 The linear slide system. 35
3.8 The block diagram of the electronic infusion system. 36
3.9 The schematic diagram of electronic infusion system. 36
3.10 The flow chart for defining input or output of the
components.
37
3.11 The directions of the lead screw determined the infusion
or diffusion process.
38
3.12 The flow chart of electronic infusion system. 39
3.13 The current measurement by using dc regulated power
supply.
41
3.14 The speed measurement by using tachometer. 42
3.15 The flow rate measurement by varied the programmed
speed.
43
3.16 The experimental setup to produce calcium alginate
microbeads.
44
3.17 The concept of producing calcium alginate microbeads
via the microfluidic device.
45
3.18 The calcium alginate microcapsules with HaCaT cells
were produced inside ESCO Class II safety cabinet.
47
4.1 Effects of outlet width to the velocity (m/s) of
microfluidic channel in simulation.
49
4.2 The relationship of velocity (m/s) and width of the
outlet channel (µm).
49
4.3 Effects of outlet width to the pressure (Pa) of
microfluidic channel in simulation.
50
xvi
4.4 The relationship of pressure (Pa) and width of the outlet
channel (µm).
50
4.5 The surface roughness of the vinyl adhesive template. 52
4.6 (a) PDMS microfluidic device with 2 inlets and 1 outlet
and (b) the fluid flow in PDMS microfluidic device.
52
4.7 The relationship of output current and operating
voltage.
53
4.8 The relationship of output current and programmed
speed.
54
4.9 The relationship of measured motor speed and
programmed speed.
54
4.10 The relationship of flow rate to the controlled motor
speed set in the microcontroller.
55
4.11 The relationship of measured volume and targeted
volume of fluid for paraffin oil.
56
4.12 The microbeads produced using different continuous
flow rate and fixed disperse flow rate at 300 µl/min.
58
4.13 The microbeads produced using different disperse flow
rate and fixed continuous flow rate at 2000 µl/min.
59
4.14 The diameter of the microbeads obtained at different
flow rates of the continuous phase.
60
4.15 The diameter of the microbeads obtained at different
flow rates of the disperse phase.
61
4.16 Phase contrast micrographs of the microbeads with an
average size of 460 µm. (Scale bar: 100 µm)
61
4.17 The size distribution of generated microbeads at a flow
rate of 5000 µl/min and 300 µl/min for continuous and
disperse phase, respectively.
62
4.18 The spectrum of calcium alginate microcapsules. 63
4.19 A phase contrast micrograph of the HaCaT cells. (Scale
bar: 100 µm)
64
4.20 DAPI staining of the HaCaT cells after 7 days of
incubation. (Scale bar: 100 µm)
64
xvii
B1 The liquid properties inserted in COMSOL
Multiphysics.
83
B2 The flow rates properties inserted in COMSOL
Multiphysics.
83
B3 The mesh setting and meshing model in COMSOL
Multiphysics.
84
D1 The control panel of the electronic infusion system 90
D2 The increase and decrease flow rate processes 90
D3 The (a) minimum and (b) maximum flow rate displayed
on LCD
91
D4 The infusion operation at flow rate of 3000 µl/min 91
D5 The diffusion operation at flow rate of 9000 µl/min 92
D6 The LCD indication message for system to stop 92
xviii
LIST OF SYMBOLS AND ABBREVIATIONS
GPC - Gas-Phase Chromatography
CE - Capillary Electrophoresis
PMMA - Poly-Methyl Methacrylate
PFA - Perfluoroalkoxyalkane
PEEK - Polyetherether Ketone
PSA - Pressure Sensitive Adhesive
PDMS - Poly-Dimethylsiloxane
CFD - Computational Fluid Dynamic
𝜌 - Density
u - Velocity
µ - Viscosity
p - Pressure
MEMS - Microelectromechanical Systems
Re - Reynolds Number
D - Diameter Of Pipe
g - Acceleration Of The Gravity
h - Height Between The Pipe And Ground
A - Area Of The Pipe
PLA - Poly-Lactic Acid
PEG - Poly-Ethylene Glycol
PS - Polystyrene
SDS - Sodium Dodecyl Sulfate
PFF - Precision Particle Fabrication
PLGA - Poly Lactic-Co-Glycolic Acid
M - β-D-Mannuronic Acid
xix
G - α-L-Guluronic Acid
Na-alginate - Sodium Alginate
CaCl2 - Calcium Chloride
Ca-alginate - Calcium Alginate
NaCl - Sodium Chloride
3D - Three Dimensional
BSA - Bovine Serum Albumin
PC12 - Pheochromocytoma of the Rat Adrenal Medulla
HN25 - Neuroblastoma
CRFK - Feline Renal Fibroblast
HaCaT - Human Keratinocyte
N2a - Mouse Neuroblastoma Cell Lines
NIH 3T3 - Fibroblast Cells
E. Coli - Escherichia Coli
M231 - Human Breast Cancer Cells
CELC - Cholesteryl Ester Liquid Crystals
PWM - Pulse Width Modulation
IDE - Integrated Development Environment
USB - Universal Serial Bus
I/O - Input and Outpu
LED - Light-Emitting Diode
DC - Direct Current
LCD - Liquid Crystal Display
DMEM - Dulbecco’s Modified Eagle’s Medium
HBSS - Hank’s Balanced Salt Solution
CO2 - Carbon Dioxide
DAPI - 4',6-Diamidino-2-Phenylindole
w/v - Weight per volume
%T - Transmittance in percentage
xx
LIST OF APPENDICES
APPENDIX TITLE PAGE
A The summary of the fabrication methods of
microfluidic device
81
B The parameters settings for COMSOL Multiphysics
software
83
C The coding of electronic infusion system 85
D The Operation of the Electronic Infusion System 89
E The DAPI staining of HaCaT cells after 7 days of
incubation
92
F Datasheet of L298N 93
1
CHAPTER 1
INTRODUCTION
1.1 Project Background
The application of microbeads for biomedical engineering research gained early
scientific community attention approximately 17 years ago [1]. Polymeric
microbeads were very popular in bioengineering, biosensor and biomedical
applications [2–6]. The microbeads made of polymeric material were applied to
encapsulate cells, implants, drugs and sensing agent [7,8]. Types of material that are
normally used for encapsulation of cells and chemical are such as alginate [9],
chitosan [10] and gelatin [11]. Encapsulation of living cells to form 3D cells is an
interesting field of research for bioengineering [12–14]. The encapsulation of cells in
microbeads provided an in-vivo microenvironment to the living cells to grow into
microtissues [15]. In close proximity, the microbeads create a confine environment
enabling them to re-establish their cell-cell adhesions. The microtissue model was
demonstrated with the potential to be applied in pharmacology testing [16].
Alginate is a natural occurring polysaccharide extracted from the brown sea
algae and comprises 1,4-linked β-D-mannuronic (M) and α-L-guluronic (G) residues
in varying proportions [17]. When alginate is used for encapsulation of cells, it
allows the exchange of catabolic and transportation of nutrients to the cells. In
addition, alginate can be easily modified via chemical and physical reactions to
obtain derivatives with various structure, properties, functions and applications [18].
Sodium alginate is soluble in aqueous solution and form gelatinous substances at
room temperature in the presence of multivalent cations such as Ca2+ through the
ionic interaction between guluronic acid groups [9]. This enables microbeads to be
formed with viable cells embedded by crosslinking in non-cytotoxic conditions.
2
Furthermore, alginate gel can be dissolved by using mild chelating agents are such as
alginate lyase in order to release the cells encapsulated [11]. There are many
applications of microencapsulation of cells that include dermal papillae (DP) cells
implant in rat ear to regenerate hair follicles [19], cartilage regeneration [18], testing
of cytokines release in microcapsules [20], insulin release in microcapsules to
streptozotocin-induced diabetic rats [21] and immobilisation of proteins encapsulated
as a biosensor to detect antigen-antibody interaction [22]. In order to achieve the
applications, engineering techniques could be designed to provide solutions to the
microencapsulation for either cells or chemical agents.
Various techniques based on emulsion [23], coacervation [21] and extrusion
[24] had been developed to fabricate microbeads in meeting the demand of the
biomedical scientist. In association with these techniques, fluid dispersion
microfluidic seemed to be an improved technique used to produce polymeric
microbeads recently [25–28]. The design of the crossed pattern of microfluidic
channels is the main part to perform emulsion in producing microbeads in which two
immiscible fluids are intercepted via different flow rates. Microfluidic is a network
of integrated channels that can be fabricated in micro or nano scales. The fabrication
of the microfluidic device has been continuously improved both in down scaling of
fluidic size and reduced complexity of fabrication [29–31]. The photolithography
associated with microfabrication is a common fabrication method because this
method enables the microchannels to scale down from micro to nano metric scales
[32–36]. Unfortunately, this technique required expensive microelectronics facilities
and the use of toxic chemicals [37–39]. The microelectronic fabrication facilities
include a class 100 clean room, spin coater, UV mask aligner, furnace and cool plate.
Based on these reasons, the search for fabricating microfluidic device using simple,
small scale and rapid methods is a continuing effort for the past ten years [38,40–45].
More environmental friendly and easy methods are always of interest to many
applications.
1.2 Problem Statements
Table 1.1 shows the major problem in fabrication microfluidic. Microfluidic device
is widely used in biomedical and bioengineering applications but the fabrication
protocol is required toxic material and clean room facilities. Photoresist such as SU-8
3
was a common used material to fabricate microfluidic mould but the material was
involving highly toxicity [35]. Although capillary [46], glass [40] and 3D object [30]
materials were discovered but the process of the fabrication required long period of
fabrication. Therefore, vinyl adhesive sheet was proposed in this research to fabricate
microfluidic mould. The fabrication technique was involving non-toxic material.
Table 1.1: The major problem in fabrication microfluidic
Fabrication method Major Problem Ref.
Photolithography This method was involving highly toxicity
material which is SU-8 as a photoresist material. [35]
Capillary Capillary device was used to replace SU-8 but
capillary device required specify manufacture
patterns which involving complex fabrication
protocol for glass material.
[46]
3D printing object 3D printing object also used to replace SU-8
which does not involve toxic material but the
roughness surface of the 3D printing object is
depending on the printing resolution.
[30]
Previous literature suggested the suitable sizes of microcapsules that could
encourage the formation of skin microtissues in the encapsulation are in the range of
350 µm [16]. Hence, the sizes of the microcapsules need to be controlled in size
ranging from 400 to 600 µm. The size control is possible if a mechatronic and
microfluidic device were designed with an aim to control the size of the microbeads
produced.
1.3 Objectives
The objectives of the research are:
To design a microfluidic device that allows the flow control of the continuous
and dispersion flow of the paraffin oil and sodium alginate solution.
To develop an electronic flow control system to actuate the high flow rate in
the microfluidic device that can fabricate calcium alginate microcapsules with
diameter ranged from 500 to 800 µm.
To develop a microcapsules with size range from 500 to 800 µm to
encapsulate Human Keratinocyte Cell Lines (HaCaT).
4
1.4 Scope
The scope of this project is planned as follow:
Development of an electronic infusion system to control the high flow rate
(1000 - 5000 µl/min) of the continuous phase.
Disperse phase of the microfluidic device will be infused with a commercial
syringe pump with flow rate ranging from 0 to 300 µl/min.
Design a crossed pattern of microchannel to perform emulsion.
Fabrication of custom-made microfluidic device with 2 inlets and 1 outlet.
Characterisation of the diameter and biochemistry content of the calcium
alginate microcapsules.
Cell embedded inside microcapsules was observed after 7 days of incubation.
5
CHAPTER 2
LITERATURE REVIEWS
2.1 Introduction
This section discussed the background information of microfluidics, fluid dynamic,
infusion pump system, materials for fabrication of microbeads, and previous
techniques in fabrication of microbeads, application of microbeads for encapsulation
of cell, and human keratinocytes. The literature review sets the foundation for the
development of this project.
2.2 Background Study of Microfluidic Device Development
A review paper written by George M. Whitesides (2006), comprehensively revealed
the history of microfluidic device [47]. The first application of microfluidic device
was used as a microanalytical method such as gas-phase chromatography (GPC),
high-pressure liquid chromatography (HPLC) and capillary electrophoresis (CE)
[47,48]. The second application of microfluidic device was in the year 1990 by
military as a sensor for chemical and biological threats. The third application of
microfluidic device was for the field of molecular biology. The microfluidic device
was claimed to provide high sensitivity, greater throughput and high resolution [47].
The microfluidic device was successfully fabricated by using photolithography and
associated technologies. The earliest fabrication of the microfluidic device was using
silicon and glass, but the materials used were unnecessary or inappropriate for
analysis of biological samples in water due to the expensive material and opaque to
visible and ultraviolet light [47]. In seeking a more biocompatible material, David C.
Duffy (1998) discovered the use of poly-dimethylsiloxane (PDMS) to fabricate
6
microfluidic device [49]. The master mould was produced by using soft lithography
which can be used to replicate microfluidic device made by PDMS [50–54]. PDMS
was not only the material that could be used to fabricate microfluidic with good
visibility. Poly-methyl methacrylate (PMMA) was proposed by Myra T. Koesdjojo
(2008) to fabricate microfluidic device for Capillary Electrophoresis (CE) with good
visibility, low cost, biocompatibility and ease of fabrication [55]. New fabrication
methods of a microfluidic device continued to be proposed after 1990’s.
Soft lithography is a commonly used method to fabricate microfluidic mould
or template on a silicon wafer because of the smooth surface of the silicon wafer and
the ability of this technique to produce very fine channels. Photolithography
technique have been used to fabricate microfluidic device over 10 years
[32,35,37,38,43–45,56,57]. However, the fabrication of the microfluidic device using
photolithography is expensive and toxic that includes the requirements of the
photoresist chemicals and facilities for microelectronics technologies. These facilities
include a clean room, spin coater, heating and cooling machine, and UV exposure
with range from 350 to 400 nm. Due to these reasons, J. Cooper McDonald (2002)
proposed a new fabrication method of microfluidic device that involved with a three
dimensional thermoplastic master mould produced using solid-object printer known
as 3D printer instead of using the photolithography technique [30]. Unfortunately,
significant surface roughness was observed for the microfluidic device fabricated
using this technique [30]. According to the paper by Won Je Jeong (2005), the
PDMS microfluidic device can be fabricated by using glass pipette as a master mould
at a diameter of 20 µm [46]. Additionally, Perfluoroalkoxyalkane (PFA) and
polyetherether ketone (PEEK) tubing were able to fabricate microfluidic device at a
diameter of 360 µm [58]. However, microfluidic that fabricated by using tubing as a
master mould could not be easily replicated and required special manufacturing
process. Therefore, Po Ki Yuen (2009) proposed an economic and straight forward
rapid prototyping method for the fabrication of microfluidic device. In this method,
the microfluidic mould was fabricated by using desktop digital craft cutter producing
patterned double-sided pressure sensitive adhesive (PSA) tape [29]. However,
microfluidic device fabricated by using PSA tape required high skill of controlling
cutting speed and thickness in order to fabricate high quality of microfluidic device
[29]. In addition, PSA tape of microfluidic was not biocompatible due to the
existence of the PSA debris. Therefore, in this thesis, a similar technique has been
7
proposed to fabricate master mould of microfluidic devices based on patterned vinyl
adhesive sheet followed by synthesis of microfluidic device using poly
dimethylsiloxane (PDMS). PDMS behaves like silicon and biocompatible for
medical application [59]. The construction process of each technique is provided in
Appendix A.
2.3 Microfluidic Simulation in COMSOL Multiphysics and Navier Stoke
Equation
COMSOL Multiphysics simulation software is a powerful tool which can be used to
simulate any model such as separating flow in microfluidic device [60], viscoelastic
fluids in rectilinear flow [61], and fluid velocity in microchannels [62]. In the
previous studies [63,64], the microfluidic device was optimised by obtaining the
result in the COMSOL Multiphysics in terms of the velocity, concentration and
pressure of the liquids. Different applications such as mixing, dispersion and
separating require different fluid flow and pressure condition. The width of the
channels and pressure are usually inversely proportional. Therefore, simulation was
extremely needed for channels that deal with nano scale in order to prevent failure in
the actual fabrication of the nanofluidic device. Finite element analysis is a numerical
method to solve partial differential equation. Therefore, finite element analysis was
applied in COMSOL Multiphysics modelling in order to mesh the spatial domain
into multiple elements [65]. In addition, finite element analysis also used to solve
several of physic and engineering phenomena in COMSOL Multiphysics. For this
research, only fluid module was used to perform laminar flow in microfluidic device.
Computational fluid dynamic (CFD) module was often simulated by other
researchers [65–68] due to the high similarity of the modelling and high accuracy
results. Unfortunately, CFD module was modelled in three dimensional which
required high performance of processor to execute simulation. In the simulation of
the COMSOL Multiphysics, Navier-stokes equation was used to analyse the motion
of the fluid and continuity equation was used to analyse the velocity of the fluid in
different area [69]. Constant fluid density, a laminar flow regime exists throughout
system and a Newtonian fluid was an assumption of the Navier-stokes equation [69].
Equation 2.1 shows the Navier-stokes equation for steady incompressible flow [65].
8
Incompressible flow is a flow in which the materials density is constant within a
fluid parcel.
𝜌 (u · ∇) u - μ ∇2 u - ∇p = 0 (2.1)
where, 𝜌 is the fluid density (kg/m3), u is the velocity (m/s), µ is the viscosity (Pa·s),
p is pressure (Pa), (∇ · u) is divergent, ∇p is gradient.
2.4 Fluid Dynamic
Fluid is a substance that deforms continuously under the application of shear stress
[70]. Fluid can be defined as a liquid or gas. The fluid is not able to resist the
deformation forces. Hence, the shape of the fluid changed according to the shearing
forces act tangentially to the surface [70]. The deformation of the fluid in an enclosed
channel is an advantage for bioengineering application which can be applied to
produce micro or nanobeads. Figure 2.1 show the liquid flow in a pipe. The liquid in
pipe was flown in different velocity due to the shearing force of the pipe wall. The
shearing forces of the liquid not only could occur on the surface of the pipe but also
could occur by applying additional gas or immiscible liquid [71]. Figure 2.2 shows
the liquid which was under the deformation due to the gas flow in a microfluidic
device [71]. To disperse the liquid, the pressure of the nitrogen gas has to be purged
higher than the pressure of the liquid. In a microfluidic device, the liquid in the
continuous phase was purged 100 times faster than the liquid in disperse phase [23].
Figure 2.1: The liquid flow in a pipe.
Shearing force
Pipe
Velocity, v
9
Figure 2.2: The depiction of the liquid sheared by the nitrogen gas. The parameter wm
is the minimal width of the liquid [71].
Two types of flow could occur for fluid flowing in a channel that includes the
laminar and turbulent flows. The characteristics of both flows in a channel are
determined by the Reynolds number (Re). Reynolds number is a dimensionless
quantity which determines the nature of fluid flow over a surface. The Reynolds
number can be calculated by using Equation 2.2:
Re = ρvD
μ (2.2)
where, ρ is the density of the fluid (kg/m3), v is the velocity of the fluid (m/s), D is
the diameter of the pipe (m) or length for non-pipe flow, μ is the viscosity of the
fluid (Pa·s).
The Reynolds number is used to calculate the initial force flow in a pipe
against the viscous force of the fluid. The Reynolds number below 2000 is defined as
laminar flow (Figure 2.3) and above 4000 is defined as turbulent flow (Figure 2.4).
Laminar flow is often used in microfluidic device for common function such as
mixing or dispersing [64]. Turbulent flow is used as a mixing purpose in microfluidic
device [54]. The transitional flow was determined when the Reynolds number is
between 2000 and 4000. The transitional flow means the flow could be neither
laminar nor turbulent. The Reynolds number was used for calculating the flow in
pipe [70]. Different situation of flow produces different Reynolds number.
0.7 bar of
nitrogen gas 10% of dish-washing
liquid in deionized
water
10
Figure 2.3: Laminar flow in a microfluidic channel.
Figure 2.4: Turbulent flow in a microfluidic channel.
Fluid dynamic in microfluidic device is important in term of understanding
the velocity, pressure, diameter of the channel and viscosity of the fluid flow.
Bernoulli’s equation is used to calculate the parameter of the fluid flow such as
pressure (N/m2), velocity (m/s), height between the pipe and ground (m), and the
diameter of the pipe (m) [72]. Viscosity of the liquid was not considered in
Bernoulli’s equation as provided in Equation 2.3:-
P1 + 𝜌1 g h + 1
2𝜌1 µ1 = P2 + 𝜌2 g h +
1
2𝜌2 µ2 (2.3)
where, P is the pressure of the fluid (N/m2), 𝜌 is the density of the fluid (kg/m3), g is
the acceleration of the gravity (m/s2) which is equate to 9.81 m/s2, h is the height
between the pipe and ground (m), µ is the velocity of the fluid (m/s).
The principal of continuity in pipe should be considered when fluid flows
from across different sizes of channels as shown in Figure 2.5. This principal is
applicable to the microfluidic device. Equation 2.4 was used to calculate the velocity
of the fluid from pipe of small to big diameter or vice versa [70]. The equation is
given as:-
𝐴1𝜇1𝜌1 = 𝐴2𝜇2𝜌2 (2.4)
where, A is the area of the pipe (m), µ is the velocity of the fluid (m/s), 𝜌 is the
density of the fluid (kg/m3).
11
Figure 2.5: The principal of continuity applied in a pipe or a microfluidic channel.
The continuity, Reynolds number and Bernoulli’s equations were important
in microfluidic technique. By using the equations, the microfluidic device can be
optimised in terms of the velocity and pressure of the fluid. The velocity and pressure
of the fluid flow in microchannels can be simulated by using COMSOL Multiphysics
software tool. For dispersion application, laminar flow is needed in the fluid flow but
turbulent flow is not necessary. Hence, Reynolds number must lower than 2000 in
order to obtain laminar flow in the microfluidic device.
2.5 Electronic Infusion System
An electronic infusion system offers advantages over manual administration of fluids,
including the ability to deliver fluids in very small volumes, and the ability to deliver
fluids at precisely programmed rates or automated intervals [73]. There are many
types of infusion pumps, including large volume, patient-controlled analgesia (PCA),
elastomeric, syringe, enteral, and insulin pumps [73]. In this thesis, an electronic
infusion system was used to deliver fluid with high velocity inside a microfluidic
device in order to perform emulsion.
In infusion pump systems, stepper motors have been used in the pump
mechanism to provide precise flow rates. Although DC motor also could be operate
as a function of stepper motor but it required additional angular-position sensors or
hall-effect sensor [74]. DC motor would not be able to have enough torque to rotate
the lead screw in order to push the syringe instead of using DC geared motor.
Therefore, stepper motor was used in this work in order to provide precise flow rate
into the microfluidic device. Motor load is affected by the position of the pump
mechanism, fluid viscosity, and flow rate. Therefore, the alignment of the lead screw
is important which could reduce the motor load [74].
µ1
µ2
12
Lead screw is one of the most important component in an electronic infusion
system in which the lead screw was used as a linkage to change the turning motion of
a motor to the linear motion [73]. Hence, the movement of the linear slider was
determined by the lead screw rotation. Therefore, a suitable lead screw mounted in
the system was determined by the application used. In some linear motion system, a
low-friction ball or roller screw provide acceptably smooth motion for many
applications but the ball or roller screw are costly [74]. Different sizes of the lead
screw was used in different applications such as 140-ton theatre stage required large
lead screw and robotic application required only small lead screw [73]. The
conversion of rotary to linear motion can be achieved by rotating the nut which
attached with lead screw. In the review paper of Orang Vahid Araghi (2009), a
micropump was developed using lead screw and nut had generated noise which
related to the friction-induced vibration of the lead screw [75]. Although the noise
can be reduced by using ball screw instead of lead screw but ball screw was an
expensive material. Therefore, lead screw was chosen for the syringe pump
mechanism which does not affect much in term of accuracy by the friction. Figure
2.6 shows structure of a ball screw and lead screw.
Figure 2.6: The different between a ball screw and lead screw.
The structures of the lead screw can be divided into three types which include
the square, Acme (known as trapezoidal) and buttress thread. Table 2.1 shows the
advantages and disadvantages of each thread. For the syringe pump system, buttress
and square threads were not considered due to the one way direction and difficult to
machine, respectively. Although square thread was able to provide high efficiency
and least friction to the system but the thread was not match with the hexagonal nut
Ball screw Lead screw
13
which will be moved with the linear slide. Therefore, the Acme thread was chosen
for the system due to the suitable criteria that match to develop a syringe pump.
However, the noise was generated from the Acme thread due to the friction-induced
vibration.
Table 2.1: The summary of three lead screw threads [76]
Type of thread Advantages Disadvantages Figure
Acme or
trapezoidal
Easy to machine. Not efficient as square
thread.
Friction occurred due to
the thread angle.
Square Most efficient.
Least friction.
Often used to carry
high power.
Difficult to machine.
Expensive.
Buttress Efficient as square
thread.
Easy to manufacture.
Can only be applied in
one direction.
Table 2.2 shows the different number of start would cause different distance
which known as lead (l). Number of start of the lead screw can be determined by
observing the length of the lead as shown in Figure 2.7. Figure 2.7 (a), (b) and (c)
shows the lead screw that in single, double and triple start, respectively. The fineness
or coarseness of a lead screw are defined by lead (l) and pitch (p). The lead was
defined as the axial distance for the screw travels in one complete revolution of the
shaft and the pitch was defined as the axial distance between the crests of adjacent
threads [77]. Double and triple start of the lead screw would perform faster rotation
speed compare to single start. Single start of lead screw was able to move the linear
motion in small distance with a complete revolution which also slower rotation speed
than double and triple start. Hence, the single start was proposed to provide precision
volume of fluid that purged from the syringe pump. Orang Vahid Araghi (2009)
reported the misalignment and nonlinearity could be due to the thread contact, load
distribution, deflection and discontinuity as a result of the backlash [75].
14
Figure 2.7: The (a) single, (b) double and (c) triple start were defined by the axial
distance of lead (l), where p is the pitch [78].
Table 2.2: Comparison of the lead screw with different start [78]
Number of start Process
Single One full rotation would cause a distance, l (referring to (a)).
Double One full rotation would cause far distance than single start
(referring to (b)).
Triple One full rotation would cause a long distance than single and
double start (referring to (c)).
Stepper motor is also an important component for an electronic infusion
system which could drive the lead screw to push or pull the syringe with sufficient
torque. A stepper motor is an electromechanical device which converts electrical
pulses into discrete mechanical movements. The shaft or spindle of a stepper motor
rotates in discrete step increments when electrical command pulses are applied to it
in the proper sequence. The motors rotation has several direct relationships to these
applied input pulses. The sequence of the applied pulses is directly related to the
direction of motor shafts rotation. The speed of the motor shafts rotation is directly
related to the frequency of the input pulses and the length of rotation is directly
related to the number of input pulses applied [78]. Table 2.3 shows the advantages
and disadvantages of the stepper motor. The hybrid stepper motor is more expensive
than the permanent magnet stepper motor but provides better performance with
respect to step resolution, torque and speed. In this thesis, the hybrid stepper motor
was used to purge high volume fluid with high precision.
Table 2.3: The advantages and disadvantages of the stepper motor [78]
Advantages Disadvantages
The rotation angle of the motor is proportional to
the input pulse.
Resonances can occur if not properly controlled.
Precise positioning and repeatability of
movement
Not easy to operate at extremely high speeds.
The motor has full torque at stand-still (if the
windings are energised).
15
2.6 Techniques to Produce Microbeads
Three different techniques that can be used to fabricate microbeads are such as
microfluidic, microextruder and spinning techniques [2,79,80]. Microfluidic
technique is one of the widely used techniques for fabricating microbeads due to the
highly controllable size and shape of microbeads. For polymer like poly-
dimethylsiloxane (PDMS), poly-lactic acid–poly-ethylene glycol–poly-lactic acid
(PLA–PEG–PLA) and polystyrene-co-poly-dimethlysiloxane (PS-co-PDMS), the
microfluidic device was built-in with heating element for polymerisation purpose
[80–82]. The flow rate for the continuous and disperse phase of the microfluidics
were controlled by using syringe pump [83]. The advantages of using microfluidic
device to produce microbeads are the size of microbeads can be very small (reach
nanometers), narrow size distribution, cost saving from the reduced consumption of
materials or reagents and safer operation [23]. From the previous research, the size of
the microbeads can be varied from 10 to 200 µm by manipulating the flow rate of the
continuous phase [82]. The consistency of the size seemed to be higher than the
suspension polymerisation technique. Figure 2.8 shows an example of the flow
focusing microfluidic device used to produce polymeric microbeads.
Figure 2.8: Flow focusing of microfluidic device to produce PDMS microbeads [82].
Electrostatic extrusion technique used to fabricate microbeads was invented
by B. G. Amsden (1997) [84]. The monomer such as alginate gel was dripped from a
micro nozzle to a reservoir which contain of a solution that could cross-linked the
monomer. The monomer that form droplet at the needle tip (inner diameter of the
Flow focusing
16
syringe = 1.19 mm) was pulled by the force from the electrostatic field. Although the
droplet was pulled early before the bigger droplet size was formed at the needle tip
but the size of the microbeads was found too large ranging from 0.78 to 1.51 mm
with a standard deviation typically in the range of 10 – 20 % [84]. This is because of
the limitation of the microextruder which was depending on the inner diameter of the
needle tip. However, the review paper by Kyekyoon “Kevin” Kim (2006) reported
that the microextruder technique was enhanced by precision particle fabrication (PPF)
system in which, continuous flow of fluid behaved as a sheath flow to minimise the
droplet size as shown in Figure 2.9 [85]. By using the precision particle fabrication
(PPF) system, the minimum size of the microbeads was smaller than the inner
diameter of the nozzle in the range of 4 and 500 µm [85].
Figure 2.9: The precision particle fabrication (PPF) system [85].
Simple polymerisation technique such as suspension, emulsion and dispersion
polymerisation also can be used to fabricate microbeads [86]. Suspension, emulsion
and dispersion polymerisation technique were a spinning technique that consists of
heating and stirring element, but these techniques involved with different sequence
of stirred and added solutions. For the suspension polymerisation technique, an
Erlenmeyer flask filled with surfactant (distilled water and stabilizer) was pre-heated
and stirred by using a magnetic stirrer. Then, the polymer solution (such as poly-
dimethylsiloxane) will be added by dripping the polymer droplet into the surfactant.
The experimental setup is simple, can produce microbeads in bulk but only available
for immiscible fluid, and the size of microbeads can be obtained in micrometers [86].
17
However, wide range of microbeads was obtained by using this technique,
approximately ranging from 40 to 1000 µm in diameter.
The process of emulsion polymerisation is similar to suspension
polymerisation. The heating and spinning element are required in the experiment.
The surfactant (distilled water and stabilizer) and initiator only (PDMS curing agent)
were stirred and heated for a few minutes for pre-warmed purpose [86]. After that,
the monomers (PDMS elastomer) was added by dripping into the surfactant [86].
Emulsion polymerisation technique was found useful to synthesise micro and nano
beads [87]. The size range of the microbeads produced using this technique is also
huge ranged from 0.1 to 10 µm.
Dispersion polymerisation technique is also one of the spinning techniques
that require heating and stirring element to the solvent which is insoluble with
polymer. The solvent was stirred with the monomers, initiator, and a stabilizing
molecule [80]. Additional added solutions were not required in this technique [88,89].
After a few hours, the polymerisation occurred and the small beads were started to
polymerise a membrane [86]. Stabilizer was used to assist the microbeads to
maintain the sphere shape before the membrane polymerised [90]. Dispersion
polymerisation technique can be used to produce microbeads in size ranged from 1 to
10 µm. Figure 2.10 shows the common experimental setup for the suspension,
emulsion and dispersion polymerisation technique. Table 2.4 shows the three
spinning techniques with different sequence of stirred and added solution.
Figure 2.10: The experimental setup for suspension polymerisation [86].
More than 10h
Polymerisation
Heated Heated
Wash/dry
Microspheres
18
Table 2.4: Three types of polymerisation techniques with different sequence of
stirred and added solution [86]
Techniques Stirred
solution
Added
solution
Weakness Strength
Suspension
polymerisation
Surfactant Monomer
and initiator
The diameter of the
microbeads could be large
during dripping.
Could prevent
massive coagulation
during spinning.
Emulsion
polymerisation
Surfactant
and initiator
Monomer Spinning speed has to be
faster in order to prevent
coagulation of microbeads
during spinning
Could possibly
produce smaller
microbead.
Dispersion
polymerisation
Surfactant,
monomer and
initiator
- Time consuming on the
process of polymerisation
to microbeads due to the
pre-heat for surfactant has
not been done early.
No additional solution
required in order to
prevent interruption
during
polymerisation.
The spinning technique presented limitation due to the time consuming
process that are more than 10 hours to complete the transformation of the polymer
from liquid to solid [31]. Therefore, microfluidic and microextruder techniques were
gaining higher popularity compared to spinning technique because of the versatility
in fluid control and save time. It was efficiently used to produce narrow size
distribution of microbeads. Polymeric microbeads such as calcium alginate
microbeads were fabricated easily by using microfluidic technique but not spinning
technique due to the different polymerisation process (calcium alginate required
cross-linked process). Therefore, most of the research applied either microfluidic or
microextruder technique to fabricate microbeads of calcium alginate microbeads
instead of spinning technique.
2.7 Previous Work of Synthesis Microbeads by using Microfluidic Device
Recently, emulsion process to synthesis microbeads is gaining interest by many
researchers in the field of bioengineering. Microfluidic device is widely used to
produce microbeads. Via the microfluidic technique, single emulsion can be
developed to produce microbeads but more complicated system of double or triple
emulsion to produce microbeads of multi cores are possible [26]. Won Je Jeong
(2005) revealed that microbeads of hydrogels were produced by using microfluidic
device with oil as a continuous carrier [46]. The previous study by P. Garstecki
(2005), described the microbeads produced using the flow rate of disperse phase was
set constant at 76 kPa and the continuous phase increased from 0.018 until 0.055
19
µl/min [91]. However, the increased flow rate of the disperse phase lead to chaotic
condition in which the stability to produce microbeads reduced significantly. The
microfluidic system required a long period of time (1 to 30 min) to become stable
under continuous flow (10-6 to 10-4 µl/min) for the production of microbeads [92].
This study indicated that a suitable flow rate must be determined for the microfluidic
system in order to produce microbeads with high stability. Table 2.5 shows the
summary of the flow rate used and dimension of microfluidic device to produce
different size of microbeads by previous studies.
Table 2.5: The different flow rate and dimension of channel influenced the size of the
microbeads
The channels
dimension Flow rate in microfluidic device Size of microbeads
Ref.
40 µm of width and
depth.
Continuous phase is 150 µl/min.
Disperse phase is 1 µl/min.
175 µm of diameter. [46]
30 µm of width and 25
µm of depth.
Continuous phase is 0.018 µl/min.
Disperse phase is 76 kPa (Pressure).
46 µm of diameter. [91]
100 µm of width and
depth.
Continuous phase is 100 µl/min.
Disperse phase is 10 µl/min.
248 µm of diameter. [58]
50 µm of width and 80
µm of depth.
Continuous phase is 50 µl/min.
Disperse phase is 10 µl/min.
48 µm of diameter. [93]
50 µm of width and
depth.
Continuous phase is 30 µl/min.
Disperse phase is 2 µl/min.
77 µm of diameter. [27]
The previous works revealed that smaller size of microbeads can be produced
by using high continuous phase with low disperse phase as an inlet of microfluidic
device. On the other hand, the size of the microbeads was affected more on the
dimension of the microchannel compared to manipulate flow rate. The previous
results showed that the size of the microbeads was difficult to become smaller size
than the dimension of the microchannels. However, the miniaturisation of the
microfluidic device provided a lot of benefits to the researchers such as lower the
cost of manufacture, reduced time of analysis and reduced consumption of the
reagents and analytes, decreased weight and volume and increased portability [43].
2.8 Polymers Used For Fabrication of Microbeads
Polymers that have been actively reported in the literature for the fabrication of
microbeads are such as calcium alginate [1,4,6,8,9,12–14,22,24,94–104], poly-
dimethylsiloxane (PDMS) [2,5,80], chitosan [105,106], gelatin [11,107], poly lactic-
20
co-glycolic acid (PLGA) [108–110], and poly methyl methacrylate (PMMA) [89,90].
Table 2.6 shows the summary of the polymers that had potential to be made into
microbeads. Polymers such as PDMS, PLGA and gelatin required certain criteria to
fabricate microcapsules; the criteria are such as PDMS microcapsules required
double emulsion technique associated with microfluidic device. Then, PLGA
microcapsules were only suitable for drug release but not able to encapsulate living
cells due to the physical and chemical properties. For the gelatin microcapsules, the
melting point of the gelatin at 37 °C was not suitable for cells encapsulation.
Therefore, Encapsulation of the living cells can be easily done by using calcium
alginate and chitosan that are biocompatible and ease for fabrication.
Table 2.6: Summary of the polymers used as microbeads
Polymer Physical and chemical
properties
Advantages Disadvantages Ref.
Calcium
Alginate
Biodegradable,
biocompatible, soft
material and fluid will be
able to flow in and out
freely.
Able to encapsulate
cells, drug delivery and
biosensor. Alginate
lyase was able to break
the alginate into small
molecules.
High stability was
required during
crosslinking process in
order to remain spherical
shape.
[8,11,
13]
PDMS Biocompatible, rubber-like
with colour clear and non-
toxic elastomer.
Hydrophobic polymer
that commonly used as
a microfluidic devices.
Complex fabrication
technique such as double
emulsion was required to
fabricate microcapsules.
[59,80
,82]
Chitosan Low toxicity,
biocompatibility,
biodegradability and
ability to adsorb or release
molecules.
Encapsulation of
enzymes and cells,
drug delivery, scaffold
in tissue engineering,
biosensor and
actuators.
Rapid disintegration
between chitosan and
tripolyphosphate (TPP)
due to the weak ionic
interactions.
[3]
Gelatin Denatured form of
collagen that cells can
bind to and degrade
through enzymatic action.
Can be dissolved in
aqueous solutions and
then reconstituted into
a hydrogel at
temperatures that are
not damaging to
proteins.
Temperature controlled
was required. The
melting point of gelatin
is near 37 °C which is
not suitable for cell
growth.
[11,10
7]
PLGA Tissue compatibility,
biodegradability, and high
stability in biological fluid
and during storage.
Widely used in drug
delivery carrier and
able to treat cancer by
combining with
chemotherapeutic
agents (nano-
formulations).
Improvement of PLGA
was required in order to
protect therapeutic
agents against enzymatic
degradation.
[111,1
12]
PMMA Crystalline and transparent
thermoplastic polymer.
Well studied in
colloidal crystals and
order-disorder
transitions.
Not suitable for
fabricating
microcapsules due to the
physical properties.
[89,90
]
21
Although there are a lot of polymers that can be used to produce microbeads
by using different techniques, calcium alginate seemed to be a polymer with good
physical and chemical properties that are possible to encapsulate living cells in
mirobeads. Alginate that biocompatible provides a good environment for the living
cells to grow in the alginate beads. Alginate microbeads can be easily fabricated with
narrow distribution of size by using the microfluidic device technique. However, the
stability of the production remains challenge without using stabilizer.
2.9 Calcium Alginate
Alginate is anionic polysaccharides composed of β-D-mannuronic acid (M) and α-L-
guluronic acid (G) as shown in Figure 2.11. Alginate is also a naturally derived
polysaccharide which is highly desirable for biomedical applications such as cells
encapsulation, tissue transplantation and controlled delivery of drugs. With the
structure of alginate, it can form hydrogels with multivalent cations such as Ca2+,
Ba2+, or Fe3+ [96].
Figure 2.11: The structural characteristics of alginates.
There were three methods that can be used for the preparation process of the
calcium alginate microcapsules which is microextruder [113], coacevation
[21,114,115], laser extrusion [8] and microfluidic technique [7,116–122]. The size of
the calcium alginate microcapsules can be easily controlled by using microfluidic
technique compare to the other techniques. Microextruder technique involved with
dripping sodium alginate solution into the calcium chloride solution to form uniforms
and spherical shaped calcium alginate hydrogel bead, but the resulted beads were
determined by the diameter of the needle tip [96]. On the other hand, microfluidic
channel was able to fabricate microbeads in smaller diameter which is depending on
22
the width of the microchannels in the microfluidic device. In the microfluidic device,
the beads produced flow from the outlet into a beaker containing calcium chloride
solution. The calcium (II) ions were released from calcium chloride solution in order
to polymerise the alginate beads. During the cross-linked process, the calcium
alginate and sodium chloride will be produced as shown in equation 2.5 [12].
2 Na-alginate + CaCl2 Ca-alginate (solid) + 2 NaCl (liquid) (2.5)
Polymer can be fabricated in 4 different ways such as linear, branched,
network (3D) and cross-linked [123]. Therefore, calcium alginate is a polymer that
used cross-linking process to polymerise from liquid to solid (gel) form. Sodium
alginate is one of the chemical products that are widely available in market. Calcium
chloride releases calcium (II) ion which is used to react with the sodium alginate at
the interface between oil and calcium chloride solution [12,13]. Figure 2.12 shows
the cross-linking process of sodium alginate and calcium (II) ion. There are three
other materials that can be used to substitute calcium chloride to obtain calcium (II)
ion that include calcium sulphate, calcium carbonate and calcium CaEDTA with
different technique [124].
Figure 2.12: The ionic crosslinking of Na-alginate and Ca2+ [125].
Calcium alginate was widely used in encapsulation such as proteins Bovine
Serum Albumin (BSA) [96], gold nanoparticles [12], pheochromocytoma of the rat
adrenal medulla (PC12) and neuroblastoma (HN25) living cells [13], and feline renal
23
fibroblast (CRFK) cells line [11]. Hence, calcium alginate microcapsule will not be a
problem to encapsulate living cell like Human Keratinocyte (HaCaT) cells. HaCaT
cells was one of the major cells types in in the epidermis of skin [126].
2.10 Calcium Alginate Used For Microencapsulation of Cells
Calcium alginate is a common material which was used to encapsulate micro or nano
particles. Encapsulation of the micro or nano particles was also known as micro or
nanocapsules which means the bead consists of shell and core. Microcapsules were
fabricated to encapsulate the particles or living cells inside the bead as shown in
Table 2.7. Although calcium alginate was used to encapsulate a lot of cell types but
encapsulating HaCaT cells to grow into 3D microtissues is scarce.
Table 2.7: The encapsulation of alginate microbeads
Types of encapsulation Application Ref.
Neuroblastoma cells (HN
25) and Pheochromocytoma
of the rat adrenal medulla
(PC 12).
PC 12 and HN 25 had been encapsulated for running vitality
test inside alginate microcapsules.
[13]
Mouse neuroblastoma cell
lines (N2a).
In vitro model systems for pharmacotoxicological
application, brain research and tissue engineering.
[14]
Fibroblast cells (NIH 3T3
and L929).
Cell viability was confirmed using live and dead cell assays
in order to show the microdevice had a significant potential of
bead or fibre-based systems for in vivo cell-based drug
delivery and tissue engineering application.
[4]
Feline renal fibroblast cell
line (CRFK).
CRFK cells were encapsulated with cell viability of 93.8 %.
Then, the cells grew and filled in the alginate core. The
multicellular spheroids were collected from the calcium
alginate microcapsules by immersing in alginate lyase within
1 min.
[11]
Anti-BCG polyclonal IgY
and anti-E. Coli polyclonal
IgG (Antibodies).
Binding affinity test for Mycobacterium bovis BCG cells and
Escherichia coli (E. Coli).
[7]
Bovine serum albumin
(BSA).
BSA release was tested with low and medium viscosity of
alginate.
[1,96]
Gold nanoparticles To verify the applicability of the microfluidic technique in
encapsulation of alginate microbeads.
[12]
Insulin was obtained as a
regular human insulin of
recombinant DNA origin
(Actrapid® 100 IU/ml).
Extracted insulin from microcapsules decreased
hyperglycemia of diabetic rats proving to be bioactive.
[21]
Human breast cancer cells
(M231).
Cells viability was tested with live and dead cells assays for 7
days. The cells were able to survive and grow.
[8]
24
2.11 Human Keratinocyte Cell Lines
Figure 2.13 shows the normal skin of human which consists of epidermis and dermis.
Human keratinocytes are epithelial cells that are found in the epidermis of human
skin [15]. Human keratinocyte cell lines (HaCaTs) are immortal cells which can be
grown for prolonged period in vitro. In addition, HaCaT had an ability to proliferate
during the wound healing process [126]. In the review paper of Chin Fhong Soon
(2013), HaCaT cells were sub-cultured on the cholesteryl ester liquid crystals (CELC)
had a good potential to grow in three dimensional (3D) microtissues [127]. The cells
were able to grow into 3D microtissues because of the compliance of the liquid
crystal that similar to the in-vivo microenvironment [15]. Moreover, HaCaT cells
that cultured on a flat surface had a limitation in the quantity of the 3D cells
produced. However, the cells were only able to self-regulate into ellipsoidal
population [128]. Therefore, Ramsey Foty (2011) presented a simple hanging drop
cell culture technique to create a spherical form of microenvironment in order to
culture 3D spheroids [129]. Hence, the interest to grow cells into 3D is growing and
microfluidic technique could be suitable to produce microencapsulation of cells
leading to the growth of 3D cells.
Figure 2.13: Normal skin of human which consists of epidermis and dermis.
Normal Skin
Epidermis
Dermis
Fatty tissue
Blood vessel
Hair follicle
69
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