||Bio-sample Pretreatment Utilizing Optically-induced Dielectrophoretic Force and Electroosmosis Effects
||Department of Engineering Science
Hydrogenated amorphous silicon
optically-induced electroosmosis (OEOF)
electrical double layer (EDL)
optically-induced dielectrophoretic (ODEP)
近年來，生物臨床樣品之前處理步驟逐漸於醫學檢測中扮演著十分重要的角色。在生醫檢測系統中，由於往往需要針對檢體當中的某種特定目標做採樣研究，這些目標微粒子可能是細胞、RNA病毒(RNA-based viruses)或是細菌等，可藉著不同種類的粒子間各種相異的特性將之區別開來，像是比重、表面性質、尺寸大小等等。一般最常使用的分離方法，即是藉著不同種類細胞之間不同的尺寸大小來做分離排列，因此，在本研究中利用光介電泳力 (optically-induced dielectrophoretic force, ODEP)可同時操作多顆粒子，也可只針對單一特定的微粒子進行挑選的優異操作能力，設計並發展出一個生物樣品前處理的平台，使得檢測的靈敏度可藉由目標微粒子的濃縮萃取而大幅提升，並可避免因繁複的人工操作步驟增加檢體的浪費跟造成汙染等問題。
因此，本研究提出一個ODEP晶片系統，利用投影機投出的影像，於光感材料非晶矽(Hydrogenated amorphous silicon)為基底的晶片上形成虛擬電極(virtual electrode)，藉由影像軟體來控制虛擬電極之圖型與移動變化，即可針對檢體中的微粒子進行任意操作。也由於虛擬電極產生的介電泳力強度可被投射出的光之顏色、線寬度以及照射強度等影響，本研究也因此設計出一個全新的分離方式，依據不同尺寸之粒子大小加以快速分離。原則上，將投影機所投影出兩條線狀圖案於晶片上形成虛擬電極，透過控制投影的光之顏色、線寬以及照射強度等變因，在這兩道光線所在的區域分別產生了不同強弱之負的介電泳力。也因此，存在於兩道光間的微粒子便受到斥力的推動。然而當這兩道光之間的距離不斷的接近時，微粒子便會因受到排斥而被推擠，當所形成之斥力區間也隨之越來越小時，位於這其中的體積較大的粒子之尺寸已超過這兩道光之間構成的斥力區間，便會因為無法容於此空間當中而被推擠。根據慣性，此時粒子會朝其所受阻力較小的方向逃出，也因此尺寸較大的微粒子便會由強度較弱的那一側彈出，最後便可使得兩種具有不同大小尺寸的粒子被區隔開在虛擬電極之兩側，因而成功達成微粒子的分離之目的。
此外，這個前處理平台除可藉著虛擬電極產生介電泳力對微粒子進行操作外，當操作頻率控制於一較低的狀態下，於照光區域會因晶片與檢體間的電雙層(electrical double layer, EDL)的存在，便會開始積聚電荷而與電場交互作用產生了庫倫力，進而帶動了整個液體之流動，此擾流則稱之為光導產生之電滲流效應(optically-induced electroosmosis, OEOF)。因此藉由這項技術，本平台亦可被發展成一個微流體混合器 (micro-mixer)進行生物檢體之混合。藉由披覆具有鍵結高專一性及靈敏性之抗體的磁珠(Antibody-conjugated magnetic beads)，經由流體所造成之擾流將目標之病毒或病原菌黏附於磁珠的表面，再藉著外加的磁場將磁珠吸附，即可將目標粒子於檢體中分離出來。藉由此步驟，目標病毒或是病原菌等奈米等級之粒子就可被快速的集中且純化。因此，本平台更解決了傳統上介電泳力很難直接對小於1 m的粒子進行有效操作的缺點。
This current study presents new methods to accurately separate micro-particles with different sizes using optically-induced dielectrophoretic (ODEP) forces. It is found that the strength of the ODEP force induced on the hydrogenated amorphous silicon surface is determined by color, line-width and intensity of the optical beams, which provide an innovative design for particle separation. Two linear-segment virtual electrodes which produced the ODEP forces were firstly defined by illuminating lights onto a photoconductive chip. One moving line and one stationary illuminated line were used to generate a stronger and a weaker ODEP force, respectively. The micro-particles were then continuously pushed forward by the stronger ODEP force. As these lines approached each other, larger micro-particles entrained by the higher ODEP forces were squeezed through the stationary electrode and subsequently separated from the smaller particles. With this approach, continuous particle separation can be automatically achieved within a few seconds. This developed method may be promising for a variety of applications such as cell-based assays and sample pre-treatment using micro-particles.
Generally, magnitude of ODEP force is not strong enough to trap particles less than 1 μm. In this study, antibody-coated magnetic beads were applied for the extraction of viruses from clinical samples prior to bead separation. Target viruses can be specifically recognized and attached onto the surface of antibody-coated beads through a mixing process for achieving incubation, then followed by isolating and separating the magnetic beads from the solution. Then this developed platform can be used as a micro-mixer utilizing optically-induced electroosmosis (OEOF) flow to generate a circulation flow in the bio-sample. This is due to the fact that the impedance of the EDL (electrical double layer) capacitor existing between the photoconductive surface and the liquid layer becomes larger and starts to accumulate charges on the surface, resulting in a Coulomb force at a lower frequency range. Therefore, this OEOF mixer can be used as a micro-mixer for bio-sample incubation. As a result, this developed platform for bio-sample pretreatment can be widely applied in any different sized micro- and nano- particles utilizing the optically-induced dielectrophoretic force and ac electroosmosis flow. In the near future, different types of viruses can be incubated and conjugated onto the magnetic beads with different sizes by using OEOF mixing effect, followed by separated and concentrated the magnetic complexes according to the sizes utilizing ODEP forces. Therefore, this developed platform can realize the biological pretreatment with multiple kinds of viruses in an automatic fashion.
摘 要 III
TABLE OF CONTENTS VII
LIST OF FIGURES IX
CHAPTER 1: INTRODUCTION 1
1.1 MEMS and Microfluidic Technology 1
1.2. Background and Literature Survey 2
1.2.1 Introduction of mechanisms for cell and micro-particle separation 2
1.2.2 Dielectrophoresis (DEP) force for particles separation 5
1.2.3 Optoelectronic tweezers (OET) system and application 6
1.2.4 Antibody-coated magnetic beads in biological applications 8
1.2.5 AC electroosmotic micro-mixer 10
1.3. Motivation and Objectives 11
CHAPTER 2: THEORY AND DESIGN 18
2.1 Principle of Optically-Induced Dielectrophoresis 18
2.2 Amorphous Silicon as a Photoconductive Material 21
2.3 Optically-induced dielectrophoresis (ODEP) Force 24
2.4 Optically-induced AC electroosmosis (OEOF) 26
2.5 Purification Utilizing Antibody-conjugated Magnetic Beads 29
CHAPTER 3: MATERIALS AND METHODS 34
3.1 Overview of the Biological Pretreatment System 34
3.1.1 Separation process utilizing spatial difference of ODEP forces 34
3.1.2 Biological incubation utilizing OEOF 36
3.2 Major Operation Parameters on the Strength of ODEP 39
3.2.1 Effect of wavelength on the strength of ODEP force 39
3.2.2 Effect of line-width and brightness on the strength of ODEP force 40
3.3 Fabrication and Experimental Setup 41
3.3.1 Formation of the ODEP and the OEOF Platforms 41
3.3.2 Sample preparation and experimental setup 42
CHAPTER 4: RESULTS AND DISCUSSION 51
4.1 Particle Separation Using Different Colors 51
4.2 Particle Separation Using Different Line-widths 56
4.3 Particle Separation Using Different Light-intensities 58
4.4 Incubation of Dengue Virus Using OEOF 59
CHAPTER 5: CONCLUSIONS AND FUTURE WORK 72
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