中文摘要
循環腫瘤細胞 (CTCs) 被視為一種良好的癌症診斷工具，其來自原發性癌症轉移至血液中經血管在人體內循環，循環腫瘤細胞的量非常的少以至於非常難以由全血中分離出來，在最近已經有期刊刊載利用微流體平台來分離血液循環腫瘤，但是為了增強循環腫瘤細胞的回收率，我們研發出新的微流體晶片包含適體 (aptamer) 可以專一性的抓取目標細胞，做到紅血球分解、負向性篩選出白血球分離，再利用正向性篩選血液循環腫瘤並萃取出來，這所有的流程將自動化沒有人為操作影響下，在單一晶片一小時內完成，比較傳統的結構篩選方法，新的微流體篩選系統可以適用於癌細胞診斷與提供更高的循環腫瘤細胞回收率，此外，高專一性的適體也許不會造成錯誤抓取的現象也在本篇文章中使用，結果將在這篇文章中報導，更重要的是，分離出的循環腫瘤與抓錯的白血球的比例，比較於傳統的方式，效率提高了非常得多。
經過篩選出來的血液循環腫瘤，將會被傳送到生物場效電晶體偵測區，在此我們將發表利用生物場效電晶體來偵測臨床血液檢體中癌細胞個數之技術，為了提高計算的準確度，我們設計出一個流體三維聚焦的結構適用於被磁珠抓取而篩選出的細胞一顆一顆的經過場效電晶體之偵測區域，此外後端設有微型轉換元件，將目標細胞與其他雜質做分離，實驗結果顯示，生物場效電晶體可以分辨磷酸鹽緩衝食鹽水（PBS對照組）緩衝液和含有癌細胞之溶液。場效電晶體已整合到微流體控制系統，使得細胞的分離，計數和收集可以自動化。為了讓細胞一個接一個通過生物場效電晶體感測區。我們設計微流道結構可聚焦流體，可以限縮流道至20 µm恰好通過感測區。因此，來自生物場效電晶體可以偵測到利用血液檢體中的癌細胞。微流體系統可利用單一晶片具有許多功能。本研究可以提供一個有效的工具，以適應臨床應用的需要。

Abstract
　 Circulating tumor cells (CTCs) are being significantly explored as a potential tool for cancer diagnosis with high sensitivity and specificity. However, the detection of CTCs is a challenge because of the difficulty in isolation from whole blood as they are shed into the vasculature from primary tumor and circulate irregularly in the bloodstream. Recently, microfluidic platforms have been utilized for the isolation of CTCs. To improve the recovery rate of CTCs, we, herein, report a new integrated microfluidic system capable of performing red blood cells (RBCs) lysis, which is a negative selection process for depleting white blood cells (WBCs) accompanied by a positive selection process for specifically capturing target cancer cells to effectively detect CTCs from the blood by cancer cell-specific aptamers. A single miniature chip has been used to perform the entire process in one hour without human intervention. Our integrated microfluidic system shows higher efficiency for the isolation of CTCs in order to effectively diagnose the cancer cells in short time when compared to the traditional negative or positive selection approach. Furthermore, all possibilities of “false positive” results could be overcome by the specificity of the aptamer. In order to improve the accuracy of the process of detecting CTCs from human whole blood after the process of isolation, a single cell captured by magnetic beads was allowed to pass through the field-effect-transistor (FET) sensing area by means of a flow-focusing device. The detected cells from the FET area were collected using a flow-switching device for subsequent operation. Experimental results showed that the spiked cancer cells could be differentiated effectively from phosphate-buffered saline (PBS, negative control) buffer. Furthermore, the FET was successfully integrated into the microfluidic control thereby making it feasible for cell isolation, cell detecting, and automated collection. In order to facilitate the passage of cells through the FET one by one precisely, a microstructure with flow focusing mode was fabricated to accurately focus a flow stream. The flow stream with a width limitation of 20 µm was designed to deliver cells to the FET area by means of a pump. Thereafter the number of cancer cells captured from whole blood was determined by analyzing the FET sensor signal. The results of this work prove that a new integrated microfluidic system is an effective alternative to detect CTCs incorporated in a single chip and it shows promise for further development as a biosensor for clinical applications in the future.

Table of Contents
Abstract I
中文摘要 III
Table of Contents IV
List of Figures VI
Nomenclature and Abbreviations x

Chapter 1 Introduction 1
1.1. Circulating Tumor Cells 1
1.2. Aptamer-Facilitated Biomarker Discovery 3
1.3. MEMS and Field-effect Transistors 4
1.4. Motivation and Novelty 6
Chapter 2 Design and Fabrication 10
2.1. Integrated Microfluidic Chip for Isolation of Circulating Tumor Cells 10
2.2. Fabrication Process 16
2.3. Integrated Microfluidic Chip and Field-effect Transistors Sensing System …………………………………… 17
Chapter 3 Materials and Methods 20
3.1. Preparation of the Experimental Materials 20
3.1.1. Preparation of Cancer Cell Line BG-1 20
3.1.2. Preparation of Specific Aptamer Coated Beads21
3.1.3. Whole Blood Pretreatment 23
3.2. RBC (red blood cell) lysis test 23
3.3. Aptamer and Antibody Specificity Test 24
3.4. Flow Focusing and Integrated Microfluidic System 27
Chapter 4 Results and Discussion 28
4.1. Results of RBC Lysis Efficiency 28
4.2. Results of Leukocyte Depletion Efficiency 31
4.3. Results of Cancer Cell Capture Recovery Rate 35
4.4. False positive and False Negative Ratio 45
4.5. Flow Focusing Test and Analysis of Field-effect Transistors Electronic Signals…….…………………………………………………………… 52
Chapter 5 Conclusions 57
References

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