本論文提出能同時檢測細胞/粒子螢光與數量計數之微流體細胞/粒子計數器以提供HIV流行病檢測之創新概念。第一種方法是利用雷射誘導螢光檢測(laser induced fluorescence, LIF)結合電阻脈衝檢測 (resistive pulse sensing, RPS) 方法來達成。電阻脈衝檢測系統檢測通過微流道細胞/粒子的總數量，同時螢光檢測系統只記錄經過特殊螢光劑染色的細胞/粒子數量。另外一種方法是利用光學的方法來達成細胞/粒子數量計數、大小與螢光檢測。當細胞/粒子通過檢測區時，雷射激發光所產生的前散射光訊號表示細胞/粒子的總數量和尺寸大小，而回傳的螢光訊號顯示螢光細胞/粒子的數量。
以上兩種檢測方法是利用150 �慆超薄的玻璃基板與軟微影製程所製造的poly-dimethylsiloxane (PDMS)結合而成的微流體晶片來完成，此一150 �慆超薄的玻璃基板作為檢測光纖和檢測樣品的檢測介面。本微流體晶片可以進一步應用於發展螢光激發自動計數與分類系統，本系統可經由整合電動流切換、螢光激發檢測和光學可視化系統來達成。因此，經由此構造簡單之微流體晶片結合外部不同的檢測系統可以達成多功能細胞/粒子計數、分類、螢光檢測和尺寸大小檢測。
在螢光和電阻脈衝檢測系統中分別利用兩種不同的訊號處理技術來改善訊號/雜訊比(S/N ratio)。電阻脈衝檢測系統利用結合金氧半場效電晶體（Metal-Oxide-Semiconductor Field-Effect Transistor, MOSFET）與差分放大器來改善訊號/雜訊比。由於MOSFET的工作電壓是介於次臨界區，此工作電壓範圍中MOSFET的作動比飽和區更為靈敏，因此有更大的放大效果。而另一種方法是利用一個單微流體通道搭配上下游之檢測微流道與2級差分放大器，其訊號/雜訊比(S/N ratio) 能有效獲得改善。在螢光檢測系統中，鎖相放大器(lock-in amplifier)能將埋入雜訊中的微小螢光訊號分離出來，而利用2級差分放大器可以取代體積龐大且昂貴的鎖相放大器能使雜訊被有效地抑制並改善訊號/雜訊比(S/N ratio)。
在本論文中，從0.9 �慆 到15 �慆不同粒徑的粒子、酵母菌細胞、白血球細胞可以經由不同的檢測技術進行分類與計數。螢光/非螢光的細胞/微粒子相對百分比可以經由分析螢光訊號與電阻脈衝或前散射光訊號之比值而求得。為了確認本檢測系統的精確性與可靠性，相同的檢測樣本均在商業流式細胞儀上進行檢測並與本論文所提出之檢測方法之檢測結果相互比較，所有的檢測結果精確度均相當高。因此，本技術具有相當高的可信度，並能有效的降低HIV檢測成本。

In this thesis, we propose a novel concept of micro flow cytometer for simultaneous cells/particles fluorescent detection and number counting for HIV diagnosis. One of the detection methods is based on a laser-induced fluorescence detection system integrated with a resistive pulse sensing system. While the resistive pulse sensing system determines the total number of the cells/particles, the concurrent fluorescence detection system determines only the number of cells/particles with specific fluorescent tag. The other cells/particles fluorescence detection, enumeration, and sizing was achieved by optical manner. The scattered light signal indicates the total number and the size of the cells/particles passing through the detection window, while the simultaneous backward fluorescence signal shows only the number of fluorescence particles.
Both detection methods are developed on a fiber free poly-dimethylsiloxane (PDMS) chip that is fabricated using soft lithography processes. The PDMS chip is bonded with a glass substrate of 150 �慆 in thickness that works as the detection interface of the detection fiber and samples. Further application of the microfluidic chip is to develop a fluorescence-activated particle counting and sorting system. The automatic sorting function is achieved by integrating electrokinetic flow switching, fluorescence excitation and detection, and an optical visualization system. Therefore, the multi-functional particle counting, sorting, sizing and fluorescence detection can be achieved by integrating different external detection systems on the simple microchip.
Different signal processing technologies have been adopted in the resistive pulse sensing systems and fluorescence detection systems for improving signal-to-noise (S/N) ratio. One of the resistive pulse sensing systems is enhanced by a metal oxide semiconductor field effect transistor (MOSFET). The MOSFET is biased to work in the sub-threshold regime, in which the MOSFET is more sensitive than in the saturation regime and the corresponging amplification factor is very high. The other one is by designing a single sensing gate with two detecting channels placed on the upstream side and downstream side of the gate, and using the two-stage differential amplification by differential amplifier, the sensitivity of RPS detection has been improved dramatically. For a fluorescent detection, lock-in amplifier is used for extracting small signal buried in the noise. An alternative means is the utilizing of a differential amplifier instead of an expensive and bulky lock-in amplifier. A two-stage differential amplification can effectively suppress the noise and consequently improve the S/N ratio significantly.
In the experiments, the particles with different diameters ranged from 0.9 µm to 15 µm. Also the yeast cells and white blood cells were discriminated and counted based on different schemes. The relative percentage of the fluorescence-labeled cells/particles was determined by the ratio of the events of fluorescence signals to forward scattered or RPS signals. In order to show the accuracy and reliability of the present detection methods, the result from the same sample test conducted using commercial flow cytometer was compared. All the detection results showed comparable accuracy to those from the commercial flow cytometer. This technique is highly promising as it could greatly reduce the cost for HIV diagnosis. This surely makes it accessible to the resource-poor developing countries.

中文摘要…………………………………………………………… Ⅰ
英文摘要……………………………………………………….... Ⅲ
誌謝……………………………………………………………… V
目錄……………………………………………………………… VI

表目錄…………………………………………………………... IX
圖目錄…………………………………………………………… X
符號說明………………………………………………………… XVI

第一章 前言…………………………………………………….. 1
1-1 研究背景…………………………………….......... 1
1-2 文獻回顧…………………………………….......... 2
1-3 研究目的…………………………………….......... 6
1-4 研究重點與本文架構…………………………….. 7
第二章 用於螢光檢測之無光纖式微流體細胞計數器……….. 16
2-1 簡介……………………………………………….. 16
2-2 微流體晶片與實驗樣品備製…………………….. 18
2-2-1 微流體晶片製作….…………………………….. 18
2-2-2 螢光微粒子……………………………………... 19
2-2-3 酵母菌螢光染色………………………………... 19
2-2-4 白血球細胞螢光染色…………………………... 19
2-3 實驗架構………………………………….............. 20
2-4 結果與討論……………………………………….. 21
2-5 結論……………………………………………….. 24
第三章 利用微流體晶片檢測淋巴球CD4+ T細胞的總數量與百分比………………………………………………… 37
3-1 簡介………………………………………………. 37
3-2 檢測樣品的製備…………...…………................... 40
3-3 實驗系統架構…..….……………………………... 41
3-3-1 MOSFET 電阻脈衝感測細胞計數…………….. 41
3-3-2 螢光檢測和系統架構…………………………... 43
3-4 結果與討論……………………………………….. 44
3-5 結論……………………………………………….. 48
第四章 微流體晶片螢光激發微粒子計數與分類系統……….. 59
4-1 簡介……………………………………………….. 59
4-2 實驗樣品與方法…………...…………................... 60
4-2-1 晶片設計與微粒子傳送分配…..….…………… 60
4-2-2 實驗系統架構..…..……………………………... 61
4-2-3 系統操作原理…………………………………... 62
4-3 結果與討論………...…………………................... 63
4-3-1 自動計數與傳送分配………………………....... 63
4-3-2 安全距離的決定………………………………... 64
4-3-3 系統的處理能力………………………………... 65
4-4 結論……………………………………………….. 66
第五章 差分放大螢光及電阻脈衝微流體細胞計數器………. 74
5-1 簡介……………………………………………….. 74
5-2 檢測系統架構…………...…………....................... 75
5-3 實驗步驟…..….…………………………………... 78
5-4 結果與討論..…..………………………………….. 80
5-5 結論……………………………………………….. 84
第六章 被動式聚焦光學式微流體細胞分類計數器…………. 93
6-1 簡介……………………………………………….. 93
6-2 晶片製作…………...…………............................... 96
6-3 實驗樣品之製備與系統架構…………………….. 97
6-3-1 實驗樣品之製備..…..…………………………... 97
6-3-2 實驗系統架構…………………………………... 98
6-4 結果與討論……………………………………….. 100
6-5 結論………...…………………............................... 106
第七章 總結與未來展望……………………………………….. 124
7-1 綜合結論…………………………………….......... 124
7-2 未來展望………………………………………….. 125

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