VIETNAM NATIONAL UNIVERSITY VNU UNIVERSITY OF SCIENCE _ Đỗ Quang Lộc DEVELOPMENT OF A CANCER CELL MANIPULATING AND SENSING MICROFLUIDIC PLATFORM (Nghiên cứu, phát triển hệ thống vi lỏng thao tác cảm biến tế bào ung thư) PhD THESIS IN PHYSICS Hanoi - 2019 VIETNAM NATIONAL UNIVERSITY VNU UNIVERSITY OF SCIENCE _ Đỗ Quang Lộc DEVELOPMENT OF A CANCER CELL MANIPULATING AND SENSING MICROFLUIDIC PLATFORM (Nghiên cứu, phát triển hệ thống vi lỏng thao tác cảm biến tế bào ung thư) Major : Radiophysics and Electronics Major code: 9440130.03 PhD THESIS IN PHYSICS SUPERVISOR: Assoc Prof Dr Chu Duc Trinh Hanoi - 2019 Pledge - Lời Cam đoan I hereby declare that this thesis is solely my own work The data in this thesis are the results of my personal research and have not been used in other publications by anyone else Tôi xin cam đoan cơng trình nghiên cứu riêng tơi Các số liệu, kết nêu luận án trung thực chưa công bố khác Người cam đoan/ Ph.D Student Đỗ Quang Lộc i Acknowledgement Foremost, I would like to express my sincere gratitude to my advisor, Prof Chu Duc Trinh, for the continuous support of my Ph.D study and research, for his patience, motivation, enthusiasm, and immense knowledge My research would have been impossible without his guidance and supports Besides, I would also like to express my appreciation to Dr Bui Thanh Tung whose office door was always open whenever I ran into a trouble spot or had a question about my research or writing He consistently allowed this paper to be my own work, but steered me in the right the direction whenever he thought I needed it My sincere thanks also goes to Dr Do Trung Kien He has offered me many good opportunities in my life and Ph.D study Further, I must express my very profound gratitude Prof Chun-Ping Jen and I would like to thank all of my friends at Department of Mechanical Engineering and Automation for their support during my internship program in National Chung Cheng University I am forever thankful to my colleagues at the Faculty of Physics for their friendship and support, and for creating a cordial working environment Last but not the least, I would like to thank my family: my mom and my sisters for encouraging and supporting me spiritually throughout my life Most importantly, I wish to thank my loving and supportive wife, Pham Thi Tuyet, and my beloved daughter, Anh Thu who provide unending inspiration ii Table of Contents Pledge i Acknowledgement ii List of Figures List of Tables Abstract 13 Introduction 17 1.1 1.2 Introduction of biological cell assay and current situation 17 1.1.1 Biological cell assays 17 Biological cell sensing systems based on microfluidic 20 1.2.1 1.3 Overview of the cancer situation and the urgency of cancer cell diagnosis 21 1.2.2 Current studies on the detection cancer cells 25 1.2.3 Material for microfluidic systems 28 Conclusion of the chapter and the direction of research 30 Biological cell manipulation methods 2.1 31 Overview of biological cell manipulation 31 2.1.1 Mechanical method 32 2.1.2 Optical method 35 2.1.3 Magnetical method 36 2.1.4 2.2 2.3 2.4 Electrical method 38 Theory and Simulation 43 2.2.1 Dielectrophoresis cell manipulation theory 43 2.2.2 Numerical Calculations and Simulations 47 Design and Experimental preparation 57 2.3.1 Fluidic chamber design 57 2.3.2 Micro-electrode structure fabrication process 57 2.3.3 Microchannel fabrication process 60 2.3.4 Cell sample preparation 63 Results and Discussions 65 2.4.1 Fabrication results 65 2.4.2 Simulation results of biological cell manipulation based on DEP 66 2.4.3 Biological cell manipulation based on DEP results 67 2.4.4 Section conclusions 69 Biological cell sensing, detection and measurement systems 3.1 3.2 3.3 3.4 70 Overview of biological cell detection and enumeration techniques 71 3.1.1 Flow Cytometry based on fluorescence activation method 72 3.1.2 Impedance Cytometry in fluidic flow 75 3.1.3 Biological cell impedance measurements in static fluidic 80 Theory and Simulation 83 3.2.1 Biological cell sensing theory 83 3.2.2 Numerical calculations 85 Design and experimental preparation 90 3.3.1 Microfluidic flow measurement and detection 90 3.3.2 Biological cell concentration and detection 95 Results and Discussions 97 3.4.1 Object detection in millimeter scale fluidic flow channel 97 3.4.2 Biological cell detection in microfluidic flow 104 3.4.3 Cell detection in fluidic chamber 112 3.4.4 Section Conclusions 118 Aptamer immobilization based on gold surface 119 4.1 Introduction 119 4.2 A549 cell line detection on aptamer immobilized gold surface 122 4.2.1 Aptamer immobilization on gold surface process 122 4.2.2 Aptamer specificity with A549 cell line experiment 123 4.3 Cell preparation results 123 4.4 A549 cell line detection on gold surface based on aptamer immobilization method 127 4.4.1 A549 cell line immobilization on gold substrate 127 4.4.2 Aptamer specification experiment with A549 cell line 128 4.4.3 Aptamer specification experiment with A549 cell line versus time 129 4.4.4 Section conclusions 130 Conclusions and Future Research 132 Publications 134 References 136 A Signal processing circuit schematic 160 B Biological cell cultivation conditions 164 List of Figures 1.1 Block diagram showing three basic steps for a CTCs cell test 21 1.2 Schematic showing the tumor metastasis through CTCs/CTM 24 1.3 Block diagram of a CTC cell separation system using magnetic nanoparticles and biological antibodies 28 1.4 Molecular structure of Polydimethylsiloxane (PDMS) 29 2.1 (A) Image scanning electron microscope system of micro gripper; (B) Bright field microscopy shows the operation of separating Ecoli K-12 bacteria from blood solutions containing red blood cells 34 2.2 Magnetic manipulation methods 38 2.3 Dielectrophoresis manipulation methods 40 2.4 Electrophoresis and dielectrophoresis: (a) The force exerted on charged particles and uncharged particles in the uniform electric field; (b) A neutral particle in a nonuniform electric field creates a total force acting on the particle because the magnitude of the electric field at the two ends of the electric dipole is different 45 2.5 Simulation program interface finite elements using COMSOL 48 2.6 Sketch view of the simulation chamber 55 2.7 Proposal of biochip sensor based on fluidic chamber 58 2.8 Process of fabricating microelectronic structure and packing with microchannel structure 59 The process of mold fabrication with SU-8 material 60 2.10 Process of manufacturing PDMS chip from SU-8 mold 61 2.9 2.11 The process of hooking up the high-precision chip to create the microchannels 63 2.12 Images of fabricated device (a) Fabricated chip; (b) working chamber; (c) fabricated electrode pattern 65 2.13 The square of the electric field (E ) on micro-electrode pairs with inward stepping electric field in a numerical simulation 67 2.14 The simulation results of the manipulation of cancer cells from a blood sample using inward (16 V of peak-to-peak voltage - MHz of frequency) stepping electric fields 68 2.15 A549 cells concentration experimental results utilizing step electric field applied to: (a) 5th electrode pair; (b) 4th electrode pair; (c) 3rd electrode pair; (d) 2nd electrode pair 3.1 69 Components and operating principles of flow cytometry in flow cell counts (photomultiplier tubes - PMT; forward scatter - FSC; side scatter - SSC) 74 3.2 Biological cell impedance sensing in fluidic flow 76 3.3 Biological cell impedance sensing in static fluidic 81 3.4 Model of coplanar capacitor 85 3.5 Meshing for fluidic chamber simulation 86 3.6 Electrical model of (a) a single-cell in suspension with complete electric circuit model; (b) single-cell in suspension with Foster and Schwan’s simplified circuit model; (c) Equivalent electric circuit of the biological cell detection structure in microfluidic channel; (d) Equivalent electric circuit of sensing system when the cell is located on one sensing electrodes pair; (e) The simplified circuit model for counting system 3.7 90 Microfluidic platform for biological cell detection in microlfuidic flow (a) Sample is mixed at mixing area (1), then separated at cell manipulation area (2) before passing through the counting area (3); (b) The detailed 3.8 design of microchip proposed for cell impedance cytometry purpose 92 Block diagram of the measurement setup 94 3.9 Block diagram of proposed dielectrophoresis microfluidic enrichment platform with built-in capacitive sensor for circulating tumor cell detection 96 3.10 Measurement setup: (a) measurement methods; (b) measurement setup and (c) controlling board 98 3.11 Block diagram design of the DC4D fluidic sensor 100 3.12 (a)The DC4D output voltage response when a plastic particle crosses electrodes water channel and salt solution channel; (b) The DC4D output voltage amplitude versus particle volume in various concentration of salt solution 100 3.13 (a) Capacitance change of sensor with three different materials; (b) Object’s diameters are 25 µm and Maximum differential capacitance output vs particle’s volume 101 3.14 Experimental result showing the output signal when air bubbles cross the sensing area 102 3.15 Working principle of PC4D sensor system 103 3.16 (a) Variation of resonance frequency when air bubbles move through channel filled with different NaCl concentrations solution; (b) Dependence of resonance frequency change on the size of: air bubble moving through DI water channel, air bubble moving through oil channel, and water droplet moving through oil channel 104 3.17 Fabricated replaceable microfluidic chip 105 3.18 Simulation results: Admittance change when a cell with radius of 10 µm moves across the sensing electrodes in: (a) Sucrose 8.6 % solution; (b) PBS 1X solution 107 3.19 Simulated admittance change according to: (a) the cell’s vertical position; (b) the cell’s radius 108 3.20 Output signal variation when an A549 cell passing over sensing electrodes (Insets are the position of the cell corresponding to the 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12(14):2560–2567 159 Appendix A Signal processing circuit schematic 160 161 Figure A.1: Schematic diagram of dielectrophoresis manipulation control circuit 162 Figure A.2: Schematic diagram of signal processing circuit for cell detection in fluidic flow 163 Figure A.3: Schematic diagram of signal processing circuit for cell detection in fluidic hamber Appendix B Biological cell cultivation conditions In the biological cell cultivation, it is often divided into two methods of culture depending on property of each cell line including adherent culture (i.e., cells often form a single layer on the surface) and suspension culture (i.e., cells cultivation in form of floating in solution) There are several characteristics in adhesion culture: suitable for most cell lines including primary culture; requires a frequent transfer (periodically) but is easy to check under a inverted microscope; cells are separated from cultivating surface by enzymes or mechanically; requires surface treated culture tools; often used in cytology and many other applications Meanwhile, in the suspension culture method, there will be some characteristics such as suitable for floating cells or some non-stick cell lines and easy to transplant but requires daily cell counting and cell viability monitoring The density of culture can be diluted to stimulate the growth of cells Cell growth is limited by cell density and can be cultured and maintained in a culture bottle/dish without needing to be handled but requires shaking/rotating for gas exchange When performing cell culture, it is necessary to pay attention to the parameters directly affecting the cell culture effective including culture medium, Serum, pH, CO2 atmosphere, culture temperature • Culture medium: Some basic culture media are used that contain amino acids, vitamins, inorganic salts, and carbon sources such as glucose This medium often needs to be supplemented with the serum to ensure cell lines grow well In low serum environments, some experiments use less serum due to their unwanted 164 effects However, in order to maintain cell growth, the basic environment must be supplemented with nutrients and animal-derived factors, thus reducing the amount of serum needed In a serum-free environment, this type of medium uses some nutrients and hormones to completely replace the serum Types of cells using this medium include: primary culture cells, cells used to produce recombinant proteins, some hybrid cell lines, insect cells, some cell lines used for production virus export • Serum: Serum plays an extremely important role as a source providing growth material, hormones, lipids, minerals, etc for cells when added to the basic culture environment There is a role in controlling the permeability of cell membranes and acting as lipid carriers, enzymes, micronutrients and a few elements for cells However, there are shortcomings such as high cost, difficulty in standardization, specificity, diversity and bring some undesirable effects such as stimulating or inhibiting the growth and/or function of several cell lines • pH: Under pH conditions of culture media, most mammalian cells grow well at pH 7.4 Some transformed cell lines grow well in mild acidic pH environments (7.0 - 7.4); while some fibroblast lines often develop well in mild alkaline environments (7.4 - 7.7) Insect cell lines normally grow at pH 6.2 In the case of the A549 cell culture, the pH of the medium is normally adjusted to 7.0 - 7.6 Meanwhile, CO2 conditions in some culture environments control pH by adding buffer solutions to the environment such as HEPES or bicarbonate (HCO3-) Thus, it can be needed to use external CO2 source with the CO2 concentration maintained in the cabinet is 5-7% Each type of environment will be suitable for specific CO2 and bicarbonate concentrations to achieve standard pH values • Temperature: Conditions of cultured temperature are also important for the cell growth process The culture temperature depends on the body temperature of the individual to which the cell is derived and from the cell origin organ The increase in heat will be much more serious than the temperature drop in culture So often set the temperature a little lower than the optimal temperature Most 165 human and mammalian cells are maintained at 30-37 ◦ C Cells from cold-blooded animals often have a wide spectrum of heat, ranging from 15 to 26 ◦ C 166 ... (Nghiên cứu, phát triển hệ thống vi lỏng thao tác cảm biến tế bào ung thư) Major : Radiophysics and Electronics Major code: 9440130.03 PhD THESIS IN PHYSICS SUPERVISOR: Assoc Prof Dr Chu Duc Trinh...VIETNAM NATIONAL UNIVERSITY VNU UNIVERSITY OF SCIENCE _ Đỗ Quang Lộc DEVELOPMENT OF A CANCER CELL MANIPULATING AND SENSING MICROFLUIDIC PLATFORM (Nghiên cứu, phát triển hệ thống vi. .. been used in other publications by anyone else Tơi xin cam đoan cơng trình nghiên cứu riêng Các số liệu, kết nêu luận án trung thực chưa công bố khác Người cam đoan/ Ph.D Student Đỗ Quang Lộc i Acknowledgement