指導教授：沈弘俊臺灣大學：應用力學研究所范育睿Fan, Yu-JuiYu-JuiFan2014-11-302018-06-292014-11-302018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/264101本篇論文是研究一個微光學元件整合的微流體晶片，並用於水珠、粒子、與多種細胞的高效能分析。這個微流體晶片是整合具有高折射率的微球透鏡於微流道晶片內，以提升螢光收集效益和平行檢測機制。在水珠檢測方面，透過一個水珠產生器在微流道上游產生水珠，並在下游將流道分枝為64個支流。在粒子和細胞檢測方面，細胞由入口處進入後，分支為32個平行流道，每個流道都有一對側向流，用以將細胞或粒子聚集成一條線排列。平行的32個流道同時檢測細胞實驗中，總效能可達每秒385,400 細胞檢測。而在這篇論文中，使用的微球形透鏡折射率為2.1，因此可以達到很短的焦距。當透鏡直接埋於流道底部，焦點仍然可以座落於流道內部。此方法提供可以使用較密集的流道，因此晶片可以較小，而同樣大小的雷射光點和相機的視野範圍，可以同時分析較多的流道。使用三維的微流道製程，可以提供每個流道都有一組側向流道，且不會增加側向流入口。這個方法解決了微流道在同時載入多流體時，需要解決的載體入口問題。而只要用一個側向流入口來供給64個側向流道、一個樣品入口來提供32個流道樣品流入、和一個出口，來達到同時32個流道檢測。This thesis reports the microfluidic devices with parallel detection channel embedded microoptics array for versatile high throughput multicolor fluorescence detection. These devices are realized by utilizing solid immersion micro ball lens arrays for high sensitivity and parallel fluorescence detection. For droplet detection, the droplets, produced by a droplet generator in the upstream of device, randomly flow into 64 parallel channels. For cell detection, the cells randomly flow into 32 channel, in which cells got aligned in the center stream. A total throughput of 358,400 cells/s has been accomplished. High refractive index (R.I.) micro ball lenses (R.I.=2.1) are embedded underneath PDMS channels close to cell detection zones in channels. This design permits patterning high N.A. micro ball lenses in a compact fashion for parallel fluorescence detection on a small footprint device. This cell detection device also utilizes 3D microfluidic fabrication to address fluid routing issues in two-dimensional parallel sheath focusing and allows simultaneous pumping of 32 sample channels and 64 sheath flow channels with only two inlets.口試委員會審定書 # 誌謝 i 中文摘要 ii ABSTRACT iii CONTENTS iv LIST OF FIGURES vi LIST OF TABLES x Chapter 1 Introduction 1 1.1 Introduction 1 1.2 Motivations and objectives 3 1.3 Method and procedure 4 1.4 Thesis structure 4 Chapter 2 Literature review 7 2.1 History of flow cytometry 7 2.1.1 Flow cytometry 7 2.1.2 Cell sorter 10 2.1.3 Fluorescence label 12 2.2 Conventional flow cytometer 13 2.2.1 Fluidic system 13 2.2.2 Optical system 17 2.2.3 Light detection 18 2.3 Microfluidic flow cytometer 23 2.3.1 History of Microflow cytomter 23 2.3.2 Flow focusing technique 24 Chapter 3 System Layout 28 3.1 Embedded Microball Lens Array 31 3.1.1 Fabrication of embedded microball lens array 31 3.1.2 Simulation of microball lens 33 3.2 Microfluidic system 40 3.2.1 Microfluidic channel fabrication for droplet detection 40 3.2.2 Microfluidic channel with 2D flow focusing 41 3.2.3 Parallel and 3D flow focusing microfluidic channel 46 3.3 Optical system 49 Chapter 4 Experimental results 52 4.1 Droplet detection 52 4.1.1 Droplet generator 53 4.1.2 Fluorescent droplet detection 53 4.2 Fluorescent bead calibration 55 4.2.1 Fluorescent bead detection 55 4.2.2 Multicolor beads detection 57 4.3 Cell detection 59 4.3.1 Protocol of cell stain 59 4.3.2 Cell flow into sample 60 4.3.3 Multiple cell detection 62 Chapter 5 Conclusions 65 REFERENCES 68 LIST OF FIGURES Figure 1.1 The 2D plot of throughput-information 2 Figure 1.2 The objective of the study is to increase throughput of flow cytometer. 4 Figure 2.1 The main structure of fluidic system [3]. 15 Figure 2.2 Some aspects of fluid flow in a commercial flow cytometer [3]. 16 Figure 2.3 The N.A. of the objective lens w/o oil immersion [3]. 18 Figure 2.4 Prism for light separation [3]. 20 Figure 2.5 Dichroic mirror for light separation [3]. 21 Figure 2.6 Three ways using dichroic mirror to separate light with different wavelength [3]. 21 Figure 3.1 The process flow for fabricating solid immersion microball lens array in PDMS. 32 Figure 3.2 (a), (b) Images of a 100×100 microball lens array across a 1.3 cm × 1.3 cm area. (c) The SEM images showing the thin PDMS layer between embedded ball lenses and a channel. 33 Figure 3.3 (a) Optical parameters of a ball lens. The simulation results show that parallel light rays propagating through a 753809120 bytesapplication/pdf論文公開時間：2019/08/25論文使用權限：同意有償授權(權利金給回饋學校)高效能高靈敏度三維微流道微透鏡陣列微流流式細胞儀thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/264101/1/ntu-103-D97543004-1.pdf