DESIGN AND DEVELOPMENT OF COLIFORM

BACTERIA DETECTION SYSTEM INTEGRATED

WITH MICROFLUIDIC AND OPTICAL

ABSORBANCE MEASUREMENT DEVICE

NURULAZIRAH BINTI MD SALIH

UNIVERSITI TUN HUSSEIN ONN MALAYSIA

UNIVERSITI TUN HUSSEIN ONN MALAYSIA

STATUS CONFIRMATION FOR MASTER’S THESIS

DESIGN AND DEVELOPMENT OF COLIFORM BACTERIA DETECTION

SYSTEM INTEGRATED WITH MICROFLUIDIC AND OPTICAL

ABSORBANCE MEASUREMENT DEVICE

ACADEMIC SESSION : 2015/2016

I, NURULAZIRAH BINTI MD SALIH, agree to allow this Master’s Thesis to be kept at the

Library under the following terms:

1. This Master’s Thesis is the property of Universiti Tun Hussein Onn Malaysia.

2. The library has the right to make copies for educational purposes only.

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FREE ACCESS

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Approved by,

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Permanent Address :

NO 6, KAMPUNG PARIT LAPIS

KADIR, 83200 SENGGARANG,

BATU PAHAT, JOHOR

Date: 29 FEBRUARY 2016

Date: 29 FEBRUARY 2016

NOTE:

** If this Master’s Thesis is classified as CONFIDENTIAL or RESTRICTED,

Please attach the letter from the relevant authority/organization stating

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This thesis has been examined on 8th December 2015

and is sufficient in fulfilling the scope and quality for the purpose of awarding

Degree of Master.

Chairperson:

PROF. MADYA IR. DR. BABUL SALAM BIN KSM KADER IBRAHIM

Faculty of Electrical and Electronic Engineering

Universiti Tun Hussein Onn Malaysia

Examiners:

PROF. MADYA DR. CHAN KAH YOONG

Faculty of Engineering

Multimedia University (MMU)

PROF. MADYA SITI HAWA BINTI RUSLAN

Faculty of Electrical and Electronic Engineering

Universiti Tun Hussein Onn Malaysia

DESIGN AND DEVELOPMENT OF COLIFORM BACTERIA DETECTION

SYSTEM INTEGRATED WITH MICROFLUIDIC AND OPTICAL

ABSORBANCE MEASUREMENT DEVICE

NURULAZIRAH BINTI MD SALIH

A thesis is submitted in

fulfillment of the requirement for the award of the

Degree of Master of Electrical Engineering

Faculty of Electrical and Electronic Engineering

Universiti Tun Hussein Onn Malaysia

FEBRUARY 2016

ii

I hereby declare that the work in this thesis is my own except for quotations and

summaries which have been duly acknowledged.

Student : ……………………………………………………

NURULAZIRAH BINTI MD SALIH

Date : …………………………………………………….

Supervisor : …………………………………………………….

DR. MOHD ZAINIZAN BIN SAHDAN

Co-supervisor : …………………………………………………….

DR. SOON CHIN FHONG

DECLARATION

29 FEBRUARY 2016

iii

For my parents

For my brothers and sisters

For my teachers

For my friends

DEDICATION

iv

ACKNOWLEDGEMENT

Alhamdulillah, I am grateful to Allah S.W.T. for the good health and wellbeing that

were necessary to complete this thesis.

I wish to express my sincere thanks to my supervisor, Dr Mohd Zainizan bin

Sahdan and my co-supervisor, Dr Soon Chin Fhong for their help and support

throughout this project. I am extremely thankful and indebted to them for the

valuable guidance and encouragement extended to me.

I take this opportunity to express my gratitude to the Microelectronics and

Nanotechnolgy - Shamsuddin Research Centre (MiNT-SRC, UTHM), Molecular

Biology Laboratory (UTHM), Institute of Nano Electronic Engineering (INEE,

UNIMAP), Low Dimensional Materials Research Centre (LDMRC, UM) and the

Faculty of Electrical and Electronic Engineering (FKEE, UTHM) for providing me

all the necessary facilities for the completion of this research. I would like to thank

all the members and staffs for their help and contribution.

Uncountable thanks to my beloved family and friends for the unceasing

encouragement, support and attention. I also place on record, my sense of gratitude

to one and all, who directly or indirectly, have lent their hand in my project.

v

ABSTRACT

The detection of coliform bacteria which contain the disease-causing microorganism

is a useful indication for water contamination. This primary indication is important

for diagnosis of infectious disease, as well as for countermeasure to potential

biological threats. Currently, the emerging of technology in molecular biology

research and industry is in demand for portable and miniaturized system. This project

involved with design and development of microfluidic and optical absorbance

measurement device for coliform bacteria detection system. Suitable microfluidic

design was simulated in the COMSOL Multiphysics software. The microfluidic

device was designed for coliform bacteria sample using optical detection approach.

The microfluidic device was fabricated with glass and polydimethylsiloxane (PDMS)

material using photolithography, replica moulding (soft lithography), and oxygen

plasma bonding techniques. Then, the optical absorbance measurement device for

coliform bacteria detection was developed based on the optical absorbance theory.

The device was constructed using 470 nm blue light emitting diode (LED), photo

detector, ARDUINO microcontroller, liquid crystal display (LCD), and mechanical

elements. This project had successfully developed a prototype which integrates the

PDMS-glass based microfluidic and optical absorbance measurement device. The

absorbance measurement from the prototype and colony number of the coliform

bacteria samples were collected and analyzed. The collected data was used for the

prototype programme. The final analysis had indicated that the developed prototype

was able to detect the coliform bacteria in suspension at the lowest detection of

13,400 CFU/ml.

vi

ABSTRAK

Pengesanan bakteria coliform yang mengandungi mikroorganisma penyebab

penyakit adalah satu petunjuk berguna untuk pencemaran air. Petunjuk utama ini

adalah penting bagi diagnosis kepada penyakit berjangkit, serta penting bagi

tindakan terhadap potensi ancaman biologi. Pada masa kini, perkembangan teknologi

dalam penyelidikan biologi molekular dan industri mempunyai permintaan untuk

sistem yang mudah alih dan kecil. Projek ini melibatkan fabrikasi menggunakan

integrasi alat cecair mikro dan alat pengukur serapan optik untuk sistem pengesanan

bakteria coliform. Reka bentuk alat cecair mikro yang sesuai telah disimulasi

menggunakan perisian COMSOL Multiphysics. Peranti ini direka bentuk untuk

sampel bakteria coliform dan pendekatan pengesanan optik. Alat cecair mikro telah

difabrikasi dengan bahan kaca dan polydimethylsiloxane (PDMS) menggunakan

teknik litografi foto, pengacuan replika (litografi lembut), dan pelekatan plasma

oksigen. Kemudian, alat pengukur serapan optik untuk bakteria coliform telah

dihasilkan berpandukan teori serapan optik. Alat tersebut telah dibina menggunakan

alat pemancar cahaya (LED) 470 nm berwarna biru, pengesan foto, mikropengawal

ARDUINO, paparan kristal cecair (LCD), dan elemen-elemen mekanikal. Projek ini

telah berjaya menghasilkan prototaip yang mengintegrasikan alat cecair mikro

berasaskan kaca dan PDMS bersama alat pengukur serapan optik. Nilai serapan

daripada prototaip dan jumlah koloni bagi bakteria coliform telah dikumpulkan dan

dianalisis. Data yang dikumpul telah digunakan bagi program prototaip tersebut.

Analisis terakhir telah mengenalpasti prototaip yang dihasilkan telah mampu

mengesan bakteria coliform di dalam sampel cecair pada had pengesanan minimum

iaitu 13,400 CFU/ml.

vii

TABLE OF CONTENTS

TITTLE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES x

LIST OF FIGURES xii

LIST OF SYMBOLS AND ABBREVIATIONS xvii

LIST OF APPENDICES xix

LIST OF PUBLICATIONS xx

LIST OF AWARDS xxi

CHAPTER 1 INTRODUCTION 1

1.1 Overview 1

1.2 Background of Study 2

1.3 Problem Statement 6

1.4 Hypothesis 6

1.5 Objective 7

1.6 Project Scope 7

viii

1.7 Research Contribution 8

CHAPTER 2 LITERATURE REVIEW 9

2.1 Introduction 9

2.2 Microfluidic Development 9

2.2.1 Historical Perspective 10

2.2.2 Fluid and Flow Properties in Micro Scale 11

2.2.3 Materials and Fabrication Techniques for

Microfluidic Device 15

2.3 Bacteria Detection 20

2.3.1 Conventional Bacteria Detection Methods 20

2.3.2 Bacteria Detection Technologies 22

2.4 Summary 27

CHAPTER 3 RESEARCH METHODOLOGY 28

3.1 Introduction 28

3.2 Microchannel Design Simulation 29

3.3 Contact Angle and Surface Tension Measurement 30

3.4 PDMS-Glass Based Microfluidic Device Fabrication 32

3.4.1 Master Mould Preparation 32

3.4.2 PDMS Microchannel Patterning 34

3.4.3 PDMS Microchannel and Glass Bonding 36

3.5 Absorbance Measurement Device Development 37

3.5.1 Circuit Design and Development 37

3.5.2 Mechanical Part Design and Development 40

3.6 Coliform Bacteria Cell Culture and Sample Preparation 41

3.6.1 Coliform Plate Cell Culture 41

3.6.2 Coliform Culture Suspension 43

3.6.3 Dilution and Colony Counting 44

3.7 Absorbance Measurement 45

3.7.1 UV-Visible Spectrophotometer Measurement 45

3.7.2 Prototype Measurement 46

3.7.3 Growth Measurement 47

CHAPTER 4 RESULT AND DISCUSSION 48

4.1 Introduction 48

4.2 Simulation Analysis 48

ix

4.2.1 Preliminary Design 48

4.2.2 Microchannel Design 49

4.2.3 Final Microchannel Design 52

4.3 Contact Angle and Surface Tension Analysis 53

4.4 PDMS-Glass Based Microfluidic 58

4.4.1 SU-8 Microchannel Master Mould 59

4.4.2 PDMS Microchannel Pattern 62

4.4.3 PDMS Microchannel and Glass Bonding 65

4.5 Absorbance Measurement Device 66

4.6 Coliform Bacteria Sample 69

4.6.1 Bacteria Culture and Suspension 69

4.6.2 Coliform Bacteria Suspension in PDMS-Glass

Based Microfluidic 71

4.7 Qualitative and Quantitative Absorbance Analysis on

Coliform Bacteria 72

4.7.1 UV-Visible Spectrophotometer Measurement

Analysis 72

4.7.2 Prototype Absorbance Measurement and

CFU Analysis 77

CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 84

5.1 Conclusion 84

5.2 Recommendations 85

REFERENCES 86

APPENDIX 101

x

LIST OF TABLES

2.1 Advantages and disadvantages of materials for

microfluidic device 20

2.2 Advantages and disadvantages of bacteria

detection methods 26

3.1 Simulation setting used in this study 29

4.1 Water flow velocity result of Figure 4.3 design

for different angle (θ1, θ2) 50

4.2 Water flow velocity result of optimized

microchannel design 51

4.3 Validation results for different value of x 52

4.4 Water flow velocity and Reynolds number result 53

4.5 Probe liquids surface tension and cosine θ on

each material 55

4.6 Critical surface tension of glass, PDMS, and

paraffin wax 56

4.7 Optimization of PDMS moulding process 63

4.8 Absorbance readings of coliform bacteria sample

using Quartz cuvette 74

4.9 Absorbance readings of coliform bacteria sample

using PDMS-glass based microfluidic device 74

4.10 Absorbance readings for coliform bacteria

sample using the developed prototype 78

4.11 Comparison of absorbance readings between the

developed prototype and UV-Visible

spectrophotometer 78

4.12 Absorbance and coliform colony number for

different dilution factor 79

xi

4.13 Absorbance and coliform colony number for

different incubation time 80

4.14 Absorbance and colony number outline for

prototype system 82

xii

LIST OF FIGURES

1.1 Agar plate indicating bacteria from polluted water

sample 1

1.2 E. coli cell culture on Chromocult agar 3

1.3 Example of ELISA plate with colour indication of

different level of antibody reactivity 4

1.4 Example of PCR machine to test poultry for

contamination 4

1.5 Microfluidic array for cell culturing 5

1.6 Example of silicon based microfluidic channels

prepared using photolithography technique 5

2.1 Gas chromatography design 10

2.2 Shear stress and shear rate for coefficient of

viscosity of fluid flow 12

2.3 Laminar and turbulent flow behaviour 13

2.4 Example of convection and diffusion behaviour

inside microchannel 14

2.5 Photolithography and etching technique on

silicon wafer 15

2.6 Capillary channels etched into Corning 7740

glass with 10 µm depth 16

2.7 Hot embossing technique for microchannel

fabrication 17

2.8 Laser ablation technique for microchannel

fabrication 17

2.9 Soft lithography technique for PDMS

microchannel fabrication 18

2.10 Conductometric immunosensor construction 22

xiii

2.11 Impedimetric detection system with impedance

analyzer and IME 23

2.12 Detection cells (Cell A, Cell B, and Cell C) in

PDMS microchip 24

2.13 Fluorescence detector device for E. coli bacteria 25

2.14 Diagram of the experimental setup of the sensing

unit with piezoelectric crystal 26

3.1 Research flow methodology process 28

3.2 Step by step simulation 29

3.3 Development of model simulation 30

3.4 Glass, PDMS, and paraffin wax samples for

contact angle measurement 30

3.5 Contact angle analyser 31

3.6 Master mould preparation process 32

3.7 Example of AUTOCAD drawing for the

microchannel mask 33

3.8 (a) SU-8 photoresist (SU-8 2075) (b) SU-8

developer 33

3.9 SU-8 spin coating on glass substrate 33

3.10 Mask aligner machine for UV expose process 34

3.11 PDMS microchannel patterning process 35

3.12 Sylgard 184 silicon elastomer and curing agent 35

3.13 PDMS mixture with master mould 36

3.14 PDMS-Glass bonding 36

3.15 Oxygen Preen System machine 36

3.16 Process flow of the absorbance measurement

system 37

3.17 Schematic drawing of the complete circuit 38

3.18 Arduino UNO R3 38

3.19 OPT101 photo detector 39

3.20 PCB layout for the circuit development 39

3.21 PCB for the hardware development 40

3.22 Hardware design drawing 40

3.23 Microfluidic holder and stage design drawing 41

xiv

3.24 Chromocult powder from Merck 42

3.25 Chromocult mixture heated in microwave 42

3.26 Solidified chromocult agar in petri dish 42

3.27 Nutrient broth powder from Becton Dickinson 43

3.28 Nutrient broth mixture in sample bottles 43

3.29 Dilution step being used in this project 44

3.30 Counting the bacteria colony using digital colony

counter device 44

3.31 UV-Visible spectrophotometer (SHIMADZU,

UV-1800) 46

3.32 Coliform bacteria suspension in cuvette and

microfluidic 46

3.33 PDMS-glass based microfluidic position in the

developed prototype for absorbance measurement 47

4.1 Initial simulation design with one inlet and one

outlet channel 49

4.2 Several models of microchannel design 50

4.3 Microchannel design with θ1 and θ2

microchannel angle 50

4.4 Optimized microchannel design specification 51

4.5 Finalized microchannel design for the prototype 53

4.6 Contact angle of probe liquids on paraffin wax

surface 54

4.7 Contact angle of probe liquids on PDMS surface 54

4.8 Contact angle of probe liquids on glass surface 54

4.9 Fox-Zisman graph of paraffin wax surface tension

analysis 55

4.10 Fox-Zisman graph of PDMS surface tension

analysis 56

4.11 Fox-Zisman graph of glass substrate surface

tension analysis 56

4.12 Microchannel design for the microfluidic

fabrication (a) preliminary trial design (b) final

design for coliform bacteria detection device 58

xv

4.13 SU-8 master mould for design in Figure 4.12 (a) 59

4.14 SU-8 master mould for design in Figure 4.12 (b) 59

4.15 Surface profiler result for SU-8 master mould in

Figure 4.13 (thickness = 96.0 µm) 60

4.16 Surface profiler result for SU-8 master mould in

Figure 4.14 (thickness = 98.0 µm) 60

4.17 SU-8 master mould thickness dependence on

deposition speed graph 61

4.18 Pre-baking or soft baking time with respect to

SU8 thickness 61

4.19 PDMS microchannel based on the master mould

design in Figure 4.13 62

4.20 PDMS microchannel based on the master mould

design in Figure 4.14 63

4.21 Surface profiler result of the prepared PDMS

microchannel depth 64

4.22 FESEM result of the prepared PDMS

microchannel depth 64

4.23 PDMS-glass microfluidic based on the master

mould design in Figure 4.13 65

4.24 PDMS-glass microfluidic based on the master

mould design in Figure 4.14 65

4.25 Absorbance measurement device 66

4.26 Dark measurement area inside the hardware

chasing 67

4.27 Movable microfluidic holder and stage 68

4.28 Movable microfluidic stage 68

4.29 Movable microfluidic holder 68

4.30 Coliform bacteria colonies on chromocult agar 69

4.31 Coliform bacteria in nutrient broth medium (1

hour to 3 hours) 70

4.32 Microscopic image of coliform bacteria in

PDMS-glass based microchannel (40X

magnification) 71

xvi

4.33 Coliform bacteria size indication (inside PDMS-

glass based microchannel) 71

4.34 Absorbance measurement for medium with

coliform bacteria and medium without coliform

bacteria 73

4.35 Absorbance measurement using Quartz cuvette

and PDMS-glass based microfluidic device 73

4.36 UV-Visible absorbance measurement (350 nm to

750 nm wavelength) of coliform bacteria

suspension in quartz cuvette for every 1 hour

incubation time 75

4.37 UV-Visible absorbance measurement (350 nm to

750 nm wavelength) of coliform bacteria

suspension in PDMS-glass based microfluidic

device for every 1 hour incubation time 76

4.38 Growth curve of coliform bacteria (Quartz

cuvette) 76

4.39 Growth curve of coliform bacteria (PDMS-glass

based microfluidic device) 77

4.40 Absorbance versus coliform colony number for

sample dilution analysis 80

4.41 Absorbance versus coliform colony number for

growth analysis 81

4.42 Absorbance versus coliform colony number for

overall analysis 82

4.43 Example of absorbance reading in the prototype

display 83

4.44 Example of colony number indication in the

prototype display 83

xvii

LIST OF SYMBOLS AND ABBREVIATIONS

θ - Angle

ρ - Density

η - Viscosity

∆P - Pressure drop across the length

d - Typical length scale/diameter

l - Length

m - Mass

t - Time

v - Volume

x - Average distance

A - Area

D - Diffusion constant

F - Force

I - Transmitted light intensity

I0 - Original light intensity

L - Capillary length/channel length

P - Pressure

Q - Volume flow

R - Radius

V - Velocity

CFU - Colony forming unit

DNA - Deoxyribonucleic acid

COC - Cyclic Olefin Copolymer

DI - Distilled

ELISA - Enzyme-linked immunosorbent assay

E. coli - Escherichia coli

FESEM - Field emission scanning electron microscope

xviii

IME - Interdigitated microelectrode

IPA - Isopropyl alcohol

LAPS - Light-addressable potentiometric sensor

LCD - Liquid crystal display

LED - Light emitting diode

MCU - Microcontroller unit

NOA81 - Norland Adhesive 81

PC - Polycarbonate

PCB - Printed circuit board

PCR - Polymerase chain reaction

PDMS - Polydimethylsiloxane

PMMA - Polymethylmethacrylate

POC - Point of care

PUMA - Polyurethane Methacrylate

Re - Reynolds number

TPE - Thermoset Polyester

UV - Ultraviolet

xix

LIST OF APPENDICES

APPENDIX TITLE PAGE

A Table A.1: Gantt’s Chart of Research Activities 101

B PDMS-glass based microfluidic fabrication flow 102

C Flowchart of the prototype system program 103

D ARDUINO coding for the prototype system

programme 104

E Prototype design drawing with full dimension 109

F Voltage and absorbance readings from prototype

measurement 111

xx

LIST OF PUBLICATIONS

CONFERENCE PAPER:

1. Advanced Material Research Conference Proceedings Journal: N. M.

Salih, U. Hashim, N. Nafarizal, C. F. Soon and M. Z. Sahdan, 'Surface

Tension Analysis of Cost-Effective Paraffin Wax and Water Flow

Simulation for Microfluidic Device', AMR, vol. 832, pp. 773-777, 2013.

2. Advanced Material Research Conference Proceedings Journal: N. M.

Salih, U. Hashim, N. Nafarizal, C. F. Soon and M. Z. Sahdan, 'Numerical

Simulation of Water Flow Velocity for Microfluidic Application Using

COMSOL Multiphysics', AMR, vol. 925, pp. 651-655, 2014.

3. IEEE Conference Proceedings Journal: N. M. Salih, N. Nafarizal, U.

Hashim, A. Tijjani, C. F. Soon and M. Z. Sahdan, 'Glass Etching for Cost-

Effective Microchannels Fabrication', Semiconductor Electronics (ICSE),

2014 IEEE International Conference on. IEEE, pp. 432-435, 2014.

4. Advanced Material Research Conference Proceedings Journal: N. M.

Salih, U. Hashim, N. Nafarizal, C. F. Soon and M. Z. Sahdan, ‘Absorbance

Analysis of Escherichia coli (E. coli) Bacteria Suspension in

Polydimethylsiloxane (PDMS)-Glass Based Microfluidic’, AMR, vol.

1133, pp. 65-69, 2015.

xxi

LIST OF AWARDS

1. Bronze Medal in Malaysia Technology Expo [MTE] 2015:

Mohd Zainizan Sahdan, Nurulazirah Md Salih, and Soon Chin Fong,

“Coliform Bacteria Monitoring System Using Integrated Microfluidic Lab-

On-Chip and Optical Sensor”.

2. Bronze Medal in Research and Innovation Festival 2013 UTHM:

Nurulazirah Md Salih, Mohd Zainizan Sahdan, and Soon Chin Fong,

“Cost-Effective Material and Process for Microfluidic Device Fabrication”.

1CHAPTER 1

INTRODUCTION

1.1 Overview

Bacteria monitoring and detection are important for diagnosis and therapy of

infectious disease, as well as for countermeasure to potential biological threats.

Infectious bacteria in water have been categorized as a considerable threat to global

health. The established finding for coliform bacteria in water includes the total

coliforms, fecal coliforms, and Escherichia coli (E. coli) [1-3]. Figure 1.1 shows the

indication of coliform bacteria on agar plate culture. The presence of coliform

bacteria is an indication of water contamination, which may contain many dangerous

microorganisms [4]. Microorganisms are primary reasons for the infectious diseases.

Therefore, the concentration of harmful bacteria should be routinely monitored to

maintain the quality of water.

Figure 1.1: Agar plate indicating bacteria from polluted water sample [5]

2

Along with the importance of bacteria analysis, suitable detection system is

required for point of care (POC) devices. This project aimed at developing

microfluidic based system for miniaturizing the analytical instrumentation and

methodology in bacteria detection. Microfluidic is a rapidly expanding scientific

discipline which deals with fluids flowing in miniaturized systems [6]. It involves

design of systems in small volumes (micro-scale) of fluids. Microfluidic offers the

ability of a system to work with smaller reagent volumes, shorter reaction times, and

the possibility of parallel operation. Microfluidic is expected able to employ different

approaches of bacteria detection technique including optical measurement method.

Optical measurement method is the most widely employed technique for

bacteria detection due to its sensitivity and selectivity. It offers a wide range of

measurement approaches including absorption, reflection, refraction, dispersion,

chemiluminescence, and fluorescence [7]. All these approaches could be specifically

selected for their suitability for different bacteria analysis. Combination of

microfluidic device with optical measurement method is expected to produce a

complete system for bacteria detection.

1.2 Background of Study

Conventional bacteria detection methods largely rely on microbiological and

biochemical analysis. Culturing bacteria on a plate is perhaps the oldest and yet the

most accurate method. Monolayer cell culture on a surface of a petri dish is widely

used in life science research for the bacteria cellular behaviour analysis. Figure 1.2

shows an example of plate culturing of E. coli bacteria on chromocult agar (Merck

KGaA) for water and food testing. However, these approaches are normally

requiring large sample volume, cost-ineffective, time-consuming, and limiting the

throughput of the cell culture-based assay works. Moreover, they are not suitable for

on-site diagnosis integration.

3

Figure 1.2: E. coli cell culture on Chromocult agar [8]

The plate cell culturing technique utilizes a significant amount of material,

tedious, labour-intensive, and required long time period in providing results [9].

Conventional cell culture that utilizes culture dishes or micro-titer plates requires

technical expertise and specific facilities to handle cell harvesting, media exchange

and cell sub-culturing procedures. It had been indicated the major drawbacks of large

surface area of plate cell culture formats is that it leads to variation in cell seeding

densities, nutrient delivery, and waste removal.

Advanced technologies in immunological methods such as enzyme-linked

immunosorbent assay (ELISA) had introduced easier and faster pathogen detection

methods, relying on the recognition specificity of antibodies as shown in Figure 1.3.

It had been discovered that immunological-based methods require less assay time

compared to traditional culture techniques [10]. However, this method is still lacking

the ability to detect microorganisms in real time. In addition, ELISA method had

been reported requires multiple steps of reagent addition and rinsing which is too

complex to be used in field [11].

The identification of bacteria through genetic analysis techniques is

becoming more conventional. Polymerase chain reaction (PCR) is one of the

methods that leverage the nucleic acid complementarity-based specificity of

pathogen detection. Figure 1.4 shows an example of commercial PCR machine for

contamination detection. PCR detection method can detect single copy of a target

DNA sequence and can be used to detect single pathogenic bacterium in sample [10].

4

Figure 1.3: Example of ELISA plate with colour indication of different level of

antibody reactivity [12]

Figure 1.4: Example of PCR machine to test poultry for contamination [13]

It had been reported that PCR assay offers the advantages of specificity,

accuracy, and capacity in detection of bacteria/pathogen compared to the standard

culture and ELISA methods. In spite of its advantages, the detection of bacteria

using PCR is expensive, complicated, and requiring skilled workers to carry out the

tests [11].

Along with the development of conventional cell culture, immunoassay, and

nucleic based method, a new technology of microfluidics has the possibility to

overcome the limitations of conventional bacteria detection method [14].

Microfluidic based detection method had been observed as a future platform in

microbiology field. A lot of research had been performed in improving and

expanding the use of microfluidic in biological and clinical applications. A

microfluidic cell culture array was produced containing 100 cell culture chambers

with integrated gradient generators as shown in Figure 1.5 [15].

5

Microfluidic had been widely used as a stand-alone device and integrated

with conventional detection method. Integration of microfluidic platform with

ELISA and PCR method had shown interesting results by reducing the complexity

and processing time [16, 17]. However, most of this integration still depends on

expensive and large size apparatus. Due to this problem, it is believed that the

integration of microfluidics with lower cost and miniaturized detection device will

benefit the industry and research. Besides the detection method, preparation process

of microfluidic devices plays a big role in overall system production and application

suitability.

In previous years, microfluidic production had revolved with different

materials which led to various fabrication techniques. The microfluidic fabrications

are mostly depend on micro-processing of silicon, glass, and polymer materials.

Figure 1.6 shows the silicon based microfluidic produced by Pal et al. group in 2009.

Figure 1.5: Microfluidic array for cell culturing [15]

Figure 1.6: Example of silicon based microfluidic channels prepared using

photolithography technique [18]

6

Each of the materials has their own advantages and disadvantages. It has

been discovered that silicon is relatively expensive and opaque, making it unsuitable

for systems that use optical detection [18]. Glass is transparent but, it is amorphous

which make the vertical sides are more difficult to etch compare to silicon [19].

Additionally, glass and silicon micromachining processes are technically demanding

and time consuming. Polymer materials had been reported to be less expensive and

offer mass production, but it still requires dedicated press equipment, a robust

mould, and suffers from a lack of convenient methods for strong bonding [20].

From these findings, it can be concluded that suitable material and fabrication

technique need to be carefully selected in tailoring specific objective of bacteria

detection in the production of microfluidic device.

1.3 Problem Statement

The colony counting on conventional plate culture and absorbance measurement

using cuvette are time-consuming, labour-intensive, and require significant amount

of sample. Moreover, the advanced conventional strategy of immunoassay (example:

ELISA) and nucleic acid based (example: PCR) detection method are commercially

developed with bulky and expensive measurement machine. It also involves with

complicated preparation procedures. These up-to-date devices have been designed

with well-equipped laboratories and trained technicians. Due to these drawbacks, a

faster approach with smaller sample volume is required to overcome the problem. In

addition, a portable and simple measurement device could be developed as

promising alternative.

1.4 Hypothesis

A microfluidic device is expected able to miniaturize the conventional cuvette and

reduce the sample volume in bacteria absorbance measurement. Then, the

development of optical absorbance measurement device can miniaturize and simplify

the bulky and complicated measurement machine. The optical absorbance

measurement device with bacteria concentration information will result in effective

detection system which eliminates the time consuming bacteria colony counting and

7

sample dilution procedures. Integration of the microfluidic and optical absorbance

measurement device will finally produce a portable prototype for coliform bacteria

detection.

1.5 Objective

This research embarks the following objectives:

i) To develop suitable microfluidic design for low sample volume of coliform

bacteria suspension.

ii) To fabricate microfluidic device with rapid fabrication process based on the

combination of glass and PDMS materials.

iii) To develop a portable and miniaturize optical absorbance measurement device.

iv) To integrate the microfluidic device with the optical absorbance measurement

device for coliform bacteria detection.

1.6 Project Scope

The scopes of this project are as follow:

i) Simulation of microfluidic water flow velocity using COMSOL Multiphysics

software.

ii) Surface tension analysis of glass and PDMS using contact angle analyser and

Fox-Zisman Theory.

iii) Fabrication of a microfluidic device for sample volume less than 1.0 ml.

iv) Preparation of SU-8 microfluidic master mould with specific microchannel

design using photolithography technique.

v) Replication of microchannel design on PDMS material using replica moulding

technique (soft lithography).

8

vi) Bonding process of PDMS microchannel and glass using oxygen plasma

treatment.

vii) Coliform bacteria suspension sample preparation in broth medium.

viii) UV-visible spectrophotometer absorbance measurement on the coliform

bacteria sample inside the PDMS-glass based microfluidic device

ix) Construction of optical absorbance measurement device using electrical and

mechanical components.

x) Absorbance measurement using the developed prototype and UV-Visible

spectrophotometer.

xi) Absorbance reading comparison between UV-Visible spectrophotometer and

developed prototype for performance evaluation.

xii) Correlation of the coliform bacteria numbers in CFU/ml with the absorbance

reading from the develop prototype.

1.7 Research Contribution

This research study contributes in production of suitable microfluidic design for low

sample volume. The microfluidic device shows good performance which make it as

a good alternative for Quartz cuvette in optical measurement analysis. Then, this

research study also contributes in production of miniaturize and portable custom-

made detection system for coliform bacteria. The prototype integrates the

microfluidic and optical absorbance measurement device. The developed prototype

will be beneficial for water quality monitoring and coliform bacteria contamination

analysis.

2CHAPTER 2

LITERATURE REVIEW

2.1 Introduction

The literature review consists of historical and research development, theory

explanation and problem discussion. Based on the purpose of the project, important

information related to the microfluidic device and bacteria detection will be

discussed in detail based on their significant for the realization of the project.

2.2 Microfluidic Development

The concept of microfluidic involves with the handling and manipulation of fluids

that are constrained to a very small volume (micro-scale) [21]. The field of

microfluidic combines engineering, physics, chemistry, micro-technology and

biotechnology with knowledge about the behaviour of fluids. It has the potential to

revolutionize the processes and products that use fluids by introducing suitable

integration approach. The attractiveness of microfluidic based systems can be seen in

its size-effect. Smaller analysis platform of microfluidic requires low consumption of

reagent and power [22]. They are also able to perform separations and detections

with high resolution and sensitivity at lower cost and faster time. The development

of microfluidic revolves with history, flow properties, materials, and fabrication

techniques.

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2.2.1 Historical Perspective

Development of microfluidic was started in the early 1980s initiated from the

realization of a miniaturized gas chromatograph by Terry et al. group on a silicon

wafer in 1979 [23]. Figure 2.1 shows the design of gas chromatography that had

been developed by the group. The system consists of injection valve, separating

capillary column. Then, the development of micro-flow sensors, micro-pumps, and

micro-valves in the late 1980s dominated the early stage of microfluidic. However,

in the 1990s, the microfluidic field has been seriously and rapidly developed since

the introduction of microfluidic by Manz et al. [24]. Microfluidic research has

emerged over the past decade, and will continue to grow in the future.

Recently, the potential role of microfluidic in point-of-care diagnostics is

widely acknowledged, and many reviews have explored its potential applications in

clinical diagnostics, personalized medicine, global health, and forensics [25-27]. In

microfluidic field, many successful commercial implementations have been

demonstrated. In order to realize the commercialization of microfluidic device,

challenges in integrating low cost and rapid production must be addressed.

Furthermore, to demonstrate the practicality of the microfluidic system, an effort

should be made to investigate the ability of the system in handling raw samples and

comparison to conventional methods.

Figure 2.1: Gas chromatography design [23]

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2.2.2 Fluid and Flow Properties in Micro Scale

In microfluidic system, the understanding of the physical phenomena that dominate

at the micro-scale is important. At the micro-scale, the effects that become dominant

includes the fluid and flow characteristics. Fluid and flow characteristics are very

important in micro-scale system design and simulation. In fluid characteristics, there

are three important parameters including density (ρ), pressure (P), and viscosity (η).

The density of liquid can be defined as the mass (m) per unit volume (v) as shown in

equation (2.1) [28].

(2.1)

The pressure in liquid depends only upon the depth, the density, and the

acceleration of gravity. The pressure is the same at each instance that having same

elevation. It is not affected by the shape of the liquid container. In micro scale

situation, the pressure differences in planar microchannel can be overlooked [29].

However, microchannel systems involve with inlets and outlets which will transfer

any pressure difference to the liquid inducing the liquid to flow. Then, it has been

discovered that the internal friction of the liquid flow is viscosity [30]. The

coefficient of viscosity (η) can be defined as the ratio of the shear stress to the shear

rate as shown in equation (2.2) and equation (2.3). F represents the force, A

represents the area, V represents the velocity, and l represents the length. Equation

(2.3) shows that the shear stress of a fluid is directly proportional to the velocity

gradient which represents the Newtonian fluid. Examples of Newtonian fluid are

water, oil, and glycerine. In non-Newtonian fluid like blood, the viscosity changes

with the shear stress [30].

(2.2)

(2.3)

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Figure 2.2: Shear stress and shear rate for coefficient of viscosity of fluid flow

The viscosity in non-Newtonian fluid is influenced by the fluid temperature

which makes the cohesive forces as a dominant role. In this situation, the increase of

temperature leads to the decrease of liquids viscosity. Figure 2.2 shows the shear

stress and the shear rate phenomena which involves in viscosity coefficient concept

of a fluid flow. The fluid flow conditions in micro scale systems can be expressed in

Reynolds number. The Reynolds number equation in (2.4) determines the relation

between magnitudes of inertia and viscous forces. In equation (2.4), ρ is density, η is

viscosity, l is the length and V is the average velocity of the moving liquid. Reynolds

number is important to describe the flow regime of a fluid whether laminar or

turbulent. From past studies, it has been discovered that Reynolds numbers higher

than 2300 are corresponding to turbulent flow regime [31].

ρlV/ η (2.4)

Under turbulent flow regime, the inertial forces become dominant. Then, the

region for Reynolds numbers lower than 2000 corresponds to the laminar flow

regime. Figure 2.3 shows the laminar and turbulent flow behaviour. Under laminar

flow regime, low Reynolds number describes a strong viscous interaction between

the wall and the fluid, and there is no turbulences occur [32]. Low Reynolds numbers

are achieved at lower velocities, smaller dimensions, smaller densities, or higher

viscosities. Therefore, in microfluidic application, the laminar flow regime is

dominant due to the small dimensions of the channels.

13

Figure 2.3: Laminar and turbulent flow behaviour

The Navier-Stokes formalism describes the theoretical framework in

analysing the fluid flow [25]. Based on the fundamental laws of conservation, it can

be combined with the consecutive equations of fluids governing viscosity and

thermal conductivity which referred as Navier-Stokes equation. Hagen-Poiseuille

flow is a solution to the Navier-Stokes equations [33]. It can be applied when a

liquid is driven through a channel using the pressure gradient. Equation (2.5) shows

the expression for volume flow (Q) in a capillary with cylindrical and spherical

cross-section. ∆v is the volume different, t is the time, R is the radius of the

capillary, L is the capillary length, η is the viscosity, and ∆P is the pressure drop

across the length.

(2.5)

The transport mechanism is another important property to be considered for

fluid flow in micro scale. There are two types of transport mechanism in microfluidic

systems which are differentiated based on the driving agent behind the transport. The

first type of the transport mechanism is the direct transport. Direct transport is

involved with fluid flow that controlled by the exerting work on the fluid which by

mean will induce a volume flow with a specific direction [34]. Convection is one of

the important direct transports in microchannel. There are few external forces to

generate the convection transport in a microsystem including capillary flow, gravity,

pressure different, and centrifugal force.

14

Figure 2.4: Example of convection and diffusion behaviour inside microchannel [35]

The second type of the transport mechanism in microfluidic is the statistical

transport. Diffusion is a dominant statistical transport for microfluidic which usually

involves with dissolved species and mixture. Diffusion can be defined as a

distribution of molecules or particles resulted from concentration gradients [34]. The

movement of molecule in diffusion transport is random and can be described using

the Einstein-Smoluchowski relation as shown in equation (2.6). From the equation,

x is the average distance, t is the time, and D is the diffusion constant. Figure 2.4

shows an example of convection and diffusion transport in microchannel.

(2.6)

In microfluidic flow, diffusion usually takes more important role compared to

convection. This situation can be determined using the Péclet number. The Péclet

number is the ratio of the convection over diffusion transport as shown in equation

(2.7). From the equation, l is the length, and V is the flow velocity. In microsystem,

Péclet number larger than 1 indicates that the diffusion has minor influence and can

be neglected [36]. Then, Péclet number less than 1 indicates that the diffusion is

dominant and the convection can be neglected.

(2.7)

15

2.2.3 Materials and Fabrication Techniques for Microfluidic Device

Initiated from the pioneering development of microfluidics by S.C Terry (1979) and

Manz et al. (1990), a lot of research and work had been done involving various

materials and fabrication techniques. Initially, microfluidic devices that analyzed

aqueous solutions were developed by several research group including Manz group,

Harrison group, and Ramsey groups [24, 37, 38, 39].

Most of these early works involved photolithography and etching techniques

on glass and silicon. Figure 2.5 shows the proses flow of micromachining on silicon

wafer using photolithography and etching technique. Manz and Harrison proposed

the machining of a miniaturized capillary electrophoresis-based chemical analysis

system on a chip using glass material [37]. Figure 2.6 shows the etched glass

channels for the electrophoresis system. This attempt shows some promising result

of flow control on microstructure. They suggest that further effort with silicon

structures is required for producing better system. Based on this preliminary finding,

Manz, Harrison, and Ramsey groups had done various studies in fabricating

microfluidic devices using silicon and glass [37, 39]. They also suggested various

designs to improve the system including manipulation of channel size, geometry, and

the accessibility of required components (injection, separation, or detection).

Figure 2.5: Photolithography and etching technique on silicon wafer

16

Figure 2.6: Capillary channels etched into Corning 7740 glass with 10 µm depth [37]

From these early studies, some problems about the suitability of the materials

and fabrication techniques were revealed. Silicon is however relatively expensive

and opaque, making it unsuitable for systems that use optical detection. Glass is

transparent, but it is amorphous which make the vertical side walls more difficult to

etch compare to silicon. Additionally, glass and silicon micromachining processes

are expensive, technically demanding, and time consuming. Based on these

problems, there has been rapid growth into many types of materials, especially

polymers. Polymers are inexpensive compared to silicon and glass. Channels can be

formed by moulding or embossing rather than etching. The device also can be sealed

thermally or by using adhesives. The development of polymer microfluidics was

mostly affected by the suitability of different type of polymer and the performance of

different fabrication techniques.

Most of the early attempt of polymer microfluidic fabrications was began

with thermoplastic polymers especially polymethylmethacrylate (PMMA),

polycarbonate (PC), and Cyclic Olefin Copolymer (COC). McCormick group

proposed injection moulding techniques for fabricating microchannel on PMMA

[40]. However, because of the complexity and high initial cost of the moulding

equipment and masters, injection moulding is rarely used for rapid prototyping.

Alternatively, hot embossing technique was introduced which was shown to be fast

and less expensive than injection moulding [41]. Figure 2.7 shows the concept of the

hot embossing technique for microchannel fabrication on plastic material. Many

common polymers had successfully hot embossed including PMMA and COC [42,

43].

17

Figure 2.7: Hot embossing technique for microchannel fabrication

Hot embossing technique is less expensive and possible for mass production.

However, it requires complicated equipment, robust mould, and lack of suitable

methods for good material bonding. Recent studies demonstrated simplified

equipment like the study done by Roy et al. and Young. They had demonstrated

improvement for the fabrication process using less expensive solid epoxy moulds

and convenient thermal bonding procedure [44, 45].

Then, in the same era, the laser ablation technique on thermoplastic polymers

including PMMA and PC was realized [46]. Figure 2.8 shows the illustration of the

laser ablation process for engraving microchannel on the plastic material. Laser

ablation technique for microfluidic fabrication was started by Roberts et al. This

technique is cost-accessible and able to produce complex 3D-multilayer structures

[47]. Some research had shown that this technique offers limited throughput and

fabricated channels showed greater surface roughness than hot embossed, or

injection moulded techniques. Recently, the works done by Huang et al. and Suriano

et al. had successfully showed reduced roughness on the sidewalls of the

microchannel [48, 49].

Figure 2.8: Laser ablation technique for microchannel fabrication

Mould

18

Even though there are many improvements in the fabrication technique,

thermoplastic polymers were observed to have severe drawbacks which limit the

range of microfluidic applications. Both PMMA and PC have opaque characteristic

which make them unsuitable for systems that require optical approach. The COC

material is unsuitable for biomedical application because it can lead to absorption of

specific compounds from biological fluids and causes clogging. Most of the

thermoplastic polymers also suffer from incompatibility with solvents used for the

biological assay and the surface chemistries are not well defined for surface

modifications.

Besides thermoplastic polymers, researcher also discovered the potential of

PDMS for microfluidic device [50, 51]. The fabrication of PDMS microfluidic was

started with the introduction of simpler soft lithography technique. Figure 2.9

describes the soft lithography process for PDMS microchannel fabrication. Soft

lithography technique on PDMS material was started by Duffy et al. [50]. Due to

this approach, PDMS has become a successful polymeric substrate material for rapid

prototyping. The advantages of PDMS for microfluidic technology can be seen in the

study done by Mac Donald et al. [51]. They had discovered that PDMS is

inexpensive, requires simple fabrication procedure, non-toxic, and able to form

multi-level microfluidic device. The study also described that PDMS material can be

sealed reversibly or irreversibly to many types of material.

Figure 2.9: Soft lithography technique for PDMS microchannel fabrication

Master mould

19

From past research works, it has been discovered that PDMS suffers from

some drawbacks including poor biocompatibility with few organic solvents. PDMS

also has unstable surface modification over time, able to absorb small molecules into

its matrix, and can deform under pressure. Several research in improving PDMS

microfluidic device was done including reducing molecule absorption and swelling

with silica particle and by sol-gel surface coating [52, 53]. Then, improvement was

performed to the PDMS microfluidic by controlling the deformation of the

microchannel [54].

Recently, a lot of researches are focusing in identifying polymers

complementary to PDMS, with similar fabrication procedures but, with higher

rigidity and better resistance to solvents. Fiorini et al. had explored the Thermoset

Polyester (TPE) [55, 56, 57]. Due to its higher rigidity, TPE is suggested as the

material of choice for application which has particularly high pressure or for

situations that demands fast flow-stabilization. The main limitation of TPE is its

unknown biocompatibility. Then, Kuo et al. had proposed Polyurethane

Methacrylate (PUMA) as a promising biocompatible material especially for micro-

devices in clinical situations [58, 59]. Bartolo et al. used Norland Adhesive 81

(NOA81) which proposed similar advantages as PUMA [60]. The limitation of

PUMA and NOA is weaker sealing strength compare to TPE but still higher than

PDMS. The channel deformation is also still visible with PUMA when pressures

increase. Researches on TPE, PUMA, and NOA show that they are promising

alternatives to PDMS for rapid prototyping that involve high pressure or

geometrically sensitive applications.

Based on the discussion of the materials and fabrication techniques for

microfluidic device, it described that each of them has their own suitability with

certain microfluidic applications and researcher should be wise in determining the

suitable materials for the microfluidic devices. More promising material and

fabrication technique should be investigated in order to produce better microfluidic

device in the future. The summarization of the reviews on microfluidic material is

shown in the Table 2.1.

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Table 2.1: Advantages and disadvantages of materials for microfluidic device

Material Advantages Disadvantages

Silicon - Good for small feature resolution

- Good surface stability

- Good solvent compatibility

- Relatively expensive and

opaque (not suitable for

optical detection)

Glass - Excellent resistance to high

pressures

- Transparent

- Good optical properties

- Amorphous and more

difficult to etch

Polymer - Cheaper and robust material

- Faster and less expensive

fabrication processes

- Some of them are also transparent

- Incompatibility with several

solvent (depending on the

application)

2.3 Bacteria Detection

This project combined the microfluidic with bacteria detection concept. Infectious

bacteria are categorized as considerable threat to human health. Effective bacteria

detection is an important requirement for prevention and treatment of infectious

diseases. Over the past years, much advancement and technology had been

investigated and applied for improving the detection and analysis of bacteria.

2.3.1 Conventional Bacteria Detection Methods

The conventional methods for bacteria detection are including the plate culture

method, immunoassay based method, and PCR. All these methods rely on specific

microbiological and biochemical identification.

Plate culture is the traditional and most reliable method for bacteria detection

[61]. The popularity of plate culture method was started by Robert Koch in the 19th

century. This method is relatively simple and found to be the standard

microbiological techniques to detect bacteria. Bacteria detection and quantification

21

using plate culture method involves with dilution and bacteria counting. In plate

culture, it is assumed that a single bacterial colony arises from a single cell. With

suitable dilution range, the individual colony can be counted and it can be used to

establish the bacteria concentration from original sample. From history, numerous

bacteria detection had been achieved using the plate culture method. Aycicek et al.

had successfully detected the salmonella, coliform and E. coli bacteria contamination

using the plate culture method [62]. From the traditional plate culture method,

researchers are still improving this method for better accuracy and possible detection

of more types of bacteria. For example, recently Deng et al. had improved the plate

culture method to detect the lactic acid bacteria [63]. From this research, the growth

and colony size of the bacteria was enhanced. Eventually, there are some drawbacks

of the traditional plate culture method including labour intensive, time consuming,

and utilizes significant amount of materials.

Immunoassay based method is a popular conventional method for

quantitative detection of bacteria. Immunoassay method is based on the specific

recognition of specific target by an antibody [64]. One of the most fundamental

immunoassay methods is the ELISA. ELISA technique can be classified into several

types including direct, competitive, and sandwich concepts. From review, the

immunology-based method has been widely used for bacteria detection. Several

researchers had successfully performed the detection of E. coli bacteria using the

immunological method [65-67]. Then, this method was also being used to detect the

salmonella bacteria [68, 69]. Even though immunological detection method had been

found to be faster and more sensitive than plate cell culture, this method is expensive

and more complicated.

Another conventional method for bacteria detection is the PCR technique.

This technique was introduced for almost 20 years ago. PCR method can detect

single bacteria based on single copy of target deoxyribonucleic acid (DNA)

sequence. This promising method detects the bacteria by amplifying target DNA

which led to more specific and sensitive detection. PCR can amplify the target DNA

to 1-million-fold in less than an hour [70]. PCR based detection method had been

used for wide range of bacteria. Choi and Lee, and Choi et al. used this method for

detection of salmonella bacteria [71, 72]. Then, Perry et al. had successfully detected

the Bacillus cereus bacteria using PCR method [7]. However, PCR method depends

22

on expensive and complicated measurement device which requires skilled workers to

carry out the analysis.

2.3.2 Bacteria Detection Technologies

In this project, it is important to suggest the most suitable detection technology that

can be integrated with the microfluidic device. For the past few years, much

advancement has been made for molecular diagnostics of infectious microorganisms.

It has been discovered that wide range of technology approaches can be used to

detect the harmful bacteria based on electrical, optical, chemical, and biological

signals [74]. Based on past works, most bacteria detection technologies are revolving

around the electro-chemical, optical, and piezoelectric properties.

Electrochemical based detection is one of the possible technologies for

detection and quantification of bacteria in microfluidic. Electrochemical detection

technique can be categorized into four methods; amperometric, potentiometric,

impedimetric, and conductometric that are respectively refer to the current, potential,

impedance, and conductance parameters. Amperometric, conductometric, and

potentiometric techniques are the most popular electrochemical technologies for

bacteria detection because these techniques have excellence sensitivity. A lot of

researchers have reported successful bacteria detection using amperometric

technique. They had successfully used amperometric biosensor technique to detect E.

coli and salmonella bacteria [75, 76]. Then, conductometric technique on the other

hand is combination of conductance and bio-recognition method. Muhammad Tahir

and Alocilja group had successfully produced a conductometric-based detector for E.

coli and salmonella bacteria as shown in Figure 2.10 [77].

Figure 2.10: Conductometric immunosensor construction [77]

23

Figure 2.10 shows the schematic diagram of the conductometric

immunosensor device developed by Muhammad Tahir and Alocilja. This device

showed a specific, sensitive, and low volume detection platform. In potentiometric

technique, the bio-recognition process is converted into a potential signal. Based on

the potentiometric detection concept, the electrical potential difference between two

electrodes at near zero current will be detected using the high impedance voltmeter.

Ercole et al. demonstrated the detection of E. coli bacteria using the potentiometric

technique on the Light-addressable potentiometric sensor (LAPS) [78]. From this

project, the potentiometric detection system was reported to be very sensitive and

fast. For the past few years, combination of impedance measurement and bio-

recognition had initiated the development of impedance biosensor for

electrochemical-based bacteria detection. The research done by Yang et al. had

demonstrated an impedance sensor using interdigitated microelectrode (IME) for

detection of salmonella bacteria [79]. Figure 2.11 shows the experimental setup of

the impedance analyzer with IME. The measurement was carried out for detection of

salmonella in milk sample. This research had shown a promising result which able to

identify impedance change for bacterial growth.

Figure 2.11: Impedimetric detection system with impedance analyzer and IME [79]

Optical detection is the most widely employed technique for bacteria

detection due to their sensitivity and selectivity. This method has diverse

measurement platforms including absorption, reflection, refraction,

chemiluminescence, fluorescence, and Raman spectroscopy. Among of all these

diversity, absorption and fluorescence platforms are the most popular for optical

24

detection approach. Absorption spectroscopy is the simplest optical detection

method. Ultraviolet (UV) and visible absorption spectroscopy is a well-established

technique in macro-scale and micro-scale diagnostics. From past research works,

changes in optical density are sufficient for diagnosis and analysis. Li et al. used

optical absorption approach on the microfluidics-based bio-sensing method. In this

work, they had successfully performed the detection of E. coli bacteria [80].

However, it has been discovered that optical absorption detection in microfluidic

devices suffers from poor detection limits. This is due to the short effective optical

path length in microchannel [81]. In the past few years, researchers had contributed

their efforts to overcome the short path length problem. For example, Ro et al. had

introduced the collimation lenses to block stray light at both input and output fiber

channels in microfluidic [82]. The collimated system is shown by Cell C of PDMS

microchip layout in Figure 2.12. From this study, the optical path length in

microchannel had been improved.

Besides absorption, fluorescence is another popular detection technique used

for microfluidic. This is due to its high selectivity and this technique is easy to

integrate with microfluidic system. Fluorescence techniques are usually applied with

microfluidic immunoassays, electrophoresis process, and polymerase chain reaction

(PCR). Xiang et al. produced a microfluidic immunoassay device with fluorescence

detection for E. coli bacteria [83].

Figure 2.12: Detection cells (Cell A, B, and C) in PDMS microchip [82]