臺灣博碩士論文加值系統

快速、靈敏、高專一性與可攜帶式之微型化生醫診斷晶片已成為近年來極受矚目的研究方向，其中更以微流體技術與電驅動力學關鍵技術為基礎者，最被認為適合於發展成微型化之檢測裝置。本論文主要以交流電場誘發的介電泳(Dielectrophoresis)力為本，架構並實現了四個主題之連續微流體晶片系統，包含生物粒子聚焦晶片、前處理分離晶片、DNA雜交檢測晶片以及光學檢測即時鑑定之生物晶片。研究內容主要包含以下四項:
(1)高流量旅波式介電泳(traveling wave dielectrophresis)分離晶片: 用以對生物粒子進行連續性的分離，設計旅波介電泳場垂直於連續流場而有效增加旅波介電泳場的作用時間進而加速分離的速度，可成功的以流量10??l/min (流速約5 mm/sec)有效的對血球細胞進行操控，相較於傳統介電泳分離晶片其速度大約增加了近一個數量級，並成功的將細菌與血球聚焦與分離至其特定的子流道，分離的效率可達將近80%以上，尺寸分佈寬廣的脂質體之微小覆膜顆粒(50 nm~6??m)亦可成功的被分選成2.3-4.6??m與 1.5-2.7??m兩個分佈。
(2)高流量生物粒子聚焦晶片: 設計兩電極陣列以雙向旅波介電泳場產生與連續流場垂直之側向移動力將粒子聚集至流道中央而使其聚焦，完全不需額外的邊梢流(sheath flow)，單純以小電壓產生之電場進行驅動且可達高流速聚焦，極適合與其他技術整合成可攜式檢測裝置，血球細胞與念珠菌可成功的於高流速(10??l/min)下僅以14 Vp-p之交流電壓有效的聚焦成一寬度約為10??m左右的粒子流，流速於10??l/min以內聚焦效率可達90 %左右。
(3)一種新穎機制用於促進DNA雜交之速度、靈敏度與選擇度: 以奈米粒子組裝造成局部電場增強所誘發之介電泳力可於連續流場中有效的操控DAN分子，並藉由高剪應力之流場與適當的介電泳力強度產生一種新穎機制將雜交反應聚集於一小點(~100??m2)並可由連場之剪應力移除大部分的非特異性吸附，可大幅增進DNA雜交之靈敏度與選擇度，而大幅增加螢光檢測的靈敏度(僅需106 molecules即可被測得)，100 pM跟 1 pM濃度之DNA檢體可分別於40秒與15分鐘內於本晶片被檢測出來。完美配對與單核酸變異的DNA序列亦可成功的於此晶片中被檢測出來，由於微管道內高剪應力的影響，可移除大部分的非特異性吸附，兩者雜合後之螢光強度差異將近八倍，可輕易的被判別。將設計稍做改變亦可成功的檢測多種不同菌種之念珠菌，達到多目標檢測的目的(multi-target detection)。
(4) 線上即時拉曼檢測之晶片: 最後，本論文也嘗試以非抗體式並且不需破壞細胞樣本之檢測方法直接進行菌種鑑定，主要於微流道上層製作一粗糙金屬表面用以進行表面增顯拉曼光譜之量測(Surface Enhanced Raman Scattering, SERS)，以介電泳力將目標菌種由血球細胞中分離出來並與濃縮聚集於晶片中的粗糙金屬表面處，再以拉曼光譜經由粗糙表面的增顯所測得之增顯拉曼訊號進行細菌指紋的菌種鑑定，增顯效果相較於一般拉曼訊號增強了一個數量級以上，綠膿桿菌與金黃色葡萄球菌可成功的於晶片中量測到可供辨識彼此的增顯圖譜，此小型化微流體生醫檢測晶片對於實驗室晶片、現場檢測裝置(field-use assay)與重點照護檢測(point-of-care)之整合將形成莫大助益.

A rapid, sensitive, high specificity, high throughput and portable bioassay kit is a popular research goal for clinical diagnosis. Microfluidics and electrokinetics are the key techniques that are very suitable for development of a miniaturized device. In this dissertation, AC dielectrophoresis (DEP) was used to manipulate bio-particles and molecules in an open-flow microfluidic chip, which achieved sample pretreatments, genetic detection and real-time identification of bacteria. There are four projects included in this dissertation as list in the following:
(1) High throughput sorting of bio-particles: Bio-particle sorting based on traveling-wave dielectrophoresis (twDEP) that provides the lateral displacements of particles in a continuous flow and long residence time for migrating particles to their specific sub-channels. The throughput is promoted four times and linear velocity is one order of magnitude increased over conventional paired-DEP sorter. Approximately 80% sorting efficiency were achieved at the flow rate of 10 ?m/min. Effective focusing and separation of red blood cells from debris-filled heterogeneous samples are demonstrated, as well as size-based separation of poly-dispersed liposome suspensions into two distinct bands at 2.3 to 4.6 ?m and 1.5 to 2.7 ?m, at the highest throughput recorded in hand-held chips.
(2) High-throughput electrokinetic bioparticle sorting: A sheathless, portable microfluidic chip capable of high-throughput focusing bio-particles based on 3D twDEP field is proposed and demonstrated. High-throughput focusing is achieved by sustained a centralized twDEP field normal to the continuous flow direction. Red blood cells and fungus cells (Candida) can be effectively focused into a narrow particle stream (~10 ?m) below a critical flow rate of 12 ?l/min. Approximately 90% focusing efficiency for blood cells can be achieved within two 6 mm electrode arrays when the flow rate is below 12 ?l/min.
(3) On-chip DNA detection: A novel mechanism enhanced sensitivity and selectivity for rapid DNA hybridization in an open-flow system has been demonstrated and investigated. Molecular dielectrophoresis (DEP) is employed to rapidly (＜ms) trap ssDNA molecules in a flowing solution to a cusp-shaped nanocolloid assembly on a chip with a locally amplified AC electric field gradient. The optimum frequency and flow rate for the corresponding hybridization rate and effective discrimination were studied in this research by tuning AC field frequency. 100 pM and 1 pM DNA concentration can be detected within 1 min and 15 min respectively. High shear rate enhanced discrimination that enables to distinguish single mismatch and perfect match DNAs within the range of optimal flow rate operated due to continuously removing almost nonspecifically bound molecules. Multi-target detection for indentifying Candia species was also achieved successfully.
(4) Label-free bacteria identification without cell destruction: A label-free on-chip surface enhanced Raman scattering (SERS) analysis is also designed that can identify pathogenic bacteria without complicated sample preparations based on their Raman spectrums as the fingerprints. Bacteria can be continuously isolated via DEP out of a sample of blood cells and concentrated to the localized roughened metal surface for SERS detection. The resulting shows very evidence peaks and very difference of Raman spectrums for discriminating Gram positive (Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa) bacteria.
These DEP-based microfluidic techniques would achieve rapid positive-negative screening for tending to a field-used assay and point-of-care diagnostic kit.

Abstract I
中文摘要 III
致謝 V
Table of contents VII
List of Tables X
List of Figures XI

Chapter 1 1
Introduction 1
1.1 Review of Bio-detection Methods 1
1.1.1 Cell separation 1
1.1.2 Identification methods of microorganism 2
1.2 Bio-MEMS applications for bio-sensing technology 3
1.2.1 Introduction of bio-MEMS 3
1.2.2 Micro-fabricated devices for bio-applications 4
1.3 Electrical cell manipulation theory 6
1.3.1 Dielectrophoresis 6
1.3.2 Travelling-wave dielectrophoresis 8
1.4 Motivations and objectives 11
1.5 Thesis organization 13
Chapter 2 16
A Continuous High-throughput Bio-particle Sorter Based on 3D Traveling-Wave Dielectrophoresis 16
2.1 Introduction and motivation 16
2.2 Material and methods 18
2.2.1 Theory 18
2.2.2 Chip design and operation 20
2.2.3 Micro-fabrication 22
2.2.4 Sample preparation 23
2.3 Results and discussions 25
2.3.1 Frequency and voltage conditions 25
2.3.2 Cell manipulations 28
2.3.3 Cell separation and liposome sorting 31
Chapter 3 35
High-throughput Electrokinetic Bio-particle Focusing Based on a Travelling-wave Dielectrophoretic Field 35
3.1 Introduction and motivation 35
3.2 Material and methods 37
3.2.1 Theory and Chip design 37
3.2.2 Sample preparation 39
3.3 Results and discussion 40
3.3.1 Frequency and voltage conditions 40
3.3.2 Travelling wave DEP Focusing of bio-particles 41
3.3.3 Focusing efficiency 46
3.3.4 Focusing and sorting bio-particles 49
Chapter 4 51
A Novel Mechanism Enhanced Sensitivity and Selectivity for Rapid DNA Hybridization 51
4.1 Introduction and motivation 51
4.2 Material and methods 53
4.2.1 Chip design and operation 53
4.2.2 Theory and the novel concepts 55
4.2.3 Micro-fabrication 59
4.2.4 Sample preparation 60
4.3 Results and discussion 61
4.3.1 Tests of on-chip hybridization and its selectivity 61
4.3.2 Frequency dependency hybridization 63
4.3.3 Flow rate effects and shearing enhanced discrimination 70
4.3.4 Multi-target detection 74

Chapter 5 79
A Dielectrophoretic Chip with a Roughened Metal Surface for On-chip SERS Analysis of Bacteria 79
5.1 Introduction and motivation 79
5.2 Material and methods 81
5.2.1 Chip design 81
5.2.2 Sample preparation 83
5.2.3 Micro-fabrication 83
5.2.4 Experimental setup 84
5.3 Results and discussions 86
5.3.1 AFM images of the roughened surface 86
5.3.2 SERS on a roughened substrate 87
5.3.3 On-chip SERS analysis 90
Chapter 6 95
Conclusions and Future Applications 95
6.1 Conclusions and overview of the dissertation 95
6.2 Future works and applications 98
References 101
Personal Information 108

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