DNA oligonucleotide synthesis in a microdevice for multiple analytical applications

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       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. i      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. ii      Table of Contents ACKNOWLEDGEMENTS················································································································ I  TABLE OF CONTENTS ················································································································· III  SUMMARY········································································································································ VI  LIST OF TABLES ························································································································· VIII  LIST OF FIGURES ·························································································································· IX  ABBREVIATIONS ··························································································································XII  CHAPTER 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 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  iii      CHAPTER 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 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 Result and discussion····························································································· 69  4.4.1 PCR primer synthesis ····················································································· 69  iv      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 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 Result and discussion····························································································· 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 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  v      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 onsite 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 vi      critical issue and can lead to failure of the synthesis. Secondly, a portable oligonucleotide synthesizer based on the developed zero deadvolume 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. vii      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  viii      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 deadvolume 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. 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. 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Microbiol., 2007. 73(1): p. 73-82. 113      Appendix A List of Components for the Portable System Oligonucleotide Synthesizer No. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Component Computer * Paintball Tank Tank on/off Valve Grip Paintball Regulator Macroline Hose Kit CompactDAQ Chassis * Sourcing Digital Output * Low Pressure Regulator Low Pressure Regulator Manifold 10 Stations Solenoid Valve Way Solenoid Valve Way 6V Battery * Micro-Boxer Pumps Inline Filter FEP Tubing Polyurethane Tubing Polyurethane Tubing Tube Fitting Tube Fitting Tube Fitting Tube Fitting HPA Fill Station Aluminum Suitcase Aluminum Rack QTY 1 1 1 1 10 10 1 19 1 1 Company Sony Carleton Custom Products Custom Products -------------NI NI Marsh Bellofram Marsh Bellofram Pneumadyne Pneumadyne Pneumadyne Powerfit Cole-Parmer Upchurch Cole-Parmer Pisco Pisco Pisco Pisco Pisco SMC PMI RIMOWA Home-made Product No. / Model VGN-P25G 6310-47416 Mini ASA Adapter Short Black -------------NI cDAQ-9172 NI 9476 962-036-000 962-083-000 MSV10-10 S10MM-20-12-3 S10MM-31-12-3 NAS30601D2VW0SC EW-79600-16 A-425 06406-60 UB 0640-20B UB 1810-20B PC 180-M5M PY 6M PLJ 6M KJS01-32 CGA 580 Remark Store argon gas Tank main valve 2000 psi to 20 psi Function as relay Fluids driven pressure Valve control pressure Normally Close Normally Open Vacuum to open valve Fluids filter Fluids tubing Pneumatic tubing Pneumatic tubing Fittings for 18 & 10 Fittings for 17 Fittings for 17 Argon tank fill station Carrying case Mounting 114      Fluorescence Detection System 26 27 28 29 Switches, cables, others PDMS microvalve chip Controlling software Universal Analog Input 30 31 32 33 34 35 36 37 38 39 40 LED Stardrive LED driver Lense Excitation Filter Emission Filter Beam Splitter Photodiode Filter cube PT100 Temp Sensor Heater Mat Aluminum Housing various 1 Self-fabricated NI NI 1 1 1 1 1 Lumileds Sumatec Edmund Optics Chroma Chroma Chroma Hamamatsu Olympus RS RS Home-made NI 9219 LXHL-PW01 1W 47343 HQ575/50x HQ645/70m-2p Z594/750rpc S8745-01 UMF-2 362-9834 245-499 Electronic accessories Microfluidic valves Control Measure signal from photodiode and PT 100 White, 1W Constant current source Built-in pre-amplifier Mounting For hybridization For hybridization Housing * Shared components that are also used in fluorescence detection system 115      Appendix B Assignment of NI 9476 Channel Connection DO0 DO1 DO2 DO3 DO4 DO5 DO6 DO7 DO8 DO9 DO10 DO11 DO12 DO13 DO14 DO15 DO16 DO17 DO18 DO19~30 DO28 DO29 DO30 DO31 A1 B1 A2 B2 A3 B3 A4 B4 A5 B5 A6 B6 A7 B7 A8 B8 A9 B9 A10 Control String Remark Washing 12 Monomer A 48 Monomer T 192 Activator 768 Monomer G 3072 Monomer C 12288 Detritylation 49152 Oxidation 196608 Argon 262144 Hybridization NOT USED Heater 805306368 ----- LED Pump 1073741824 2147483648 Vacuum ----- Pin Layout of NI 9476 – Digital output module Remarks: A1 is solenoid valve mounted on aluminum manifold A to control the operation of microfluidic valve; B1 is solenoid valve 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. 116      Ap ppendiix C Sp pectrum m of Optical P Parts for f Fluorrescencce Deteection Figure C-1 Relative in ntensity vs. wavelength h of the wh hite color L LED (Luxeo on Emitters, LXHL-PW W01). Light that can pass throughh the excitaation filter is highligh hted. (From m Technical D Datasheet DS25, D Lumilleds) Figure C-2 Spectrum of o the excitaation filter, emission fiilter and beaam splitter. (Data from m www.chrom ma.com) 1177      Figure C-3 Spectral reesponse of the t photodio ode. The reegion that overlaps witth the Texass ngth is highlighted. (Frrom Datasheeet of Si Phhotodiode with w Preamp p Red emission wavelen H ATSU Photonics) S8745-01, HAMAMA Excitation Emission Waveleength (nm)) Figure C-4 Excitation and emissio on spectrum m of Texas Red R fluorophhore 1188      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 119    [...]... 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... 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. .. dimethylformamide xiii      CHAPTER 1 INTRODUCTION 1    Chapter 1 Introduction  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... 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... 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... hydrogel, sol-gel, or paraffin due to temperature, pH, or light >2s Hydrogel [75-77], Sol-gel[78], paraffin [79, 80] 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 22    ... 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 20    Chapter 2 Literature Review 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... 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... 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 . 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. 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 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