DEVELOPMENT OF A MICROFLUIDIC DEVICE AND ELECTRONIC...

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.

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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.

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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

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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

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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

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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).


100 µm of width and

depth.




50 µm of width and 80

µm of depth.




50 µm of width and

depth.




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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