DNA OLIGONUCLEOTIDE SYNTHESIS IN A MICRODEVICE FOR ... · dna oligonucleotide synthesis in a...

DNA OLIGONUCLEOTIDE SYNTHESIS IN A MICRODEVICE FOR

MULTIPLE ANALYTICAL APPLICATIONS

WANG CHEN

(B. Eng, Zhejiang University)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DIVISION OF BIOENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2010

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Acknowledgements

First of all, I would like to express my sincere gratitude to my supervisor, Dr. Dieter Trau

for his guidance and support over the course of my Ph.D. study. He has taught me how to

think critically and conduct research independently. His financial support during the last

year of my study helped me pass the most difficult time.

I would like to thank all my lab mates in Nanobioanalystics Lab. Especially, Dr. Mak

Wing Cheung and Ms. Cheung Kwan Yee have taught me a lot of research techniques at

the early stage of my study and gave me a lot of valuable suggestion through the course

of my study. Mr. Bai Jianhao helped me revise my manuscript and suggest me several

good experiments. Ms. Lee Yee Wei helped me purchase everything used in my study.

I would also like to thank Dr. Partha Roy for providing me the photolithography facilities

and A/P Zhang Yong for allowing me use the oxygen plasma machine. I also thank Dr.

Shakil Rehman for his guide on optical detection.

I would also like to thank National University of Singapore for providing me Research

Scholarship for the first four years of my study.

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I would also like to thank Ministry of Defence of Singapore. This project is fully

supported by research grant provided by Ministry of Defence. I was also financially

supported by the research grant in the last year of my study. Without this support, this

research cannot be accomplished.

Last but not the least; I would like to thank my family who has always been there for me.

I appreciate the continuous love and encouragement from my parents. Above all, I thank

my wife Wu Liqun for her patience, understanding and unending love. During my most

difficult period, she encouraged me to continue and not give up. She also gave me

unconditional support for all decision I made. Without her accompanying me, this study

could not be completed.

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Table of Contents

ACKNOWLEDGEMENTS ················································································································ I

TABLE OF CONTENTS ················································································································· III

SUMMARY ········································································································································ VI

LIST OF TABLES ························································································································· VIII

LIST OF FIGURES ·························································································································· IX

ABBREVIATIONS ·························································································································· XII

CHAPTER 1 INTRODUCTION ······································································································· 1

1.1 Background ············································································································· 2

1.1.1 DNA oligonucleotide ························································································ 2

1.1.2 Microfluidic device ··························································································· 4

1.1.3 Combine oligonucleotide synthesis with microfluidic technologies ················· 5

1.2 Scope and specific aims of study ············································································· 5

CHAPTER 2 LITERATURE REVIEW··························································································· 7

2.1 DNA oligonucleotide synthesis ··············································································· 8

2.1.1 Synthesis chemistry ·························································································· 8

2.1.1.1 Phosphodiester approach ········································································· 10

2.1.1.2 Phosphotriester approach ········································································· 10

2.1.1.3 H-phosphonate approach ········································································· 11

2.1.1.4 Phosphoramidite approach ······································································· 12

2.1.2 Solid phase synthesis ······················································································ 13

2.1.2.1 Solid supports for oligonucleotide synthesis ············································ 15

2.1.2.2 Oligonucleotide synthesizer ····································································· 17

2.1.2.3 In situ oligonucleotide synthesis ······························································ 18

2.2 Microfluidic valve ································································································· 20

2.2.1 Passive and active microvalves ······································································· 20

2.2.2 Pneumatically actuated microvalves ······························································· 22

2.2.2.1 Pneumatic diaphragm microvalve ···························································· 23

2.2.2.2 Pneumatic in-line microvalve ·································································· 26

2.3 Oligonucleotide synthesis in a microfluidic device ················································ 28

2.3.1 Microfluidic device reactor ············································································· 28

2.3.2 Microfluidic oligonucleotide synthesizer ························································ 29

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CHAPTER 3 ZERO DEAD-VOLUME MICROVALVE ···························································· 31

3.1 Introduction ··········································································································· 32

3.2 Microvalves design and fabrication ······································································· 35

3.2.1 Zero dead-volume microvalve design ····························································· 35

3.2.2 Microvalve working principles ······································································· 37

3.2.3 Microvalve fabrication ···················································································· 37

3.2.3.1 Master fabrication and PDMS micromolding ·········································· 37

3.2.3.2 PDMS membrane fabrication ··································································· 38

3.2.3.3 Microvalve fabrication ············································································· 40

3.3 Result and discussion····························································································· 43

3.3.1 Microvalve characterization ············································································ 43

3.3.1.1 Microvalve operation ··············································································· 43

3.3.1.2 Leakage pressure ····················································································· 46

3.3.2 Zero dead-volume ··························································································· 47

3.3.3 Minimal cross contamination tested by PCR ·················································· 49

3.4 Conclusion ············································································································· 53

CHAPTER 4 OLIGONUCLEOTIDE SYNTHESIS IN A PORTABLE SYNTHESIZER ···· 54

4.1 Introduction ··········································································································· 55

4.2 Portable oligonucleotide synthesizer ····································································· 57

4.2.1 Software ········································································································· 57

4.2.2 Hardware ········································································································ 59

4.2.3 System integration ·························································································· 60

4.2.4 Microvalve assignment ··················································································· 62

4.3 Materials and methods ··························································································· 62

4.3.1 Chemicals ······································································································· 62

4.3.2 Synthesis reactor ····························································································· 63

4.3.2.1 Reactor for primer synthesis on CPG ······················································· 63

4.3.2.2 Reactor for probe synthesis on glass slide ················································ 64

4.3.3 Surface modification of microscope glass slides ············································· 65

4.3.4 Oligonucleotide synthesis protocol ································································· 66

4.3.5 PAGE and PCR ······························································································· 67

4.3.6 Probe deprotection and hybridization ····························································· 68


4.4.1 PCR primer synthesis ····················································································· 69

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4.4.2 Probe synthesis ······························································································· 72

4.4.2.1 Probe for oligonucleotide hybridization ··················································· 72

4.4.2.2 Probe for single-base mismatch differentiation ········································ 73

4.5 Conclusion ············································································································· 75

CHAPTER 5 PORTABLE GENERIC DNA HYBRIDIZATION BIOASSAY SYSTEM ······ 76

5.1 Introduction ··········································································································· 77

5.2 System design ········································································································ 78

5.2.1 Texas Red fluorescence detection system ······················································· 78

5.2.2 Integration of fluorescence detection and oligonucleotide synthesis system ··· 79

5.2.2.1 Modified microfluidic chip ······································································ 80

5.2.2.2 Reactor for oligonucleotide synthesis and hybridization ·························· 81

5.2.2.3 Portable DNA bioassay system ································································ 83

5.3 Materials and methods ··························································································· 85

5.3.1 Hybridization probe immobilization on glass slide ········································· 85

5.3.2 Asymmetric PCR ···························································································· 85

5.3.3 DNA hybridization ························································································· 86


5.4.1 Fluorescence detection system ········································································ 86

5.4.1.1 System characterization ··········································································· 86

5.4.1.2 Detection of complementary and single-base mismatch DNA hybridization ····························································································································· 88

5.4.2 Bacterial differentiation ·················································································· 90

5.5 Conclusion ············································································································· 93

CHAPTER 6 CONCLUSION ·········································································································· 94

6.1 Conclusion and original contributions ··································································· 95

6.2 Technical challenges ······························································································ 98

6.3 Future works ·········································································································· 99

BIBLIOGRAPHY ···························································································································· 101

APPENDIX A LIST OF COMPONENTS FOR THE PORTABLE SYSTEM ······················ 114

APPENDIX B ASSIGNMENT OF NI 9476 ················································································· 116

APPENDIX C SPECTRUM OF OPTICAL PARTS FOR FLUORESCENCE DETECTION ····························································································································································· 117

APPENDIX D LIST OF PUBLICATIONS ·················································································· 119

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Summary

DNA Oligonucleotide, a short piece of DNA, is one of the most commonly used materials

in biomolecular applications. Nowadays, most oligonucleotides are synthesized in

commercialized oligonucleotide synthesizers. Due to the complexity of the synthesis

process, these synthesizers are bulky in size. Recently, there is an emerging need of on-

site applications of oligonucleotides, such as in civil defense to immediately detect

biological attacks. However, due to the size limitation of the available oligonucleotide

synthesizers, only pre-made oligonucleotides with certain sequence can be brought to the

field. It is practically impossible to get oligonucleotide of any sequence on demand in the

field. In this thesis, a solution is offered by proposing and building a portable

oligonucleotide synthesizer based on microfluidic technology.

Firstly, a microfluidic chip with integrated microvalves is developed. The microvalves

have zero dead-volume characteristic that is attributed to the design of zigzag shaped

main channel and special position of each microvalve. This design removes the

connecting channels between the microvalve and the main channel that are usually found

in traditional designs. Therefore, reagents cannot be trapped within the microfluidic

device and cross contamination can be effectively minimized. The zero dead-volume

characteristic is proven by detection of trace amount of DNA template molecules trapped

in the device. This contamination free characteristic is extremely important for

applications, such as DNA oligonucleotide synthesis, in which cross contamination is

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critical issue and can lead to failure of the synthesis.

Secondly, a portable oligonucleotide synthesizer based on the developed zero dead-

volume microchip is built. The portable synthesizer has the ability to synthesize

oligonucleotide as either primer or probe. The sequence and biological functionality of

the synthesized oligonucleotides are proven by polyacrylamide gel electrophoresis, PCR

and DNA hybridization experiment. The portable synthesizer has the ability to synthesize

oligonucleotide of any sequence on demand. To the best of our knowledge, this is the first

reported portable oligonucleotide synthesizer.

To further improve the function of the portable system and realize generic DNA bioassay

within it, a DNA hybridization and fluorescence detection unit is presented and integrated

into the portable synthesizer. The fully integrated system, a portable generic DNA

hybridization bioassay system, is successfully used to distinguish different bacteria

strains based on in situ DNA oligonucleotide synthesis, hybridization and fluorescence

detection. As far as we know, this is also the first portable generic DNA bioassay system

reported. As the system has the potential to detect DNA target of any sequence in the field,

we envisage that the system could help to enable fast responses to emerging bio-threats

for homeland security and in pandemics.

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List of Tables

Table 2-1 Reaction scheme of different oligonucleotide synthesis approaches, their inherent problems and their contributions to the development of oligonucleotide synthesis chemistry ............................................................................................................................. 9

Table 2-2 Categorization of active microvalves ............................................................... 22

Table 4-1 Reagents and reaction time for oligonucleotide synthesis ................................ 67

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List of Figures

Figure 2-1 Chemical structures of deoxyribonucleotide phosphoramidites. The protecting groups are labeled in different colors: Red color: acid-labile DMT group to protect 5’-OH. Blue color: 2-cyanoethyl group to protect phosphite. Green color: isobutyryl or benzoyl group to protect exocyclic amino group. .......................................................................... 13

Figure 2-2 Scheme of solid phase oligonucleotide synthesis. Four reactions are involved in one synthesis cycle: detritylation, coupling, oxidation and capping. ........................... 15

Figure 2-3 Schematic illustrating the working principle of the pneumatically actuated diaphragm microvalve. (a) The microvalve is closed when positive pressure is applied through the pneumatic port. (b) The microvalve is opened when negative pressure is applied, causing the formation of a connecting channel between the thin membrane and the valve seat. .................................................................................................................... 24

Figure 2-4 Schematic illustrating the working principle of pneumatically actuated inline microvalve. (a) Microvalve is open at rest. (b) Microvalve is closed when positive pressure is applied through the pneumatic channel. The thin fluidic layer is pushed down to close the valve. .............................................................................................................. 27

Figure 3-1 Traditional design of multiple valves integrated microfluidics (a) All inlets are distributed along the main channel. (b) All inlets merge into the main channel at one point. ........................................................................................................................................... 33

Figure 3-2 Virtual valve designed by Braschler and co-workers. The process of switching from reagent B to reagent A. No cross contamination from reagent B is observed after fully switching to reagent A. ............................................................................................. 34

Figure 3-3 Design of the microvalves integrated microfluidic device with zero dead-volume characteristic. The main channel is zigzag shaped. The microvalves are positioned at each turning point of the main channel. ...................................................... 36

Figure 3-4 Thickness of PDMS membranes vs. spin speed. 2 ml PDMS was applied on the silicon wafer. ............................................................................................................... 40

Figure 3-5 Schematic diagram depicting the fabrication process of the microvalve (a) PDMS fluidic layer fabricated by micromolding and fabricated PDMS thin membrane. (b) Permanent bonding of fluidic layer and thin membrane by treating them with oxygen plasma. The valve seat is covered to avoid bonding with the thin membrane. (c) Peal off the fluidic layer-thin membrane complex after baking and then permanent bonding with pneumatic layer (fabricated also by micromolding) by treating with oxygen plasma. (d) The device is ready to use after baking for overnight. ...................................................... 42

Figure 3-6 Fabricated microvalves integrated microfluidic chip ...................................... 43

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Figure 3-7 Photograph of the microvalve. (a) The microvalve is closed by external positive pressure through pneumatic channel. (b) The microvalve is opened by external negative pressure through pneumatic channel. ................................................................. 45

Figure 3-8 Relationship between valve closing pressure and leakage pressure of the fabricated microvalve ........................................................................................................ 47

Figure 3-9 Photograph of the microvalve showing the zero dead-volume characteristic; two colored liquids (green and red) are used for illustration. (a) The microvalve that controls green liquid was opened. (b) The microvalve that controls green liquid was closed and another microvalve (located at upstream of the main channel) that controls red liquid was opened. No contamination of the red liquid from the green liquid was observed. ........................................................................................................................................... 49

Figure 3-10 Trace amount of DNA template molecules detection by PCR and gel electrophoresis. Lane 1: 100 bp DNA ladder; Lane 2-5: positive control with PCR starting from 1000, 100, 10 and 0 template molecules; Lane 6-8: PCR with effluent after washing for 10 s, 20 s and 30 s. (a) Our microvalve design (b) Traditional microvalve design ..... 52

Figure 4-1 Screen shots of the software for controlling oligonucleotide synthesis. The software was programmed under LabVIEW. (a) The information input interface (b) The synthesis monitoring interface. ......................................................................................... 58

Figure 4-2 System design and photograph of portable oligonucleotide synthesizer (a) Schematic diagram illustrating the complete system, including electrical and electronic parts, fluidic parts and pneumatic parts. Only four microvalves are shown on the diagram for simplicity. (b) Photograph of the portable oligonucleotide synthesizer: (1) PC (2) Argon tank (3) USB-DAQ (4) Solenoid valves and manifold (5) Pressure regulators (6) Vacuum pump (7) Batteries (8) Microfluidic chip (9) Synthesis reactor (10) Reagents. . 61

Figure 4-3 Assignment of microvalves to oligonucleotide synthesis reagents (1) Washing (2) Argon (3) Activator (4) A (5) T (6) G (7) C (8) Detritylation (9) Oxidation. .............. 62

Figure 4-4 Reactor for oligonucleotide primer synthesis. (1) PDMS cube (2) Glass fiber filter (3)(4) FEP tubing (5) CPG. ...................................................................................... 64

Figure 4-5 Reactor for oligonucleotide probe synthesis. (1) Top acrylic cover with tubing access holes (2) PDMS microchannel (3) Surface modified microscope glass slide (4) Bottom acrylic case. .......................................................................................................... 65

Figure 4-6 Scheme of surface modification of microscope glass slide for synthesis of oligonucleotide probe. ....................................................................................................... 66

Figure 4-7 PAGE image for synthesized oligonucleotide length confirmation. Lane 1: oligonucleotide ladder. Lane 2: HPLC purified commercial oligonucleotide with the same sequence. Lane 3: oligonucleotide synthesized by the portable oligonucleotide synthesizer. ........................................................................................................................................... 70

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Figure 4-8 The gel electrophoresis image of PCR product. Lane 1: 50bp DNA ladder. Lane 2: positive control with both forward and reverse primer purchased. Lane 3: PCR product amplified by the synthesized oligonucleotide in our portable synthesizer as reverse primer. ................................................................................................................... 71

Figure 4-9 Fluorescence image of 6-FAM labeled complementary DNA hybridized to synthesized probe .............................................................................................................. 72

Figure 4-10 Fluorescence image of the synthesized probe hybridizing with Texas Red labeled oligonucleotide. (a) Complementary sequence. (b) Single-base mismatch. ........ 74

Figure 5-1 (a) Schematic diagram of the Texas Red fluorescence detection system (b) Photograph of the Texas Red fluorescence detection system. .......................................... 80

Figure 5-2 Microfluidic chip for oligonucleotide synthesis and DNA hybridization. (a) Microvalve assignment (1) Washing (2) Argon (3) Activator (4) A (5) T (6) G (7) C (8) Detritylation (9) Oxidation (10) Hybridization. (b) Fabricated microfluidic chip with 10 microvalves integrated. ..................................................................................................... 81

Figure 5-3 Design of oligonucleotide probe synthesis and hybridization reactor. (1) Top acrylic cover with tubing access hole (2) PDMS microchannel (3) PT100 temperature sensor (4) Surface modified glass slide (5) Bottom acrylic case (6) Heating unit with light access hole. The whole assembly was held together by screw and then fixed inside the fluorescence detection box. ............................................................................................... 83

Figure 5-4 Photograph of the portable system: (1) PC with control software (2) Argon tank (3) NI-DAQ (4) Solenoid valves and manifold (5) Pressure regulators (6) Vacuum pump (7) Batteries (8) Microfluidic chip (9) Synthesis and hybridization reactor (10) LED (11) Photodiode (12) Lenses, filters and beam splitter (13) Injection port for hybridization related reagents. .......................................................................................... 84

Figure 5-5 Calibration curve for Texas Red fluorescence detection system. The lower range of the calibration curve (corresponding to amount of Texas Red fluorophore from 0.5 fmol to 12.5 fmol) is enlarged. .................................................................................... 88

Figure 5-6 Fluorescence image of hybridization to immobilized probes for test of fluorescence detection system. (a) Complementary hybridization (b) Single-base mismatch hybridization. .................................................................................................... 90

Figure 5-7 3D bar graph showing the fluorescence signal from specific and cross hybridization between PCR products of two different bacteria strains and synthesized strain specific probes. ........................................................................................................ 92

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Abbreviations

PCR polymerase chain reaction

PDMS poly(dimethylsiloxane)

DMT dimethoxytrityl

CPG controlled-pore glass

PGA photo-generated acid

PFPEs perfluoropolyethers

FEP fluorinated ethylene propylene

MOSFET metal oxide semiconductor field-effect transistor

PFOTCS 1H,1H,2H,2H-perfluorooctyl-trichlorosilane

IPA isopropyl alcohol

ddH2O double distilled water

dA 5'-dimethoxytrityl-N-benzoyl-2'-deoxyadenosine,3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite

dC 5'-Dimethoxytrityl-N-benzoyl-2'-deoxycytidine,3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite

dG 5'-Dimethoxytrityl-N-isobutyryl-2'-deoxyguanosine,3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite

dT 5'-Dimethoxytrityl-2'-deoxythymidine,3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite

PAGE polyacrylamide gel electrophoresis

SSC saline-sodium citrate

6-FAM 6-carboxyfluorescein

xiii

TxRd Texas Red

LED light emitting diode

NI National Instruments

DAQ data acquisition

APTES 3-Aminopropyltriethoxysilane

DSS disuccinimidyl suberate

DMF dimethylformamide

1

CHAPTER 1

INTRODUCTION

Chapter 1 Introduction

2

1.1 Background

A DNA oligonucleotide is a single stranded, short fragment of DNA. The length of DNA

oligonucleotide usually ranges from a few bases to 50 bases. However, oligonucleotides

with length around 20 bases are more widely used in biomolecular applications and

studies. Applications for DNA oligonucleotides are widely found in biological studies

such as polymerase chain reaction (PCR) which uses DNA oligonucleotide as a primer to

initiate the PCR reaction [1], DNA microarray which uses DNA oligonucleotide as a

probe for DNA hybridization [2], or gene synthesis which uses DNA oligonucleotide as

the basic building blocks to form the target gene [3]. To obtain oligonucleotide with

desired sequence, chemical synthesis of oligonucleotide by polymerizing individual

nucleotide precursors is commonly used.

1.1.1 DNA oligonucleotide

It has been more than 50 years since the first dimer oligonucleotide was chemically

synthesized [4]. Since then, the research on oligonucleotide synthesis chemistry has been

extensively conducted due to numerous applications for DNA oligonucleotides.

Nowadays, the phosphoramidite method has become the ‘golden standard’ approach for

oligonucleotide synthesis [5]. The essence of this method is polymerizing nucleoside

phosphoramidite one by one according to the required sequence. Several different

chemical reactions, which form a synthesis cycle, take place sequentially. One nucleoside


3

phosphoramidite is added onto the growing chain in one cycle. The synthesis of an

oligonucleotide with specific length and sequence requires the repetition of this synthesis

cycle for a number of times. In Chapter 2, various oligonucleotide synthesis chemistries

and recent improvement will be reviewed.

Nowadays, the oligonucleotide synthesis process has been fully automated. Most

oligonucleotides are synthesized in commercialized oligonucleotide synthesizers [6, 7].

However, due to the complexity of the synthesis process, these synthesizers are bulky in

size. Recently, there is an emerging need of “in the field applications” for

oligonucleotides, such as in civil defense to immediately detect biological attacks.

However, due to the size limitation of the available oligonucleotide synthesizers, only

pre-synthesized oligonucleotides with certain sequence can be brought to the field. It is

practically impossible to get any sequence on demand in the field. This is because, for

example, for a 20-mer oligonucleotide, there are totally 420 (=1.1× 1012) possible

sequences, the cost for synthesizing and storing all the possible sequences is tremendous

and increases exponentially with the increase in the length of the oligonucleotide.

Therefore, only selected oligonucleotides can be prepared and brought in the field. As a

consequence, no immediate detection and response to artificially manipulated or naturally

mutated agents harmful to human in the field is possible. Thus, it is absolutely necessary

to develop an oligonucleotide synthesizer in a portable format in order to obtain

oligonucleotide with any required sequence in the field.


4

1.1.2 Microfluidic device

Microfluidic device or Lab-on-a-chip has gained a lot of attention and applications in the

past two decades. Its broad applications in biological and chemical studies such as DNA

analysis [8], cell separation [9], cell culture [10], and material synthesis [11] make it a

dynamic research area. The decrease of fluidic devices in dimension to micrometer scale

leads to the dramatic reduction of the volume to microliter or nanoliter. The small volume

of microfluidic devices reduces the consumption of chemicals and production of waste,

which is environment friendly and cost saving. Furthermore, many reactions can be

carried out simultaneously on a single chip, which increases the throughput [12]. The use

of silicon-based polymers in biological studies such as poly(dimethylsiloxane) (PDMS)

also makes the fabrication process of microfluidic device much easier and cheaper

compared to the use of glass or silicon.

Complicated systems based on microfluidic technologies have been reported [8, 12]. In

these microfluidic systems, often microvalves are an important fundamental element,

which implements the fluids control and manipulation in such devices. By integrating a

number of valves in one microfluidic chip, multiple liquid reagents can be controlled in

sequential or parallel manner as needed. Therefore, applications which require adding a

number of different reagents in a certain sequence can be accomplished within

microfluidic system. In chapter 2, various microvalves based on different actuation

strategies will be reviewed, with more details on pneumatically controlled microvalve.


5

1.1.3 Combine oligonucleotide synthesis with microfluidic technologies

As described, DNA oligonucleotide synthesis is such an application in which a number of

reagents are involved. These reagents need to be added into the reactor chamber in a

sequential manner for a series of reactions to take place. As microfluidic device has the

ability to control and manipulate a number of fluids with the aid of microvalve

technologies, combination of microfluidic technologies and DNA oligonucleotide

synthesis chemistry should provide an easy and efficient way to synthesize DNA

oligonucleotide. The microfluidic system can be then packed into a portable format to

form a portable oligonucleotide synthesizer, which can be used for in the field

applications.

1.2 Scope and specific aims of study

As the project is funded by Ministry of Defence of Singapore (MOD), the overall aim of

the research is to develop a portable DNA oligonucleotide synthesizer for MOD for their

in the field applications. The portable synthesizer should have the ability to synthesize

DNA oligonucleotide of any sequence on demand and the synthesized oligonucleotide

can be used as either primer or probe. Furthermore, the function of the portable system

can be extended to conduct DNA hybridization based bioassay. These aims will be

achieved by accomplishing the following four objectives:


6

1. Develop a microvalves integrated microfluidic device which can control the flow of a

number of reagents, the microfluidic device should minimize cross contamination

among reagents within the device;

2. Integrate the microfluidic device with the electronic and pneumatic controlling units,

and pack them into a portable format to form the portable oligonucleotide synthesizer;

3. Synthesize both DNA oligonucleotide primers and probes within the portable

synthesizer;

4. Design a hybridization and fluorescence detection unit and integrate it into the

portable oligonucleotide synthesizer to conduct generic DNA hybridization based

bioassay.

7

CHAPTER 2

LITERATURE REVIEW

Chapter 2 Literature Review

8

2.1 DNA oligonucleotide synthesis

DNA oligonucleotides are one of the most widely used materials in biomolecular

applications and studies. Till now, almost all oligonucleotides used in those applications

are chemically synthesized. However, researchers spent more than 25 years to find the

satisfactory method to synthesize oligonucleotide. As the development of the computer

technology and process automation, oligonucleotide synthesis has been fully automated

and is carried out in commercialized oligonucleotide synthesizers. In this section,

different oligonucleotide synthesis approaches will be reviewed first, followed by recent

advances in solid phase synthesis and oligonucleotide synthesizer design.

2.1.1 Synthesis chemistry

It has been more than 50 years since the first synthesis of a dithymidine dinucleotide was

reported by Michelson and Todd in 1955 [4]. Since then, extensive research has been

conducted on the chemistry of oligonucleotide synthesis due to numerous applications of

DNA oligonucleotides [2, 13-15]. Different approaches have been invented, including

phosphodiester approach, phosphotriester approach, H-phosphonate approach and

phosphoramidite approach. Among these methods, phosphoramidite approach has

become the standard method and has been dominantly used in the past 20 years. However,

it is necessary to understand all these methods and their respective contribution to the

development of the oligonucleotide synthesis chemistry and inherent problems. Table 2-1


9

shows a brief summary of all these approaches and they will be reviewed in more details

in the following.

Table 2-1 Reaction scheme of different oligonucleotide synthesis approaches, their

inherent problems and their contributions to the development of oligonucleotide synthesis

chemistry

Approach Coupling Reaction Schemes Problems Contributions

Phosphodiester O

OO

OR

B1P

DMTrO

O

O

B2

O-

+

DMTrO

O

OH

B2

O

O-

O-

O

PO

OR

B2

Branched product, tedious purification

Base and 5’-OH protection, elucidation of genetic code

PhosphotriesterO

OO

O

B1P

DMTrO

O

O

B2

RO

Support

+

DMTrO

O

O

B2

RO O-

O

POH

O

O

B2

Support

Low coupling yield

Phosphate protecting group, initiate solid phase synthesis

H-phosphonate O

OO

O

B1P

DMTrO

O

O

B2

H

Support

+

DMTrO

O

O

B2

H O-

O

POH

O

O

B2

Support

Side reaction, incomplete oxidation

Easy way to get oligonucleotide analogues

PhosphoramiditeO

O

O

B1P

DMTrO

O

O

B2

RO

Support

+

OHO

O

B2

Support

P N

DMTrO

O

O

B2

RO

Water sensitive

Standard approach, automated process,synthesis of long oligonucleotide


10

2.1.1.1 Phosphodiester approach

In the second half of 1950s, Khorana and his co-workers developed the phosphodiester

approach [16-18]. In the phosphodiester approach, phosphomonoester and the formed

internucleotide phosphodiester are not protected during the coupling reaction, leading to

the problem that oligonucleotides could branch at the internucleotide phosphate.

Therefore, tedious purification is required. Due to these reasons, the phosphodiester

approach was not in use since the 1970s. However, the use of dimethoxytrityl (DMT) as 5’

hydroxyl protecting group [19] and benzoyl and isobutyryl as base protecting group [20]

are very important improvement. These protecting groups are still used in modern

oligonucleotide synthesis (see phosphoramidite approach below). Another contribution

that Khorana has made is the elucidation of genetic code by using this approach to

synthesize short oligomers [21, 22].

2.1.1.2 Phosphotriester approach

In the 1960s, phosphotriester approach was reinvestigated after first reported in 1955 by

Michelson and Todd [4]. Its key difference from the phosphodiester approach is that the

internucleotide phosphate is protected. Therefore, oligonucleotides could not branch at

the internucleotide phosphate. A few protecting groups have been used for protecting the

internucleotide phosphate, including 2-cyanoethyl group [23, 24]. Nowadays this group is

still used in phosphoramidite approach (see below). The problem with this method is that


11

the coupling efficiency is relatively low, which therefore limits its use in synthesizing

only short oligonucleotide [5]. However, the phosphotriester approach was the first

method used in solid phase synthesis of oligonucleotide [23]. Thereafter, massive

research in this area was initiated and automatic synthesis of oligonucleotide is made

possible due to the success of solid phase synthesis. Nowadays, most of the synthetic

oligonucleotides, if not all, are synthesized by the solid phase approach.

2.1.1.3 H-phosphonate approach

Although first reported by Hall and co-workers in the 1950s [25], this method gained

more attention thirty years later since it was applied to solid phase synthesis of

oligonucleotide [26, 27]. In H-phosphonate approach, only one oxidation reaction is

carried out to convert all the phosphonate diesters to the desired phosphodiesters at the

end of the synthesis (different from phosphoramidite method that oxidation is carried out

in each cycle, see below). The advantage of this method is that only two steps are

involved in one synthesis cycle and oligonucleotide analogues can be easily obtained

during the final oxidation by using different nucleophiles other than water [28, 29].

However, undesired side reaction during coupling and incomplete oxidation in the final

step may lower the final yield of the synthesis. Therefore it is generally accepted that

phosphoramidite approach is superior to H-phosphonate approach in terms of synthesis

efficiency.


12

2.1.1.4 Phosphoramidite approach

In the 1970s, phosphite triester approach, a revolutionary development in oligonucleotide

synthesis chemistry, was reported [30]. This method was further developed to

phosphoramidite approach a few years later by the introduction of nucleoside

phosphoramidites by Beaucage and Caruthers in 1981 [31]. In this approach, the

phosphite triester linkage [P(III)] is formed first during the coupling reaction and then

oxidized to the desired phosphate triester linkage [P(V)]. This is because that P(III) is

much more reactive than P(V) and therefore more efficient in the formation of

internucleoside linkage [30]. Nowadays, the phosphoramidite approach has become the

‘standard’ method and is dominantly used for oligonucleotide synthesis due to its high

coupling yield (98%) [32]. Almost all automated synthesizers are specially designed for

using phosphoramidite approach. Figure 2-1 shows the chemical structure of

deoxyribonucleotide phosphoramidites which are the basic building blocks used in

phosphoramidite approach. The protecting groups for protection of various functional

groups during oligonucleotide synthesis are labeled in different colors.


13

2.1.2 Solid phase synthesis

Since first reported by Letsinger and Mahadevan in 1965 [23], enormous research was

carried out on synthesizing oligonucleotide on solid support. In the past 20 years, almost

all commercial oligonucleotides were synthesized in solid phase. In solid phase synthesis

O CH3

OCH3

CN

NH

N

N

NH

O

O

P

O

O

O N

O

N

N-2-isobutryl-deoxyguanosine

phosphoramidite (G)

CH3NH

N

O

O

O

O

O

O CH3

OCH3

PO N

N

Deoxythymidine

phosphoramidite (T)

O

C

N

N

N

N

NH

O

O

O

O CH3

OCH3

PO N

N

N-6-benzoyl-deoxyadenosine

phosphoramidite (A)

N

N

NH

O

O

C

O

O

O

O CH3

OCH3

PO N

N

N-4-benzoyl-deoxycytidine

phosphoramidite (C)

Figure 2-1 Chemical structures of deoxyribonucleotide phosphoramidites. The

protecting groups are labeled in different colors: Red color: acid-labile DMT group to

protect 5’-OH. Blue color: 2-cyanoethyl group to protect phosphite. Green color:

isobutyryl or benzoyl group to protect exocyclic amino group.


14

of oligonucleotides, one nucleotide is added onto a support-bonded oligonucleotide at a

time till the desired sequence is obtained. Phosphoramidite approach is the most widely

used method for solid phase synthesis of oligonucleotide. In solid phase synthesis by

phosphoramidite approach, a number of reactions forming a synthesis cycle are required

to extend one nucleotide. These reactions include detritylation, coupling, oxidation, and

capping, Excess reagents are usually added in each reaction to achieve higher synthesis

yield and then washed off from the solid support after the reaction is completed [5]. A

point worth noting is that the synthesis direction is from 3’ end to 5’ end, which is

different from the direction of chain extension in natural DNA replication or PCR (5’ end

to 3’ end). The solid phase synthesis technique is a rapid and efficient approach as excess

reagents can be easily removed and thus no tedious and time-consuming purification is

needed after each step. A practical importance of the success of solid phase synthesis is

that it makes the automation of oligonucleotide synthesis become possible. Figure 2-2

shows the reaction scheme of solid phase synthesis of oligonucleotide by using the

phosphoramidite approach.


15

2.1.2.1 Solid supports for oligonucleotide synthesis

Various materials have been used as the solid support materials in oligonucleotide

synthesis, such as silica gel, controlled-pore glass (CPG) and polystyrene [33-35]. Among

these materials, CPG [34, 36] is still the most widely used support material nowadays.

The pore size of CPG ranges from below 10 nm to several hundred nm. The nucleoside

Figure 2-2 Scheme of solid phase oligonucleotide synthesis. Four reactions are involved

in one synthesis cycle: detritylation, coupling, oxidation and capping.

P

DMTrO

O

O

B1

1. Detritylation

P

OOH

O

B1

NNH

N N

P

OO

O

B1

P

DMTrO

O

O

B2

O

N

P

Abz

Cbz

Gib

or T=

P = Solid Support, e.g., CPG

Start

Reaction Cycle

B1,B2,Bn,Bn+1

P N

DMTrO

O

O

B2

O

N

N

O

OO

O

B1

P

DMTrO

O

O

B2

O

P

O

OO

O

B1

2. Coupling

3. Capping

4. Oxidation

OO

OH

B1

O

P

OO

O

Bn

O-

O

P

OOH

O

O-

n

Bn+1

Cleavage and deprotection


16

loading decreases with increasing pore size, as the surface area on the CPG decreases

with increasing pore size. However, longer oligonucleotide can be synthesized with larger

pore size due to retaining of high coupling yield when growing strand became longer [37].

The commercialized CPG usually has the first nucleotide attached on the support (see

Figure 2-2 ‘start’). The linkage between CPG and the first base is cleavable [38, 39].

Upon completion of the synthesis, the linkage is cleaved and the oligonucleotide is

released from the CPG. The synthesized oligonucleotide bears 3’ end hydroxyl group,

which is important for oligonucleotide primer to initiate PCR. There are four basic CPG

(A, T, G, or C) available to start the synthesis and one must choose the CPG with the

correct first base according to the 3’ end base of the required sequence (because synthesis

direction was from 3’ end to 5’ end). This undoubtedly causes trouble and increases

possibility of using the wrong CPG. Later on, universal linker chemistry was reported by

various researchers [40-42]. The CPG with universal linker (universal CPG) does not

have the first nucleotide attached on it. Therefore, the same solid support can be used

regardless of the sequence to be synthesized, which is more convenient and reduces

human error possibility. The universal CPG has the similar efficiency for oligonucleotide

synthesis and no modification of synthesis chemistry is needed for using them on

commercial oligonucleotide synthesizer.


17

2.1.2.2 Oligonucleotide synthesizer

Oligonucleotide synthesis has been fully automated through the development of

oligonucleotide synthesizers. In solid phase synthesis of oligonucleotides, a number of

reagents are added and removed from the solid support in a sequential manner. Therefore,

a valve system which controls the flow of different reagents is the most important part of

an oligonucleotide synthesizer. A column with solid support packed within it is used as

the synthesis reactor. However, the number of oligonucleotide that can be synthesized in

parallel is limited.

Later on, due to the high demand for oligonucleotides in genome-wide studies [43, 44],

high throughput oligonucleotide synthesizers were developed. In the high throughput

synthesizer, 96- and 384-well microplates were both successfully used as the synthesis

reactor [7, 45]. A system consisting of four 384-well microplates has also been developed

to further increase the throughput and has the ability to produce 1536 oligonucleotide at

the same time [7]. However, for such highly parallel synthesis, well-to-well quality

variation of the synthesized oligonucleotides remains a concern. Due to the high

productivity and complexity of system design, all of these synthesizers are bulky in size.

Currently, there are some research conducted on applying microfluidic technology on

oligonucleotide synthesis, this will be reviewed later.


18

2.1.2.3 In situ oligonucleotide synthesis

In situ oligonucleotide synthesis emerged through the development of DNA microarray

technology. In situ oligonucleotide synthesis has the following advantages. Firstly,

oligonucleotide synthesized on the substrate can be directly used for DNA hybridization;

therefore, no immobilization of pre-synthesized oligonucleotide is required. Secondly, a

large number of different sequences can be synthesized simultaneously at high surface

density.

Several techniques have been used in in situ synthesis of oligonucleotides, including ink-

jet printing [46, 47], micro-contact printing [48], and photolithography [49, 50]. Ink-jet

printing technology has been successfully applied in fabricating microarray [46].

Phosphoramidites were delivered to dedicated location on a glass surface by the ink-jet

printing process. An array of 23,965 oligonucleotides representing human genes was

obtained on an area of 25 × 75 mm2 with 100 µm in diameter per spot. However, the

coupling efficiency was estimated to be 94% to 98%, slightly lower than that of

conventional oligonucleotide synthesis.

Xiao and co-workers used PDMS micro-contact printing technology to synthesize

oligonucleotide microarrays [48]. The synthesized oligonucleotide spots had a feature

size of 90 µm in diameter and 2500 probes were produced in an area of 1 cm2. As many

as 168 different microstamps were made for fabricating the 20-mer oligonucleotide array.


19

Extra attention must be paid on alignment of the microstamps and the substrate during the

contact printing process to guarantee the quality of the synthesized oligonucleotides. The

solvent compatibility of PDMS with oligonucleotide synthesis reagent was not mentioned

in this study.

In photolithography approach, the 3’ end hydroxyl is deprotected by light instead of acid

as in the phosphoramidite approach. Therefore, a photo-labile protecting group replaced

the traditional acid-labile DMT group. Photomasks [49] or micromirror array [50] were

used to selectively produce the 3’ end hydroxyl at specific location. The synthesized

oligonucleotide spot had a feature size smaller than 25 µm in diameter. The

commercialized Affymetrix® DNA microarray chip is fabricated by this approach.

However, the limitation of this method is that the synthesis efficiency is lower than that

of traditional phosphoramidite chemistry, excluding its use in synthesizing longer

sequences.

To solve the lower synthesis efficiency problem, Gao and co-workers invented the

photogenerated acid (PGA) approach [51, 52]. In this method, standard phosphoramidite

chemistry, i.e., the acid-labile DMT protecting group, is used. The acid to remove the

DMT group is generated by illumination of an acid precursor at selected reaction

chambers. The generated acid then selectively removes the DMT group at specific

chambers. The synthesis must be carried out in a microfluidic device with thousands of


20

micro chambers integrated instead of on flat substrate surface. When removing the DMT

group, acid diffusion among different reaction chambers must be prevented, otherwise,

the reaction will occur at wrong reaction chambers, leading to undesired oligonucleotide

sequences.

2.2 Microfluidic valve

With the development and aid of microfabrication and micromachining technologies,

microfluidic systems found applications in biological studies and material synthesis [10,

53, 54]. The microfluidic valve is an important component in microfluidic devices. With

microvalves integrated into the microfluidic system, it is possible to manipulate and

control fluid flow within microfluidic devices. In the following sections, the microfluidic

valve technology will be reviewed and the focus is on two types of pneumatically

actuated microvalves, which are widely used in microfluidic biological systems.

2.2.1 Passive and active microvalves

Microvalves can be categorized into two main classes: passive valves and active valves.

Passive valves, or check valves, only open to forward pressure [55-59]. This kind of

valves has diode-like characteristics. The advantage of passive valves is that their design

is relatively simple. However, its main disadvantage is that a leakage flow exists even

under low pressure. This disadvantage excluded the use of such valves in applications


21

where leakage or cross contamination between reagents is a critical issue and must be

avoided.

Unlike passive valves, active valves can be operated by external means. Therefore, the

valves behavior is not dependent on direction the fluid flow. It is generally accepted that

active microvalves can be categorized into three subgroups according to their actuation

means [60]: mechanical active microvalves, non-mechanical active microvalves and

external active microvalves.

For mechanical active microvalves, the movable membranes are coupled to mechanical

moving parts which can be actuated by different methods such as magnetic, electric,

piezoelectric or thermal means. In contrast, for non-mechanical microvalves, the movable

membranes are actuated by means other than mechanical, e.g., electrochemical reaction

or phase-change materials. For external active microvalves, they are usually actuated by

external systems such as pneumatic pressure (more details in section 2.2.2). A brief list of

the categorization of active microvalves and their respective operation time are

summarized in Table 2-2.


22

Table 2-2 Categorization of active microvalves

Category Actuation Method

Mechanism Valving Time References

Mechanical

Magnetic Magnetic force 10 ms [61-64]

Electric Electrostatic force ---- [65, 66]

Piezo-electric

Piezoelectric effect < 2 ms [67, 68]

Thermal

Thermal bimetallic actuation or volumetric thermal expansion (thermopneumatic)

bimetallic 0.2 s thermopneumatic > 5 s

bimetallic [69, 70], thermo-pneumatic [71, 72]

Non-mechanical

Electro-chemical

Electrolysis to generate O2 pressure

> 1 min [73, 74]

Phase change

Phase change of hydrogel, sol-gel, or paraffin due to temperature, pH, or light

> 2 s Hydrogel [75-77], Sol-gel[78], paraffin [79, 80]

External Pneumatic External pneumatic pressure

< 5 ms [81-85]

2.2.2 Pneumatically actuated microvalves

Pneumatically actuated microvalves are suitable for biological and chemical applications.

This is due to the reasons that pneumatically actuated microvalves have the advantage of


23

fast actuation, ease of large-scale integration, no leakage flow and ease of fabrication.

Two types of pneumatically actuated microvalves were previously reported [81, 85], they

are the pneumatic diaphragm microvalve and the pneumatic in-line microvalve.

2.2.2.1 Pneumatic diaphragm microvalve

The pneumatically actuated diaphragm microvalve was first reported by Vieider and co-

workers in 1995 [85]. This microvalve has a three-layer structure. In this design, a

deflectable thin membrane is sandwiched in between a fluidic layer and a pneumatic layer.

The microfluidic channel (on fluidic layer) is interrupted by a valve seat. The working

principle of this type of microvalve is shown in Figure 2-3. The thin membrane is pressed

against the valve seat if a positive pressure (relative to atmospheric pressure) is applied

on the membrane through the pneumatic channel. Under positive pressure, the valve is

closed and liquid is stopped behind the valve seat. On the other hand, if a negative

pressure is applied, the membrane is deflected away from the valve seat and a connecting

channel is formed in between. Under negative pressure, the valve is opened and liquid

can flow over the valve seat.


24

Figure 2-3 Schematic illustrating the working principle of the pneumatically actuated

diaphragm microvalve. (a) The microvalve is closed when positive pressure is applied

through the pneumatic port. (b) The microvalve is opened when negative pressure is

applied, causing the formation of a connecting channel between the thin membrane and

the valve seat.

The material of the deflectable thin membrane is a key issue in the design of such

microvalve. As the membrane is the only moving part in the microvalve, it should be

flexible, durable and readily sealable against other materials (glass, silicon or other

polymers which are used for fabricating the fluidic and pneumatic layer). The materials

that have been used as the membrane includes silicone membrane [86], 3M tape sheet [87]

and PDMS membrane [88, 89]. The PDMS membrane has been a good choice due to its


25

low Young’s modulus [90], excellent sealing property [91] and good biocompatibility

[92]. However, a disadvantage is the incompatibility of PDMS with some organic

solvents [93]. Therefore, other materials including perfluoropolyethers (PFPEs) [94] and

commercially-available fluorinated ethylene propylene (FEP) have been introduced as

microvalve membrane materials [83, 84].

The pneumatically actuated diaphragm microvalves have been successfully used in many

applications. A microfluidic gradient generation and perfusion system was reported [95].

Microvalves were used to control 16 different inlets. A cell culture chamber can be fed by

81 possible combinations of reagents and switching among different combination can be

done within seconds.

Li and co-workers presented a micro-mixer with a fabricated pneumatic diaphragm

microvalve array [96]. Chambers of defined volumes were isolated by the microvalve

array. The microdevice allowed for storing and precisely mixing aqueous solutions at

different mixing ratios. The precision of mixing was confirmed by quantitative analysis

of the mixing between fluorescein and deionized water. The device has potential

applications in miniaturized diagnostic assays as well as in cell-based assays such as drug

screening and enzyme-based biomolecule detection.

The working principle of the microvalve also shows perfect analogy to metal oxide


26

semiconductor field-effect transistor (MOSFET). The membrane serves as the gate that is

controlled by external pneumatic pressure, and the inlet and outlet work as the source and

drain [97]. Microfluidic integrated circuits with a pressure gain of 32.0 dB [86] and

microfluidic digital logic circuit with computing capability were demonstrated [98]. An

advantage of microfluidic circuits is that they are immune to electromagnetic interference.

A micro pump formed by serial connection of three diaphragm microvalves was also

reported by Grover and co-workers [82]. The pumping rate of the fabricated micro pump

ranging from 1 nl/s to over 100 nl/s was achieved. A microfluidic system for amino acid

analysis, with parts-per-trillion limit of detection, was demonstrated by integration of a

micro pump into the system [99].

2.2.2.2 Pneumatic in-line microvalve

The pneumatic in-line microvalve also has a three-layer structure: pneumatic layer,

fluidic layer and bottom substrate layer. The fluidic layer is cast thin and no valve seat is

present in the microfluidic channel, different from the pneumatic diaphragm microvalve

[81]. The working principle of the in-line microvalve is shown in Figure 2-4. The valve is

opened at rest (normally open). When positive pressure is applied through the control line,

the thin fluidic layer is pushed down to the bottom substrate and the valve is closed. To

avoid any leakage flow when the valve is closed, the shape of the fluidic channel must be


27

round instead of rectangular. Otherwise, the valve cannot be fully closed near the side

walls of the rectangular fluidic channel. PDMS is still the commonly used material for

fabricating such microvalves.

Figure 2-4 Schematic illustrating the working principle of pneumatically actuated inline

microvalve. (a) Microvalve is open at rest. (b) Microvalve is closed when positive

pressure is applied through the pneumatic channel. The thin fluidic layer is pushed down

to close the valve.

Large-scale integration of pneumatic in-line microvalves have been successfully

demonstrated by Quake’s group [100]. An application of this large-scale integration is in

PCR [101]. In this work, totally 2860 microvalves were integrated on a microfluidic chip.

400 PCR reactions were carried out simultaneously within isolated 3 nl compartments


28

formed by the microvalves. The work presented is pretty useful in biochemical

experiments in which precious reagents are involved.

Marcus and co-workers reported a microdevice for single cell mRNA isolation and

analysis [8]. This system integrated the function of cell isolation, cell lysis, mRNA

purification, cDNA synthesis and cDNA purification. The device was scaled up to

process 50 different samples (50-plex) in parallel.

2.3 Oligonucleotide synthesis in a microfluidic device

Applying microfluidic technologies in oligonucleotide synthesis has been reported by

only a few researchers. In some studies, the microfluidic device offered micro reactors for

highly parallel synthesis [52, 102-104]. In other studies, a complete microfluidic

synthesizer was fabricated [105, 106].

2.3.1 Microfluidic device reactor

Oligonucleotide synthesis has been carried out in a microfluidic reactor. Hua and co-

workers developed a chemical-resistant microvalve array by coating parylene on PDMS

[102]. The microdevice was connected to a commercial oligonucleotide synthesizer and

the microvalve array was used to distribute the oligonucleotide synthesis reagents to


29

different reaction channels for synthesizing oligonucleotide probes. However, no

characterization of the synthesized oligonucleotide was presented in this work.

Zhou and co-workers designed a microfluidic device with around 3600 reaction chambers

of picoliter volume [52, 103]. Oligonucleotides with different sequence were synthesized

in individual chambers by the PGA approach. The synthesized oligonucleotide was

successfully used for distinguishing single-base mismatch hybridization and assembly of

gene fragment with 712 and 714 bp in length.

Moorcroft and co-workers used PDMS as the solid support and directly synthesized an

oligonucleotide within the PDMS microchannel [104]. Although PDMS has the weakness

of incompatibility with certain organic solvents, this work proved that with substitution of

certain organic solvents used in oligonucleotide synthesis, PDMS can still be used as a

substrate material for oligonucleotide synthesis and potentially for fabricating DNA

microarray.

2.3.2 Microfluidic oligonucleotide synthesizer

Oligonucleotide synthesis in microfluidic reactors was usually done by connecting a

microfluidic device directly to a commercial oligonucleotide synthesizer. Therefore, there

is no fluidic control within the microfluidic device. The first real microfluidic


30

oligonucleotide synthesizer was reported by Huang and co-workers [105]. In this work,

in-line microvalves were fabricated in PFPE solvent-resistant polymer to control the flow

of synthesis reagents. A 20-mer oligonucleotide was obtained in the device and its length

was confirmed by gel electrophoresis.

Extended research was conducted later by Lee and co-workers to simultaneously

synthesize 16 different oligonucleotides in the microfluidic synthesizer [106]. The

synthesized oligonucleotides were used for assembly of a gene fragment from Bacillus

cereus of 230 bp in length without further purification. The reported microfluidic

oligonucleotides synthesizer is a great progress in the development of oligonucleotide

synthesis instruments.

31

CHAPTER 3

ZERO DEAD-VOLUME MICROVALVE

Chapter 3 Zero Dead-volume Microvalve

32

3.1 Introduction

Microvalves integrated microfluidic devices have been used in applications where

sequential switching of a number of reagents is needed [105, 107, 108]. In some

applications, it is desirable to minimize intermixing and cross contamination among

reagents as the result can be badly affected by the purity of the reagents. In traditional

designs of microfluidic device with multiple inlets and integrated microvalves, all inlets

are distributed along the main channel or merge into the main channel at the beginning

(Figure 3-1). Connecting channels are usually placed in between the microvalves and the

main channel. During operation, small volumes of reagents can be trapped within the

connecting channels and lead to contamination of the reagent flow in the mainstream

channel. For many applications this contamination problem is negligible. However, for

some applications such as DNA synthesis, PCR and other analytical applications [109,

110], it is absolutely necessary to minimize intermixing and cross contamination among

reagents.


33

Figure 3-1 Traditional design of multiple valves integrated microfluidics (a) All inlets are

distributed along the main channel. (b) All inlets merge into the main channel at one point.

A few studies have been conducted to solve the cross contamination problem [95, 111,

112]. Braschler and co-workers have designed a virtual valve by incorporating a bypass

channel to each inlet channel side by side [111] (as shown in Figure 3-2). When a reagent

is selected (by increasing the corresponding inlet pressure), it flows towards the main

channel. At the same time, the higher inlet pressure causes reagents from other inlets to

be diverted to its bypass channel. In other words, the selected reagent washes all the other

reagents out into their respective bypass channels. Therefore, cross contamination is

avoided. However, due to the presence of bypass channels and absence of real valves,

reagents keep flowing and are wasted all the time, which is not cost effective.

(a) (b)


34

Figure 3-2 Virtual valve designed by Braschler and co-workers. The process of switching

from reagent B to reagent A. No cross contamination from reagent B is observed after

fully switching to reagent A.

Researchers from other groups tried to solve the cross contamination problem by adding

adjacent purging channels to each inlet [95, 111, 112]. In such designs, the reagents left in

the system can be washed out by the washing reagent from their corresponding purging

channels. However, this design increases the complexity of the system and more space is

required for placing the purging channels.

In this chapter, we offer a solution for the cross contamination problem encountered in

microfluidics. A pneumatic microvalves integrated microfluidic system with zero dead-

volume between each valving site and the main channel for minimal cross contamination


35

of reagents is demonstrated. The main channel is zigzag shaped and the microvalves sit

exactly at each turning point of the main channel, hence creating a zero dead-volume

between each valving site and the main channel. This zero dead-volume allows any

reagent entering the main channel to be thoroughly washed out after use and not to

remain entrapped within the microfluidic device. Therefore contamination of the reagent

used in the current step to subsequently added reagents in the next step is minimized. To

prove the zero dead-volume characteristic, PCR is used to detect trace amount of DNA

template molecules left in the device after washing.

3.2 Microvalves design and fabrication

3.2.1 Zero dead-volume microvalve design

The design of the zero dead-volume is shown in Figure 3-3. (NB: The term “zero dead-

volume” in this work refers to the volume between each valving site and the main

channel is zero and hence no reagents can be trapped. However, there is always leftover

reagent in the main channel and washing is always needed to remove these reagents from

the main channel.) All microvalves are positioned at the turning points of a zigzag shaped

main channel; except the outermost left valve which is used to control the main channel

flow itself. The novel characteristic of the design is that the connecting channel between

the main channel and each valve site, which was commonly used in previous studies [105,

107, 108], is removed in this design. The distance between each valve site and the main


36

channel is visibly reduced to zero, hence creating a zero dead-volume between each valve

site and the main channel. This zero dead-volume allows any reagent entering the main

channel to be thoroughly washed out after use and not to remain entrapped within the

microfluidic device. The zero dead-volume can effectively minimize cross contamination

among reagents as no previous reagents can be trapped in the device. All branch channels

are aligned with their downstream direction of the main channel to aid reagents to flow

towards the outlet. In our design a total number of 9 valves are integrated; however, more

valves could be integrated if necessary.

Figure 3-3 Design of the microvalves integrated microfluidic device with zero dead-


37

volume characteristic. The main channel is zigzag shaped. The microvalves are

positioned at each turning point of the main channel.

3.2.2 Microvalve working principles

The working principle of the valve is shown in Figure 2-3. When positive pressure is

applied through the pneumatic port, the membrane is pressed against the valve seat and

the valve is closed. Liquid flow stops behind the valve seat (Figure 2-3(a)). On the other

hand, when negative pressure is applied, the thin flexible membrane is lifted away from

the valve seat and a connecting channel is formed between the valve seat and the

membrane. The valve is opened and the liquid can flow over the valve seat (Figure

2-3(b)).

3.2.3 Microvalve fabrication

3.2.3.1 Master fabrication and PDMS micromolding

The microvalves integrated microfluidic chip was fully fabricated in PDMS (Sylgard 184

Silicone Elastomer kit, Dow Corning, US). The chip has a fluidic-membrane-pneumatic

three-layer structure. The fluidic layer and pneumatic layer were both fabricated by

PDMS micromolding and the membrane was made by spin-coating PDMS on silicon

wafer.


38

The masters for PDMS micromolding were fabricated by photolithography. SU-8 2050

negative photoresist (Microchem, US) was applied on silicon wafers by spin-coating (~50

µm thickness), followed by a two-step soft bake at 65 C for 3 minutes and 95 C for 6

minutes. The resist coated wafer was then exposed to UV light at 365 nm through a

transparency film mask, followed by a post exposure bake at 65 C for 1 minute and 95

C for 5 minutes. The exposed photoresist was finally developed in SU-8 developer for 6

minutes.

To micromold PDMS, the fabricated master was first exposed to 1H,1H,2H,2H-

perfluorooctyl-trichlorosilane (PFOTCS) vapor under vacuum at room temperature for 15

minutes. Fully mixed PDMS base and curing agent (10:1 ratio) was poured onto the

master and degassed under vacuum for 30 minutes. The PDMS was then baked at 65 C

in an oven for at least 2 hours. After baking, the PDMS microchip was peeled off from

the master, washed with isopropyl alcohol (IPA) and double distilled water (ddH2O),

blown dry with nitrogen and ready to use.

3.2.3.2 PDMS membrane fabrication

To fabricate the PDMS membrane, the PDMS was spin-coated on a silicon wafer. Firstly,

2 ml of fully mixed PDMS was directly poured on the center of a 4-inch PFOTCS coated

silicon wafer. Then the wafer was spun at 2000 rpm for 30 seconds and followed by


39

baking for 1 hour at 65 C on a hot plate. The PDMS membrane was then washed with

IPA, ddH2O, blown dry with nitrogen and ready to use. The fabrication process of the

PDMS membrane should be completed in a clean room environment as dust can affect

the functionality of the fabricated PDMS membrane.

The thickness of the fabricated PDMS membrane was measured by using a microscope

(Olympus BX41). A strip of the membrane was cut out and removed from the silicon

wafer. The cutting edge of the membrane that still stuck on the wafer was observed under

the microscope. First, the top surface of the membrane was focused and the reading of the

micrometer on the fine focus knob was taken. After that, the surface of the silicon wafer

was focused by adjusting the fine focus knob and the reading was taken again. By

calculating the difference of the two readings, the thickness of the PDMS membrane was

obtained.

Figure 3-4 shows the relationship between the thickness of the fabricated PDMS

membrane and the spinning speed. As the spinning speed increases, the thickness of the

PDMS membrane decreases. It is also worth to note that as the spinning speed increases,

the rate of thickness decreasing becomes smaller. An empirical equation for the

relationship between polymer thickness (t) and spinning speed ( ), solution

concentration (C) and molecular weight (η) was given before:


40

where K is an overall calibration constant. α, β and γ are exponential factors [113].

In this study, the PDMS membrane was spun at 2000 rpm and the corresponding

thickness is 34.5±0.8 µm.

Figure 3-4 Thickness of PDMS membranes vs. spin speed. 2 ml PDMS was applied on

the silicon wafer.

3.2.3.3 Microvalve fabrication

The fabrication process of PDMS microvalves is shown in Figure 3-5. This process

involves two steps of oxygen plasma treatment. First, the fluidic layer and the PDMS

20

30

40

50

60

70

80

1000 1500 2000 2500 3000

Mem

bran

e T

hick

ness

(μm

)

Spin Speed (rpm)


41

membrane were treated by oxygen plasma (March Instrument Incorporated, radio

frequency generator power 200 W, plasma treatment period 30 s, oxygen pressure 450

mTorr) and then bonded together for 2 hours at 65 ºC. After baking, the PDMS fluidic

layer and the thin PDMS membrane were bonded permanently. As the PDMS membrane

was the moving part of the microvalve, it should not bond with the fluidic layer at the

valve seat regions. Therefore, during oxygen plasma treatment, the valve seat regions on

the fluidic layer must be covered from exposure to plasma. The valve seat was covered

by using an oil-based permanent marker pen (Zebra, Japan) to directly mark on the target

areas. Only oil-based marker pen instead of water-based one can be used here. This is

because PDMS is very hydrophobic [114], as a consequence, water-based mark does not

stick to the raw PDMS surface well and droplets can be easily formed.

The bonded two-layer PDMS assembly was then peeled off from the flat silicon wafer.

Next, the PDMS pneumatic layer and the other surface of the thin PDMS membrane were

bonded together by treating with oxygen plasma again. Alignment of the valve seats with

the pneumatic controlling channels was done under a stereo microscope. Finally the

three-layer PDMS sandwich was baked at 65 ºC overnight. To remove the valve seat

protective marking, IPA was used to flush through the microvalves following by washing

with ddH2O. After drying in the oven, the microchip was ready to use.


42

Figure 3-5 Schematic diagram depicting the fabrication process of the microvalve (a)

PDMS fluidic layer fabricated by micromolding and fabricated PDMS thin membrane. (b)

Permanent bonding of fluidic layer and thin membrane by treating them with oxygen

plasma. The valve seat is covered to avoid bonding with the thin membrane. (c) Peal off

the fluidic layer-thin membrane complex after baking and then permanent bonding with

pneumatic layer (fabricated also by micromolding) by treating with oxygen plasma. (d)

The device is ready to use after baking for overnight.

valve seat marked with

oil-based marker

The fabrica

fluidic chan

however, th

Figure 3-6

3.3 Result

3.3.1 Micro

3.3.1.1 Mic

Figure 3-7

conditions.

ated microv

nnel was fi

he size of th

Fabricated m

and discus

ovalve char

crovalve ope

shows tw

When a po

M

valves integ

lled with g

he whole chi

microvalve

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racterizatio

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wo working

ositive press

Microvalve

grated micr

green color

ip can be fu

s integrated

on

states of

sure (7 psi)

Inlets

s

Chapter

rofluidic ch

liquid. The

urther reduc

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the fabrica

) was applie

r 3 Zero De

hip is show

e whole chip

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ed through

Pneumatic

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wn in Figur

p is 7 cm i

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the pneuma

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Microvalve

43

re 3-6. The

in diameter;

ement.

er different

atic channel

e

3

e

;

t

l


44

to the thin membrane, the membrane temporarily sealed with the valve seat and the valve

was closed (Figure 3-7(a)). Under normal conditions, i.e. without external pressure, the

design of the valve structure would allow the microvalve to remain closed. This occurs as

both the valve seat and the membrane are fabricated in PDMS and the reversible sealing

of PDMS to PDMS closes the valve. However, due to the weak bonding of PDMS-PDMS

reversible sealing, the temporary sealing can be broken by pressure of magnitude larger

than 1 psi. Therefore, an external positive pressure is intentionally used to ensure that the

valve is closed. In this microvalve design, dust or large particles cannot be tolerated. This

is because that these contaminants could be stuck in between the membrane and the valve

seat, causing the valve not to be fully closed. For this reason, in-line filters were placed in

between the reagent reservoirs and the microchip inlets in this study to avoid malfunction

of the microvalves.

On the other hand, when a negative pressure (-4.2 psi) was applied, this negative pressure

applied would overcome the reversible bonding and separate the membrane and the valve

seat. The flexible membrane would be sucked towards the pneumatic channel and a

connecting channel between the membrane and the valve seat was formed. This formed

channel provides a fluidic path for liquid to enter the main channel (Figure 3-7(b)). After

the valve is opened, in theory, less negative pressure is needed to maintain the open state

of the valve. The time needed to open the valve is estimated to be less than 17 ms (liquid

can pass through the valve when the controlling solenoid valve is energized for 25 ms to


45

allow vacuum to be applied on the membrane. However, the solenoid valve needs 8 ms to

be energized).

(a)

(b)

Figure 3-7 Photograph of the microvalve. (a) The microvalve is closed by external

positive pressure through pneumatic channel. (b) The microvalve is opened by external

negative pressure through pneumatic channel.

500 µm

Pneumatic channel

Valve seat

Main channel


46

3.3.1.2 Leakage pressure

The leakage pressure of the fabricated microvalve is defined as the maximum liquid

driven pressure the valve can retain without leakage at certain valve closing pressure.

Figure 3-8 shows the relationship between the valve closing pressure and the leakage

pressure. As expected, the microvalve can retain higher liquid driven pressure as the

valve closing pressure increases.

From Figure 3-8, we can see that when the valve closing pressure is 0, the valve can still

retain a pressure of ~1 psi. This is due to the weak reversible sealing of PDMS to PDMS.

It was reported previously that the reversible PDMS-PDMS bonding can retain as much

as 5 psi pressure [115]. In that study, both PDMS layers were thick. Therefore, the

elasticity is negligible. However, in our case, the pressure that can be retained is much

lower because the thin PDMS membrane is very flexible; as a consequence, a weaker

pressure is able to bend it and causing separation of the membrane and the valve seat.

The valve closing pressure used in this study is 7 psi. At this pressure, the leakage

pressure is as high as 9.8 psi. However, the liquid driving pressure used in this study is

only 2 psi. Therefore, under normal conditions, it can be guaranteed that there is no

leakage of reagents through the microvalve.


47

Figure 3-8 Relationship between valve closing pressure and leakage pressure of the

fabricated microvalve

3.3.2 Zero dead-volume

The fabricated microfluidic device with integrated microvalves has zero dead-volume

between each valving site and the main channel. As shown in Figure 3-9(a), when the

valve controlling green reagent (green valve) was opened, the green reagent entered

directly into the main channel and flowed toward the outlet. After switching off the green

valve, the main channel was flushed with washing reagent which was controlled by the

left most valve (see Figure 3-3). The green reagent can be completely flushed out of the

device due to the absence of the connecting channel between the green valve and the

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8

Lea

kage

Pre

ssur

e (p

si)

Valve Closing Pressure (psi)


48

main channel, which is different from previous designs [105, 107, 108]. The complete

removal of green liquid minimized the possibility of its contamination to following

reagents entering the main channel. It can be seen in Figure 3-9(b) that the red reagent,

which was controlled by an upstream valve, flowed through the main channel without

mixing with green reagent after the green valve was closed.

In the design of the microfluidic device, the main channel is zigzag shaped and

microvalves sit exactly at each turning point of the main channel. The distance between

each valving site and the main channel was visibly reduced to zero, hence creating a zero

dead-volume between each valving site and the main channel. This zero dead-volume

allowed any reagent entering the main channel to be thoroughly washed out after use and

not to remain entrapped within the microfluidic device.


49

(a)

(b)

Figure 3-9 Photograph of the microvalve showing the zero dead-volume characteristic;

two colored liquids (green and red) are used for illustration. (a) The microvalve that

controls green liquid was opened. (b) The microvalve that controls green liquid was

closed and another microvalve (located at upstream of the main channel) that controls red

liquid was opened. No contamination of the red liquid from the green liquid was observed.

3.3.3 Minimal cross contamination tested by PCR

PCR was used as a detection tool to demonstrate minimal cross contamination of the zero


50

dead-volume microvalve as PCR is a very sensitive assay for detection of specific DNA

templates. With appropriate primer design and optimized amplifying conditions, as few as

10 DNA template molecules can be detected (Figure 3-10).

PCR template (plasmid DNA, 4142 bp, Promega) with a concentration of 1×106

molecules/µL in 1X TE buffer was controlled and fed into the main channel by one of

these microvalves. The microvalve was opened for 5 seconds followed by TE buffer

washing for either 10, 20 or 30 seconds. TE buffer was controlled by the left most

microvalve. After washing for desired time, another 20 µL of the washing buffer was

injected into the device and the effluent was collected at the outlet. The total volume of

the PDMS main channel is 1.8 µL and flow rate is 5 µL/s.

This effluent containing the DNA template molecules was mixed with other PCR

components (PCR core system II, Promega, US) including nucleotide mix, polymerase,

primers, buffers and water to a final volume of 50 µL and a concentration described

previously [116]. 35 PCR amplification cycles were completed in PCR thermal cycler

(PTC-200, MJ Research) and the product (323 bp) was analyzed by 1.5% agarose gel

electrophoresis. The gel electrophoresis was run at 120 V for 45 minutes and stained with

SYBR® Safe dye. (Invitrogen, US). The gel was imaged with G:BOX gel documentation

system (Synoptics, UK).

As shown in Figure 3-10(a), after washing with TE buffer for 10 seconds (Lane 6), the


51

effluent produced a band intensity which was stronger than that of PCR product amplified

from 1000 template molecules (Lane 2), indicating that the amount of template molecules

was more than 1000 in the 20 µL effluent after 10 seconds of washing. However, after 30

seconds of washing (Lane 8), the effluent produced a band intensity which was between

the intensity of PCR product amplified from 100 (Lane 3) and 10 (Lane 4) template

molecules; equating to 0.5 to 5 template molecules per µL. By comparing with the

original concentration of the PCR template stock solution (1×106 molecules/µL), there

was a 10-6 reduction factor in the concentration. Extended washing can further reduce the

concentration of the template molecules in the effluent.

For comparison, the same experiment was conducted by microvalve system of traditional

design as shown in Figure 3-1(a). The PCR and gel electrophoresis image is shown in

Figure 3-10(b). It can be seen that after washing for 30 seconds, the 20 µL effluent still

contained more than 1000 DNA template molecules, which indicates that the washing

was not as efficient as in our design. This could lead to cross contamination of reagents

that cannot be tolerated in certain applications.

In summary, it has been demonstrated that cross contamination of reagents in our

microdevice design can be minimized to part in a million (ppm) levels after washing for

30 seconds.

Figure 3-1

electrophor

from 1000,

for 10 s, 20

0 Trace am

resis. Lane 1

100, 10 an

0 s and 30 s.

mount of D

1: 100 bp D

nd 0 templat

. (a) Our mi

(a

(b

DNA temp

DNA ladder;

te molecule

icrovalve de

Chapter

a)

b)

plate molec

; Lane 2-5: p

s; Lane 6-8

esign (b) Tr

r 3 Zero De

cules detect

positive con

8: PCR with

aditional m

ad-volume

tion by PC

ntrol with P

h effluent af

microvalve d

Microvalve

52

CR and gel

PCR starting

fter washing

design

e

2

l

g

g


53

3.4 Conclusion

In this chapter, we designed and fabricated a microvalves integrated microfluidic device

for minimal cross contamination among the reagents. The zigzag shape of the main

microfluidic channel and the position of each microvalve removed the dead-volume

between the common pathway and each valving site, which effectively minimized the

contamination of currently used reagent to subsequently added reagents. The microfluidic

device presented in this chapter is a good candidate to be used in chemical synthesis and

biological studies where reagents need to be added into a reaction chamber in a serial

manner and cross contamination among reagents is an important issue and must be

avoided.

54

CHAPTER 4

OLIGONUCLEOTIDE SYNTHESIS

IN A PORTABLE SYNTHESIZER

Chapter 4 Oligonucleotide Synthesis in a Portable Synthesizer

55

4.1 Introduction

DNA oligonucleotides are one of the most common materials used in biomolecular

research and applications nowadays. Every year, hundreds of thousands of

oligonucleotides are synthesized. Through the development of the phosphoramidite solid

phase synthesis of oligonucleotide, the process has been fully automated and can be

completed in oligonucleotide synthesizers. The oligonucleotide synthesizer has been

commercialized for many years and there are several companies offering this product.

However, due to the complexity of the synthesis chemistry in which a number of

synthesis reagents are involved, those synthesizers are bulky in size. The limitation on the

size of the commercialized oligonucleotide synthesizers makes them only suitable to be

used in laboratory and excludes their potential in the field applications.

Nowadays, due to the continuous threat from terrorism, in the field monitoring and

detection of biological warfare and infectious disease agents such as bacteria and viruses

become absolutely necessary. State of the art DNA based bioassays for the detection of

these biological substances make use of oligonucleotides that function as either probes

for hybridization or primers for PCR [117]. However, due to the lack of portability of

existing commercialized oligonucleotide synthesizing instruments, pre-made

oligonucleotides with known sequences need to be prepared and then brought to the

places where bioassays are carried out. As a consequence, it is impossible to conduct

bioassay with oligonucleotides of completely new sequences. In such case, no immediate


56

in the field detection of artificially manipulated or naturally mutated agents harmful to

human is possible. Therefore, there is an emerging need for new instrument that can

complement the state of the art DNA bioassay technologies by immediately synthesizing

oligonucleotides as either primers or probes with any sequence on demand in the field.

Microfluidic is a promising technology due to its small size and ease of integration. The

invention of microfluidic valves enables microfluidic devices to manipulate and control

fluids flow and therefore dramatically extended their use [81, 85]. Oligonucleotide

synthesis is an application where sequential switching of a number of reagents is needed.

Therefore, microfluidic technology is a potential solution for building a portable

oligonucleotide synthesizer. As far as we know, a few studies have been conducted on

developing microfluidic device for oligonucleotide synthesis [105, 106]. However, no

work has been done aiming to develop an oligonucleotide synthesizer in a portable format

that can be potentially used for in the field applications.

In this chapter, a microfluidics based portable oligonucleotide synthesizer is proposed and

built. The zero dead-volume microvalves integrated microfluidic chip (designed and

reported in pervious chapter) is the core component of the portable synthesizer. By the aid

of other peripheral components (including electronic & electrical, fluidic, and pneumatic

part), the portable oligonucleotide synthesizer was successfully used to synthesize both

oligonucleotide primers and probes. The sequence and biological functionality of the


57

synthesized oligonucleotide primers were analyzed and confirmed by polyacrylamide gel

electrophoresis (PAGE) and PCR. The sequence and biofunctionality of the synthesized

oligonucleotide probes were proven by DNA complementary and single-base mismatch

hybridization experiments.

4.2 Portable oligonucleotide synthesizer

4.2.1 Software

The synthesis controlling software was programmed under LabVIEW (Version 8.5, NI,

US). The software is run on a notebook computer (VGN-P25G, SONY, Japan). Two

screen shots of the programmed software are shown in Figure 4-1. There are two main

interfaces in the software: the information input interface and the synthesis process

monitoring interface.

In the information input interface (Figure 4-1(a)), users are allowed to manually input the

sequence information of an oligonucleotide to be synthesized or load it directly from a

text format file. The direction of the sequence is from 3’ to 5’, same as the direction of the

synthesis process. The reaction time for each synthesis step can be input in the time

control frame. If nothing is input, the default reaction time is used. The time control

frame is still accessible after synthesis starts. Therefore, parameters can still be changed

when the synthesis is in progress. Figure 4-1(b) shows the synthesis monitoring interface.


58

The information about the overall synthesis progress and the current reaction in each

synthesis cycle can both be observed.

(a)

(b)

Figure 4-1 Screen shots of the software for controlling oligonucleotide synthesis. The

software was programmed under LabVIEW. (a) The information input interface (b) The

synthesis monitoring interface.


59

4.2.2 Hardware

The PDMS microchip with integrated microvalves was operated through pneumatic ports

by three-way normally open solenoid valves (Pneumadyne, US) that were mounted on a

multiple-station aluminum manifold. The microvalves were closed under positive

pressure (7 psi, relative to atmospheric pressure) and opened under negative pressure (-

4.2 psi). All reagents were driven by positive pressure (2 psi) that was controlled by two-

way normally closed solenoid valves mounted on another aluminum manifold.

The positive pressure used in the system was provided by pressurized argon gas stored in

a paintball tank (6310, Carleton Technologies Inc, US). The tank output pressure 450 psi

was first regulated by a paintball regulator (shorty style regulator, Custom Products, US)

to 20 psi. After that, the 20 psi pressure was further regulated down to the necessary

pressures for closing the valve (7 psi) and driving the reagents (2 psi) by two miniaturized

pressure regulators (Type 91, Marsh Bellofram, US). The negative pressure for opening

the microvalves was provided by a micro-boxer pump (Cole-Parmer, US). The solenoid

valves and vacuum pump were controlled by a 32-Channel sourcing digital output card

(9476, NI, US) functioning as relays. The digital output card was mounted on a USB

CompactDAQ Chassis (cDAQ-9172, NI). The electronic and electrical parts used in the

system were all powered by 12 V DC, which was provided by two 6 V lead acid

rechargeable batteries.


60

4.2.3 System integration

The complete portable oligonucleotide synthesizer comprises three main parts: the

electrical and electronic controlling part, the pneumatic controlling part, and the fluidic

part. Figure 4-2(a) shows the schematic diagram of the portable oligonucleotide synthesis

system. The three main parts are marked with different colors. Figure 4-2(b) shows the

photograph of the portable oligonucleotide synthesizer.

All parts forming the portable synthesizer were mounted on a home-made aluminum rack.

The rack was divided into two main compartments. The left compartment was for

pneumatic parts and electrical and electronic parts. The right compartment was for liquid

reagents related parts including the microfluidic device and reagent reservoirs and waste

bottle. The size of the aluminum rack is 42 cm × 19 cm × 28 cm and the weight is around

6 kg. The whole rack can be inserted into an aluminum suitcase for carrying purpose. The

portability of the whole system makes it easy to carry and suitable for being used in the

field. A detailed list of all components used to build the portable oligonucleotide

synthesizer can be seen in Appendix A.


61

(a)

(b)

Figure 4-2 System design and photograph of portable oligonucleotide synthesizer (a)

Schematic diagram illustrating the complete system, including electrical and electronic

parts, fluidic parts and pneumatic parts. Only four microvalves are shown on the diagram

for simplicity. (b) Photograph of the portable oligonucleotide synthesizer: (1) PC (2)

Argon tank (3) USB-DAQ (4) Solenoid valves and manifold (5) Pressure regulators (6)

Vacuum pump (7) Batteries (8) Microfluidic chip (9) Synthesis reactor (10) Reagents.

4.2.4 Micro

Each valve

the valves

oligonucleo

reagents ca

Figure 4-3 A

(2) Argon (

4.3 Materi

4.3.1 Chem

Oligonucle

tetrazole so

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Assignment

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62

signment of

etonitrile in

hat all other

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acetonitrile

chloroacetic

ma-Aldrich

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2

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.


63

Phosphoramidites including 5'-dimethoxytrityl-N-benzoyl-2'-deoxyadenosine,3'-[(2-

cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite (dA), 5'-Dimethoxytrityl-N-benzoyl-2'-

deoxycytidine,3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite (dC), 5'-

Dimethoxytrityl-N-isobutyryl-2'-deoxyguanosine,3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-

phosphoramidite (dG), and 5'-Dimethoxytrityl-2'-deoxythymidine,3'-[(2-cyanoethyl)-

(N,N-diisopropyl)]-phosphoramidite (dT), were purchased from Glen Research (US). All

reagents were directly used as received without further purification.

4.3.2 Synthesis reactor

4.3.2.1 Reactor for primer synthesis on CPG

Oligonucleotide primer was directly synthesized on CPG. The reactor for primer

synthesis is shown in Figure 4-4. A hole with 1 mm in diameter was punched through a

PDMS cube. A glass microfiber filter (Schleicher & Schuell, Germany) was inserted into

the punched hole. Two FEP tubings were then inserted into the hole from both ends. The

glass fiber filter was thus fixed in between the two tubings. To load CPG into the reactor,

they were first suspended in acetonitrile. Then the FEP tubing was connected to a syringe.

The CPG (around 1 mg) was sucked into the reactor and stayed above the glass filter,

while liquid can pass through the filter. The reactor was directly connected to the outlet of

the microfluidic chip and ready for synthesis.


64

Figure 4-4 Reactor for oligonucleotide primer synthesis. (1) PDMS cube (2) Glass fiber

filter (3)(4) FEP tubing (5) CPG.

4.3.2.2 Reactor for probe synthesis on glass slide

Oligonucleotide probe was synthesized on a surface modified microscope glass slide. The

synthesis reactor (Figure 4-5) was formed by reversibly sealing the glass slide with a

PDMS microfluidic channel. The microchannel (16 mm length × 500 m width × 50 m

height) with one inlet and one outlet was fabricated by micromolding as previously

described (Section 3.2.3.1). The formed reactor had a volume of 0.4 L. Oligonucleotide

probe was then synthesized on the glass slide surface within the formed channel. The

PDMS-glass sandwich was held firmly by an acrylic case to prevent leakage. The inlet of

the formed reactor was directly connected to the outlet of the microfluidic chip.


65

Figure 4-5 Reactor for oligonucleotide probe synthesis. (1) Top acrylic cover with tubing

access holes (2) PDMS microchannel (3) Surface modified microscope glass slide (4)

Bottom acrylic case.

4.3.3 Surface modification of microscope glass slides

Oligonucleotide was synthesized on microscope glass slides as probe for DNA

hybridization. The microscope glass slides were modified as previously reported [118].

They were first treated with 2% N-(3-triethoxysilylpropyl)-4-hydroxybutyramide in 95%

ethanol for 4 hours at room temperature. After that, the slides were thoroughly rinsed

with ethanol and then cured in vacuum oven at 120 °C for 1 hour. The treated slides were

stored in dehumidifying cabinet if not immediately used. The scheme of the surface

modification reaction is shown in Figure 4-6.


66

2% in EtOH, RTGlass Slide

OH OH OH OH OH OH

Synthesis

OH

CNH

OOOSi

Et Et Et

O

Glass Slide

OH

CNH

OO

OSi

Et Et

O

OH

CNH

OO

OSi

Et Et

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Glass Slide

OH

CNH

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Si

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Vacuum

120 oC Glass Slide

O

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OSi

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CNH

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Si

O

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Si

O

DMT

O

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B1

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N

DMT

O

P

OO

O

B1

O

N

DMT

O

P

OO

O

B1

O

N

Figure 4-6 Scheme of surface modification of microscope glass slide for synthesis of

oligonucleotide probe.

4.3.4 Oligonucleotide synthesis protocol

The phosphoramidite method was used to synthesize oligonucleotides in this study (the

principle of synthesis chemistry can be seen in Figure 2-2). The detritylation reagent was

substituted by 3% dichloroacetic acid in anhydrous nitromethane [119], as PDMS is

badly swelled by dichloromethane [93]. The oxidation reagent was replaced by 0.1 M

iodine in 9:1 pyridine/acetic acid (v/v) [104].


67

For the coupling step, there are two reagents involved. These two reagents, activator and

phosphoramidite, can be only mixed right before the reaction. Therefore, these two

reagents were injected into the microfluidic chip in an alternating fashion for a number of

times. This injection manner can improve the mixing of these two reagents in the

microfluidic chip, which is important for achieving high coupling efficiency. The

reagents and reaction time for each step are summarized in Table 4-1. The total time for

one synthesis cycle (adding one nucleotide) is 9 minutes.

Table 4-1 Reagents and reaction time for oligonucleotide synthesis

No. Reaction Reagent Time

(minutes)

1 Detritylation 3% dichloroacetic acid in nitromethane 1.5

2 Washing Acetonitrile 1

3 Coupling

0.45 M tetrazole in acetonitrile

0.1 M phosphoramidite in acetonitrile (dA, dT,

dG or dC)

3


5 Oxidation 0.1 M iodine in 9:1 pyridine/acetic acid (v/v) 1.5


4.3.5 PAGE and PCR

Oligonucleotide synthesized on universal CPG was used as primer for PCR. 15% PAGE

was used to confirm the length of the synthesized oligonucleotide. The ratio of

acrylamide and bis-acrylamide is 29:1 (Bio-rad, US). The prepared gel was pre-run in 1X


68

TBE buffer for 30 minutes at 200 V. The samples were heated to 95 °C for 2 minutes,

then immediately chilled by transferring onto ice. After that, the samples were added into

the wells and the electrophoresis was run for 1.5 hours at 200 V. Then the gel was stained

with SYBR® Gold dye and imaged with a G:BOX gel documentation system (Synoptics,

UK).

The synthesized oligonucleotide was used as reverse primer to amplify E. coli 16S

ribosomal RNA. The 596 bp PCR product was run in 1.7% agarose gel for 45 minutes at

120 V. The gel was also imaged with the G:BOX gel documentation system.

4.3.6 Probe deprotection and hybridization

Oligonucleotide probe was synthesized on a surface modified glass slide for hybridization.

After synthesis, the slide was treated with ethylenediamine:ethanol (1:1) for 2 hours at

room temperature to remove the protecting groups. It was then washed thoroughly with

ethanol followed by ddH2O and finally blown dry with nitrogen.

For hybridization, 5 M Texas Red labeled oligonucleotide was dissolved in 0.5X SSC

buffer containing 0.1% SDS. 15 L of the fluorescence labeled oligonucleotide was

spotted on the glass slide and then covered with cover slide. The hybridization was taken

place at 40 °C for 2 hours. After that, the glass slide was washed with 1X SSC buffer and


69

0.5X SSC buffer at 37 °C. The glass slide was then blown dry with compressed nitrogen.

The fluorescence image was taken by using a CCD color digital camera, Retiga 4000R

(QImaging), connected to a system microscope (Olympus BX41) with a mercury arc

(Olympus HBO103W/2) excitation source.

4.4 Result and discussion

4.4.1 PCR primer synthesis

To demonstrate whether oligonucleotide could be synthesized by the portable

oligonucleotide synthesis system and used as a primer for PCR, a 16-mer oligonucleotide

(sequence 5’-GGTTGCGCTCGTTGCG-3’) was synthesized on universal CPG. The

synthesized oligonucleotide was then cleaved from CPG by concentrated ammonium

hydroxide (28% in water) at 65 °C overnight. The synthesized oligonucleotide was used

as the reverse primer for amplifying E. coli 16S ribosomal RNA.

The oligonucleotide synthesized in the portable synthesizer was analyzed by PAGE.

Figure 4-7 shows the PAGE image of the synthesized oligonucleotide. From the PAGE

image, it can be seen that main product of the synthesized oligonucleotide is the 16-mer

full length oligonucleotide (lane 3), which has the same mobility as comparing with the

HPLC purified commercial oligonucleotide of the same sequence (lane 2). The main

impurity in the product is n-1 mer. By measuring the band intensity, the final yield of the


70

full length product was about 70%, corresponding to a step wise yield of 98%. This yield

was satisfactory and comparable with synthesis yield reported before [105]. The

synthesized primer of such yield is suitable for applications such as PCR without further

purification.

Figure 4-7 PAGE image for synthesized oligonucleotide length confirmation. Lane 1:

oligonucleotide ladder. Lane 2: HPLC purified commercial oligonucleotide with the same

sequence. Lane 3: oligonucleotide synthesized by the portable oligonucleotide synthesizer.

The synthesized 16-mer oligonucleotide was then used as reverse primer in PCR to

amplify E. coli 16S ribosomal RNA. The agarose gel electrophoresis image of the 596 bp

PCR product is shown in Figure 4-8. The sequence of the forward primer was 5’-


71

CCAGCAGCCGCGGTAA-3’. Lane 2 shows the positive control with both primers

purchased. For lane 3, the reverse primer was synthesized by the portable synthesizer.

The gel electrophoresis image shows that the target DNA template was successfully

amplified when using the oligonucleotide synthesized in our synthesizer as the reverse

primer. Therefore, the sequence and biological functionality of the synthesized

oligonucleotide was successfully demonstrated.

Figure 4-8 The gel electrophoresis image of PCR product. Lane 1: 50bp DNA ladder.

Lane 2: positive control with both forward and reverse primer purchased. Lane 3: PCR

product amplified by the synthesized oligonucleotide in our portable synthesizer as

reverse primer.


72

4.4.2 Probe synthesis

4.4.2.1 Probe for oligonucleotide hybridization

To further test the function of the portable oligonucleotide synthesizer, an oligonucleotide

probe (5’-GGTTGCGCTCGTTGCG-3’) was synthesized on surface modified glass slide.

After synthesis, the probe was hybridized with a complementary DNA which is labeled

with 6-carboxyfluorescein (6-FAM) fluorophore (5’-CGCAACGAGCGCAACC[6-

FAM]-3’). The fluorescence image was taken by using fluorescence microscope. The

fluorescence image in Figure 4-9 shows that the complementary DNA was successfully

hybridized to the synthesized oligonucleotide probe, which also confirmed the sequence

and biofunctionality of the synthesized oligonucleotide.

Figure 4-9 Fluorescence image of 6-FAM labeled complementary DNA hybridized to

synthesized probe

500 m


73

4.4.2.2 Probe for single-base mismatch differentiation

Probes were also synthesized for single-base mismatch differentiation in order to confirm

the function of the portable oligonucleotide synthesizer. The sequence of the synthesized

probe on the modified slide was 5’-GCTACAACTGCTTATG-3’. The complementary

oligonucleotide for hybridization with Texas Red labeled at the 3’ end (5’-

CATAAGCAGTTGTAGC[TxRd]-3’) was purchased. For comparison, a probe with

single-base mismatch was also synthesized. The sequence of oligonucleotide with single-

base mismatch was 5’-GCT ACC ACT GCT TAT G-3’.

The fluorescence image in Figure 4-10(a) shows that the dye labeled complementary

strand successfully hybridized with the probe synthesized on the glass slide. Figure

4-10(b) shows the fluorescence image of the hybridization to the probe with single-base

mismatch. From the images, it can be observed that the fluorescence intensity of the

complementary hybridization is much higher than that of hybridization with single-base

mismatch (the ratio of the fluorescence intensity is about 8 by measuring with ImageJ

software), indicating the higher hybridization efficiency between complementary

sequences. Therefore, the sequence and biological functionality of the oligonucleotide

probes directly synthesized on surface modified microscope glass slide are successfully

proven.


74

(a)

(b)

Figure 4-10 Fluorescence image of the synthesized probe hybridizing with Texas Red

labeled oligonucleotide. (a) Complementary sequence. (b) Single-base mismatch.

500 m

500 m


75

4.5 Conclusion

In this chapter, we presented a microfluidic device based portable oligonucleotide

synthesizer. To the best of our knowledge, this is the first portable oligonucleotide

synthesizer reported. The core component of the portable synthesizer is the zero dead-

volume microvalves integrated microfluidic chip which was designed in the previous

chapter. The portable oligonucleotide synthesizer was successfully used to synthesize

oligonucleotide primers. The length of the synthesized primer was confirmed by PAGE.

The primer was successfully used to amplify bacteria ribosomal RNA gene, therefore

proving the sequence and biofunctionality of the synthesized primer. Oligonucleotide

probe was also successfully synthesized on a flat glass surface by the portable synthesizer.

The sequence and biological functionality of the synthesized oligonucleotide probes were

demonstrated by DNA complementary and single-base mismatch hybridization

experiments. The time for synthesizing one base is 9 minutes and therefore 3 hours is

needed for a oligonucleotide with typical length of 20mer. The portable synthesizer

reported here has the potential capability of synthesizing oligonucleotide of any sequence

in the field.

76

CHAPTER 5

PORTABLE GENERIC DNA

HYBRIDIZATION BIOASSAY SYSTEM

Chapter 5 Portable Generic DNA Hybridization Bioassay System

77

5.1 Introduction

Bioassay based on DNA hybridization has been developed for many years for different

applications such as detection of organism and diagnosis of diseases [120, 121]. When

conducting these bioassays, the DNA targets are usually labeled with fluorescent

molecules for detection purpose. Therefore, it is important to accurately measure the

fluorescence signal in such bioassays.

As stated previously, state of the art DNA based bioassays for the detection of biological

warfare and infectious disease agents such as bacteria and viruses make use of pre-made

oligonucleotides that function as either probe or primer [117]. In the previous chapter, we

have developed a portable oligonucleotide synthesizer based on microfluidic technology.

The portable synthesizer has the potential to synthesize oligonucleotide of any sequence,

thus complementing state of the art DNA based technologies for in the field application.

However, to detect the result of DNA based bioassays in the field, for example DNA

hybridization, the detection system must also be fabricated in a low cost and portable

format.

In many systems, expensive laser excitation source and sophisticated detector are used,

which make them not suitable for in the field applications [122]. There are several studies

using cheaper light sources or simple photo detectors such as light emitting diode (LED)

and photodiode [123-125]. However, some studies focused on fluorescence detection in


78

liquid phase instead of solid phase [123, 124]. In the other studies, DNA probes were

directly immobilized on the photodiode surface, limiting the reusability of the detector

[125].

In this chapter, we present the first portable generic DNA hybridization based bioassay

system. The system integrates the portable oligonucleotide synthesizer presented in the

previous chapter and a Texas Red fluorescence detection system based on a LED and

photodiode. The fluorescence detection system can detect Texas Red fluorophore as low

as 0.5 fmol and was successfully used for differentiating complementary and single-base

mismatch hybridization. The complete generic portable bioassay system was successfully

used to distinguish DNA from two different bacteria strains. The portable system has the

potential to detect any DNA sequence in the field.

5.2 System design

5.2.1 Texas Red fluorescence detection system

Figure 5-1(a) shows the schematic of the Texas Red fluorescence detection system. A

white color Luxeon Emitters (LXHL-PW01, Lumileds Lighting, US) was used as the

light source for excitation light. The LED was powered by a constant current Stardrive

LED driver (1 Watt, Sumatec Ltd, UK). The white light was first collimated by a lens

(f=30 mm, Edmund Optics, US) and then passed through an excitation filter (HQ575/50x,


79

Chroma, US). The filtered light was reflected by a beam splitter (Z594/750rpc, Chroma,

US) and focused onto the sample. The fluorescence emitted from the sample passed

through the emission filter (HQ645/70m-2p, Chroma, US) and was focused by a lens on a

high sensitivity, low noise photodiode detector with built-in pre-amplifier. (S8745-01,

Hamamatsu photonics, Japan). The photodiode was powered with ±6 V and the signal

from the pre-amplifier was directly measured with an NI 9219 Analog Input card

(National Instrument, US) without further amplification. All optical parts were fixed into

a home-made light tight aluminum housing. The photograph of the detection system is

shown in Figure 5-1(b). Spectrum of the various optical parts including LED, excitation

and emission filters, beam splitter, and photodiode can be seen in Appendix C.

5.2.2 Integration of fluorescence detection and oligonucleotide synthesis system

To build the portable generic DNA bioassay instrument, the fluorescence detection

system was further integrated into the portable oligonucleotide synthesizer developed in

previous chapter. As on-the-chip DNA hybridization is required, the microvalve

integrated microfluidic chip and the oligonucleotide probe synthesis reactor (as shown

previously in Figure 4-5) were both redesigned to incorporate the new function, as

described in the following.


80

(a)

(b)

Figure 5-1 (a) Schematic diagram of the Texas Red fluorescence detection system (b)

Photograph of the Texas Red fluorescence detection system.

5.2.2.1 Modified microfluidic chip

The microfluidic chip used in the portable oligonucleotide synthesizer has totally 9

microvalves integrated. Each microvalve was assigned to one oligonucleotide synthesis

reagents. To

microfluidi

and washin

shown in

integrated

washing bu

plate of the

Figure 5-2 M

Microvalve

Detritylatio

microvalve

5.2.2.2 Rea

The oligonu

o realize the

ic chip to c

ng buffer (v

Figure 5-2

is shown

uffers) can

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Microfluidi

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Chapter 5

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81

ded into the

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82

modified to realize the on-site DNA hybridization. Comparing with the previous reactor,

the current one has two more elements, the temperature sensing and the heating parts.

The temperature sensor (PT100, RS) was sandwiched in between the top acrylic cover

and the microscopy glass slide on which the probe is synthesized (part 3 in Figure 5-3).

Thus the temperature measured is at the top surface of the glass slide and it is also the

surface on which the hybridization occurs. Therefore, the temperature measured is much

more accurate than placing the sensor elsewhere.

The heating unit was placed under the bottom acrylic case. A heater mat was placed on

the bottom surface of an aluminum piece (part 6 in Figure 5-3). The aluminum piece

directly contacted with the synthesis glass slide. Heat can be evenly distributed over the

aluminum piece, which can guarantee the uniform heating of the glass slide and increase

the temperature controlling accuracy. A hole was drilled into the aluminum piece to allow

excitation and emission light to pass through for fluorescence detection.


83

Figure 5-3 Design of oligonucleotide probe synthesis and hybridization reactor. (1) Top

acrylic cover with tubing access hole (2) PDMS microchannel (3) PT100 temperature

sensor (4) Surface modified glass slide (5) Bottom acrylic case (6) Heating unit with light

access hole. The whole assembly was held together by screw and then fixed inside the

fluorescence detection box.

5.2.2.3 Portable DNA bioassay system

The photograph of the complete portable generic DNA bioassay system is shown in

Figure 5-4. The fluorescence detection system was fixed inside the right partition

chamber of the original portable oligonucleotide synthesizer. The probe synthesis and

hybridization reactor was fixed on the upper wall inside the fluorescence detection device

(see part 9). The inlet of the reactor was directly connected to the outlet of the


84

microfluidic chip by FEP tubing. After probe synthesis, the hybridization reagent can be

manually injected into the reactor using a syringe through the port which was extended

outside the aluminum rack (see part 13, this injection port was controlled by valve 10 on

the microfluidic chip).

Figure 5-4 Photograph of the portable system: (1) PC with control software (2) Argon

tank (3) NI-DAQ (4) Solenoid valves and manifold (5) Pressure regulators (6) Vacuum

pump (7) Batteries (8) Microfluidic chip (9) Synthesis and hybridization reactor (10)

LED (11) Photodiode (12) Lenses, filters and beam splitter (13) Injection port for

hybridization related reagents.

42 cm

28 cm

19 cm


85

5.3 Materials and methods

5.3.1 Hybridization probe immobilization on glass slide

Hybridization probes were immobilized on glass slide for testing the function of

fluorescence detection unit. The glass slide was first cleaned with piranha solution

(sulfuric acid : hydrogen peroxide, 7:3) at 120 ºC for 30 minutes followed by thoroughly

washing with ddH2O. The cleaned glass slide reacted with 2% 3-

aminopropyltriethoxysilane (APTES) in acetone at room temperature for 2 hours and was

then baked in vacuum oven at 120 ºC for 1 hour. The glass surface was bearing amine

groups now. After that, 20 L of 50 mM disuccinimidyl suberate (DSS) in

dimethylformamide (DMF) was spotted on the amine functionalized glass slide and

covered with cover slide for 1 hour at room temperature. After washing with acetone,

1L of amine labeled oligonucleotide probe (10 M in 50 mM phosphate buffer pH 7.4)

was spotted on the activated surface for 2 hours. After washing, the slide was ready to use

for hybridization.

5.3.2 Asymmetric PCR

For bacteria strains differentiation, the bacteria genomic DNA (Bacillus cereus ATCC

14579 and Salmonella enterica subsp. enterica ATTC 13311) was first amplified by

asymmetric PCR. One primer is labeled with Texas Red fluorophore, thus the PCR

product containes the fluorophore for hybridization detection. The ratio of Texas Red


86

labeled primer and non-labeled primer was 10:1. Therefore, more fluorescence labeled

single-strand target DNA can be produced for hybridization and the competition between

the hybridization probe and the target-complementary strand can be reduced.

5.3.3 DNA hybridization

The hybridization of the Texas Red labeled PCR product to in situ synthesized probes

took place at 45 C for 1 hour in 5X SSC buffer containing 0.1% SDS. The temperature

was controlled by the integrated hybridization reactor. After hybridization, washing was

carried out with 0.1X SSC at 37 C for 5 minutes twice.

5.4 Result and discussion

5.4.1 Fluorescence detection system

5.4.1.1 System characterization

To characterize the Texas Red fluorescence detection system, a serial dilution of Texas

Red labeled oligonucleotide in water ranging from 1 nM to 500 nM was prepared. Then

0.5 μL of each dilution was spotted on glass slide (corresponding to 0.5 fmol to 250 fmol).

As the size of the spot was smaller than the size of the excitation light spot, all

fluorophores can be excited.


87

Texas Red fluorophore was chosen as the detection target because of the following three

reasons. Firstly, Texas Red is one of the most commonly used fluorophore for labeling

DNA. Secondly, the peak wavelength intensity of the excitation light source (LED)

matches with Texas Red’s peak excitation wavelength (589 nm). Thirdly, at the peak

emission wavelength of Texas Red (615 nm), the photo sensitivity of the photodiode is

higher (comparing with its sensitivity at peak emission wavelength of 6-FAM 520 nm)

(refer to Appendix C).

Figure 5-5 shows calibration curve of the Texas Red detection system. It can be seen that

the detection system can detect Texas Red fluorophores ranging from 0.5 fmol to 150

fmol with good linearity (R2=0.989). The system is saturated when the amount of

fluorophore is above 200 fmol. It is worth noting that at the lower range (0.5 fmol to 12.5

fmol), the linearity of the detection system is even better (R2=0.996) and this range is

coincident with the amount of fluorophores from DNA hybridization observed in this

study (shown in the following).


88

Figure 5-5 Calibration curve for Texas Red fluorescence detection system. The lower

range of the calibration curve (corresponding to amount of Texas Red fluorophore from

0.5 fmol to 12.5 fmol) is enlarged.

It was reported that the density of DNA probe immobilized on commercial ammine-

reactive glass slides was above 1012 probes/cm2 [126], corresponding to 1.66 ×10-5

fmol/µm2. Thus, by assuming 100% hybridization efficiency, our system can detect

hybridization to such probe surface with diameter as small as 200 µm.

5.4.1.2 Detection of complementary and single-base mismatch DNA hybridization

The fluorescence detection system was then tested by DNA hybridization to a surface


89

immobilized probe. Two amine labeled oligonucleotide probes (5’-

GCTACAACTGCTTATG[Amine]-3’ and 5’-GCTACCACTGCTTATG[Amine]-3’) were

immobilized on modified glass slide through DSS linker as described. These two

oligonucleotides have one base difference in the middle of the sequence. A Texas Red

labeled oligonucleotide (5’-CATAAGCAGTTGTAGC[TxRd]-3’), complementary to the

first oligonucleotide but with single-base mismatch to the second, was then hybridized to

both immobilized probes. After proper washing, the hybridization was measured by the

fluorescence detection system.

From the result of our system, the signal for complementary hybridization was 0.190 V.

However, the signal for single-base mismatch was just 0.018 V. The ratio of these two

signals is as large as 10.6, indicating that the fluorescence detection system successfully

discriminate the fluorescence signal from complementary and single-base mismatch

hybridization. A point to note is that both signals fall in the range where the system has

higher linearity.

For comparison, the fluorescence images of the complementary and single-base mismatch

hybridization were also taken by our fluorescent microscope system (Qimaging CCD

camera Retiga 4000R, and Olympus BX41 microscope) (Figure 5-6). The fluorescence

intensity was measured by using ImageJ software. The ratio of the fluorescence intensity

is 8.4, which is lower than the ratio of the signals measured by our fluorescence detection

system. Th

system is co

Figure 5-6

fluorescenc

mismatch h

5.4.2 Bacte

The functi

differentiati

illness from

ribosomal

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91

was used to amplify 16S rRNA of the two strains (5’-CCTACGGGAGGCAGCAGT-3’

and 5’-[TxRd]CGTTTACGGCGTGGACTAC-3’). The reverse primer was labeled with

Texas Red at 5’ end to generate the fluorescence signal for detection. Two probes

(Bacillus 5’-CGCGCAGGTGGTTTCTTAA-3’ and Salmonella 5’-

GGTCTGTCAAGTCGGATGTG-3’), each is specific to one strain, were synthesized on

surface modified glass slides. The synthesis process was same as reported in previous

chapter. Each probe was then hybridized with both Texas Red labeled PCR product and

the hybridization results were examined by the fluorescence detection system.

As shown in Figure 5-7, Texas Red labeled PCR products from two different bacteria

strains hybridized only to the synthesized probe that is specific to its strain. The

fluorescence signals from specific hybridization were much stronger than that from cross-

hybridization. Therefore, these two bacteria strains were successfully differentiated by

our portable generic bioassay instrument.


92

Figure 5-7 3D bar graph showing the fluorescence signal from specific and cross

hybridization between PCR products of two different bacteria strains and synthesized

strain specific probes.

It can also been seen that the fluorescence signals from specific hybridization were not as

high as that from previous hybridization between oligonucleotide and oligonucleotide

(5.4.1.2). This could be due to the fact that the PCR product has a length of 475-mer. The

hybridization efficiency can be affected by the length of the hybridization target [127].

Longer PCR product can lower the hybridization efficiency, leading to smaller

fluorescence signals. However, the signals are still within the detection range where the

system has higher linearity.


93

5.5 Conclusion

In this chapter, we have developed a portable and generic DNA bioassay instrument. To

the best of our knowledge, this is the first portable system developed for in situ synthesis

of oligonucleotide probe and integrated detection of DNA hybridization. The portable

oligonucleotide synthesizer is capable of synthesizing probe of any sequence on demand.

The Texas Red fluorescence detection device, based on a LED excitation light source and

a photodiode detector, can detect Texas Red fluorophore of the amount down to 0.5 fmol

and is able to discriminate complementary and single-base mismatch DNA hybridization.

The complete system was successfully used to distinguish different bacteria strains by

synthesizing strain specific probes and detecting the hybridization signals within the

portable instrument. The system presented in this work has the potential to detect any

DNA sequence in the field. We envisage that our system could help to enable fast

responses to emerging bio-threats for homeland security and in pandemics.

94

CHAPTER 6

CONCLUSION

Chapter 6 Conclusion

95

6.1 Conclusion and original contributions

This work focused on the development of a portable oligonucleotide synthesizer and

generic DNA hybridization based bioassay instrument due to the emerging need of in the

field synthesis of DNA oligonucleotide and in situ detection of specific DNA sequence.

In the following paragraphs, the important findings that were obtained in this work are

outlined.

The first part of this study was concerned with designing a microvalves integrated

microfluidic chip. The distinct advantage of the developed microchip is that it has a zero

dead-volume characteristic. This property is attributed to the special design of the zigzag

shaped main channel and the microvalves were located exactly at each turning point of

the zigzag shaped main channel. This design removed the connecting channels which are

found in previous designs. The absence of the connecting channels eliminated the

possibility of liquid reagents trapped within the microdevice, which could lead to cross

contamination to subsequently added reagents. The zero dead-volume characteristic of

the microdevice was proven by using PCR to detect trace amount of DNA template

molecules left in the device after washing. The result showed that cross contamination of

reagents in our microdevice design can be minimized to ppm levels after 30 seconds

washing, which is more efficient when comparing with traditional design. We believe that

the microdevice developed here is extremely useful for applications in which cross

contamination is a critical issue and lead to unpredictable results. DNA oligonucleotide


96

synthesis is such an application in which cross contamination among reagents makes

successful synthesis of oligonucleotides problematic.

The second part of this work concentrated on developing a portable oligonucleotide

synthesizer with the zero dead-volume microfluidic device that was obtained in the first

part of this work as the core component. The portable synthesizer consists of three main

parts: electronic & electrical part, pneumatic part and fluidic part. The synthesis

controlling software was developed with LabVIEW to control and monitor the whole

synthesis process. All components forming the portable synthesizer were fixed within a

home-made aluminum rack. The rack had a dimension of 42 cm × 19 cm × 28 cm and 6

kg of weight and can be inserted into a suitcase for ease of carrying. The function of the

portable oligonucleotide synthesizer was proven by synthesizing both oligonucleotide

primers and probes. The primers synthesized were analyzed by PAGE to confirm the

length. The sequence and biological functionality of the synthesized primers were

successfully demonstrated by amplifying E.coli 16S ribosomal RNA. The synthesized

probes were successfully used for DNA hybridization and had the ability to differentiate

complementary and single-base mismatch sequence, which further confirmed the

sequence and biological functionality of the synthesized probes. In summary, the

developed portable synthesizer is capable of synthesizing an oligonucleotide with any

sequence on demand as either primer or probe. As far as we know, this is the first portable

oligonucleotide synthesizer reported.


97

The third part of this work was concerned with developing a portable generic DNA

hybridization based bioassay system. A fluorescence detection unit was designed and

integrated into the portable synthesizer to form the generic bioassay system. The

fluorescence detection unit, using an ultra bright LED as the excitation source and a high-

sensitivity photodiode as the detector, was capable of detecting Texas Red fluorophore of

amounts ranging from 0.5 fmol to 150 fmol. The detection unit had a good linearity at

this range of Texas Red fluorophore amount and even a better linearity at the lower range,

which was also coincident with the amount of fluorophores from DNA hybridization

experiments. The Complementary and single-base mismatch hybridization were easily

discriminated by using the fluorescence detection unit. The complete bioassay system,

formed by integrating the portable oligonucleotide synthesizer and the fluorescence

detection system, was successfully used to differentiate two bacteria strains by in situ

synthesis of strain specific probes, hybridization to Texas Red labeled amplicons and then

on site detection of the fluorescence signals. To the best of our knowledge, no such

generic and portable DNA hybridization bioassay system was reported previously. As the

system has the potential to detect DNA target of any sequence for in the field applications,

we envisage that our system could help to enable fast responses to emerging bio-threats

for homeland security and in pandemics.


98

6.2 Technical challenges

PDMS is one of the widely used polymeric materials for fabrication of microfluidic

device. However, it can be badly swelled by organic solvent used in oligonucleotide

synthesis such as dichloromethane in detritylation reaction and tetrahydrofuran in

oxidation reaction. To overcome this problem, these traditionally used reagents were

replaced. For detritylation reaction, dichloroacetic acid was dissolve in nitromethane,

which does not swell PDMS, instead of dichloromethane. For oxidation reaction, iodine

was dissolved in pyridine/acetic acid mixture instead of THF/Pyridine/H2O mixture. The

replace of traditional oligonucleotide synthesis reagents does not affect the synthesis

yield as the step wise yield is 98%, which is comparable to yield reported before [105].

In building the portable oligonucleotide synthesizer, the size of each component is critical.

As microvalve and liquid reagents are both controlled by argon gas pressure, a gas tank

must be integrated into the system. However, the pressurized gas tank used in laboratory

is too big and too heavy to integrate into the portable system. We finally adapted the gas

tank which is used in paintball game into our portable system. The Output pressure from

the paintball tank (~450 psi) is first regulated to 20 psi by a paintball tank regulator and

then regulated to pressures needed in the portable system. The use of paintball tank in the

system dramatically reduces the size and weight of the complete system.


99

6.3 Future works

In the present work, single oligonucleotide was synthesized at one time in current system

design. However, the system can be easily extended due to the flexibility of the system

design and modular configuration of electronic and pneumatic hardware. One future work

could be simultaneously synthesizing multiple oligonucleotides within the portable

oligonucleotide synthesizer, which can significantly improve the throughput of the

portable system.

This task could be done by designing a new synthesis reactor in which microvalves are

integrated. The microvalves within the synthesis reactor can be used to selectively deliver

synthesis reagents to certain reaction chambers (especially different phosphoramidite

monomers during coupling reaction. All other reactions are universal regardless of the

sequence to be synthesized, thus no selective delivery of reagents is required). The

coupling reactor will occur in the chambers where coupling reagents are delivered.

Therefore, multiple oligonucleotides with different sequence could be obtained by the

proposed new reactor.

Another possible way to simultaneously synthesize multiple oligonucleotides, especially

for oligonucleotide probe, is to adopt a photochemical synthesis approach (reviewed in

2.1.2.3). A selective light excitation unit, such as an addressable LED array, could be

designed and incorporated into the system. The excitation unit could selectively activate


100

certain regions on the flat solid support surface to remove the 5’-OH protecting group.

The surface area with proper activation could then couple with the incoming

phosphoramidite. Therefore multiple in situ synthesized oligonucleotide probes could be

obtained.

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101

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114

Appendix A List of Components for the Portable System

No. Component QTY Company Product No. / Model Remark

Oligonucleotide Synthesizer

1 Computer * 1 Sony VGN-P25G 2 Paintball Tank 1 Carleton 6310-47416 Store argon gas 3 Tank on/off Valve 1 Custom Products Mini ASA Adapter Tank main valve 4 Grip Paintball Regulator 1 Custom Products Short Black 2000 psi to 20 psi 5 Macroline Hose Kit 1 -------------- -------------- 6 CompactDAQ Chassis * 1 NI NI cDAQ-9172 7 Sourcing Digital Output * 1 NI NI 9476 Function as relay 8 Low Pressure Regulator 1 1 Marsh Bellofram 962-036-000 Fluids driven pressure 9 Low Pressure Regulator 2 1 Marsh Bellofram 962-083-000 Valve control pressure 10 Manifold 10 Stations 2 Pneumadyne MSV10-10 11 Solenoid Valve 2 Way 9 Pneumadyne S10MM-20-12-3 Normally Close 12 Solenoid Valve 3 Way 10 Pneumadyne S10MM-31-12-3 Normally Open 13 6V Battery * 2 Powerfit NAS30601D2VW0SC 14 Micro-Boxer Pumps 1 Cole-Parmer EW-79600-16 Vacuum to open valve 15 Inline Filter 10 Upchurch A-425 Fluids filter 16 FEP Tubing 1 Cole-Parmer 06406-60 Fluids tubing 17 Polyurethane Tubing 1 Pisco UB 0640-20B Pneumatic tubing 18 Polyurethane Tubing 1 Pisco UB 1810-20B Pneumatic tubing 19 Tube Fitting 19 Pisco PC 180-M5M Fittings for 18 & 10 20 Tube Fitting 1 Pisco PY 6M Fittings for 17 21 Tube Fitting 2 Pisco PLJ 6M Fittings for 17 22 Tube Fitting 1 SMC KJS01-32 23 HPA Fill Station 1 PMI CGA 580 Argon tank fill station 24 Aluminum Suitcase 1 RIMOWA Carrying case 25 Aluminum Rack 1 Home-made Mounting

115

26 Switches, cables, others various Electronic accessories 27 PDMS microvalve chip 1 Self-fabricated Microfluidic valves 28 Controlling software 1 NI Control

Fluorescence Detection System

29 Universal Analog Input 1 NI NI 9219 Measure signal from photodiode and PT 100

30 LED 1 Lumileds LXHL-PW01 White, 1W 31 Stardrive LED driver 1 Sumatec 1W Constant current source 32 Lense 3 Edmund Optics 47343 33 Excitation Filter 1 Chroma HQ575/50x 34 Emission Filter 1 Chroma HQ645/70m-2p 35 Beam Splitter 1 Chroma Z594/750rpc 36 Photodiode 1 Hamamatsu S8745-01 Built-in pre-amplifier 37 Filter cube 1 Olympus UMF-2 Mounting 38 PT100 Temp Sensor 1 RS 362-9834 For hybridization 39 Heater Mat 1 RS 245-499 For hybridization 40 Aluminum Housing 1 Home-made Housing

* Shared components that are also used in fluorescence detection system

116

Appendix B Assignment of NI 9476

Channel Connection Control String Remark DO0 A1

3 Washing DO1 B1 DO2 A2

12 Monomer A DO3 B2 DO4 A3

48 Monomer T DO5 B3 DO6 A4

192 Activator DO7 B4 DO8 A5

768 Monomer G DO9 B5

DO10 A6 3072 Monomer C

DO11 B6 DO12 A7

12288 Detritylation DO13 B7 DO14 A8

49152 Oxidation DO15 B8 DO16 A9

196608 Argon DO17 B9 DO18 A10 262144 Hybridization

DO19~30 NOT USED DO28

Heater 805306368 ----- DO29 DO30 LED 1073741824 ----- DO31 Pump 2147483648 Vacuum

Remarks: A1 is solenoid valve 1 mounted on aluminum manifold A to control the operation of microfluidic valve; B1 is solenoid valve 1 mounted on aluminum manifold B to drive oligonucleotide synthesis reagent; Heater is connected to both DO28 and DO 29 as working current of the heater is larger than the maximum current allowable in a single channel.

Pin Layout of NI 9476 – Digital output module

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Appendix D List of Publications

1. Chen WANG and Dieter TRAU, A portable generic DNA bioassay system based

on in situ oligonucleotide synthesis and hybridization detection. Biosens.

Bioelectron., 2011. 26(5): p. 2436-2441.

2. Chen WANG and Dieter TRAU, Zero dead-volume microfluidic valves for

minimal cross contamination and its application in oligonucleotide synthesis.

(Submitted)

3. Chen WANG and Dieter TRAU, Microfluidic device for oligonucleotide synthesis

in the field. Joint 6th Singapore International Symposium On Protection Against

Toxic Substances and 2nd International Chemical, Biological, Radiological &

Explosives Operations Conference, 8-11 December 2009, Singapore

4. Chen WANG and Dieter TRAU, Microfluidic device for oligonucleotide synthesis

and bioassays in the field. 20th Anniversary World Congress on Biosensors, 26-28

May 2010, Glasgow, UK

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