蝴蝶蘭是臺灣最具有重要性的經濟作物之一。為了避免或抑制大量蘭花病毒疾病的大流行，因此快速、準確且能即地偵測蘭花病原體十分重要。傳統上，病原體偵測的方式相當耗時，且需要在有精密的儀器的實驗室裡，經由訓練過的專業人員進行操作，因此難以在田野間進行檢測。本研究開發一個整合光纖的微流體系統以用來偵測蝴蝶蘭病毒。首先，可藉由修飾有特異性核酸探針之微型磁珠進行病毒核糖核酸之純化，；接著，使用反轉錄恆溫環狀擴增法放大病毒的核糖核酸。成功增幅的反轉錄恆溫環狀擴增反應會導致沉澱物焦磷酸鎂生成，而反應物的濁度改變，可以直接用肉眼觀察。此外，光纖式偵測模組以及微型混合元件成功地被整合於微流體晶片以直接在晶片上偵測反轉錄恆溫環狀擴增反應之產物。使用微型混合元件，反轉錄恆溫環狀擴增反應之產物可於混合步驟後均勻地分布。因此，測量隨著濁度改變而變化之光學訊號可代表增幅的情形。此方法也提高光學的靈敏度以及偵測極限。此整合型微流體系統應用在番椒黄化病毒的偵測極限為25 飛克 (約為5000拷貝數)，而東亞蘭嵌紋病毒的偵測極限則為25 皮克 (約為5000000拷貝數)，上述兩種病毒的偵測靈敏度皆與其他目前已知或傳統的大型精密方法接近。因此，光纖式整合型微流體系統已經被成功開發，並可在65分鐘內，自動化地完成具有高靈敏度且同時兼具快速、準確的方式偵測蝴蝶蘭裡病毒性的病原體，除了避免傳統上大型且複雜的儀器使用，更可減少不必要的能源、試劑損耗、減少系統體積，在方便攜帶的情況下，更有利田野間進行檢測。

Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Rapid, accurate, and on-site detection of pathogens from these orchids is therefore of critical importance for preventing or suppressing costly disease outbreaks. Traditional pathogen detection methods are relatively time-consuming, require well-equipped laboratories with well-trained personnel, and cannot be conducted in field. In this study, a microfluidic system integrated with buried optical fibers was developed to detect viral pathogens of Phalaenopsis spp. Briefly, virus-specific ribonucleic acid (RNA) purification was achieved by using a pre-treatment incubation with magnetic beads surface-coated with specific nucleotide probes, and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was used subsequently to amplify the viral RNA. Positive RT-LAMP reactions resulted in the precipitation of magnesium pyrophosphate, which caused a change in turbidity that could be seen by the naked eye. Alternatively, a buried optical fiber-based detection module and a micro-stirring device were then integrated into the microfluidic chip to detect the RT-LAMP reaction product directly on the chip in situ. With the micro-stirring device, the RT-LAMP product became uniformly distributed after the stirring step. Therefore, the situation of amplification can be detected by measuring the change of the optical signals caused by the turbidity change. The optical sensitivity and limit of detection of the optical detection system were also improved by this approach. The limit of detection for this system was found to be 25 femtograms (about 5000 target copies) for capsicum chlorosis virus and 25 picograms (about 5000000 target copies) for cymbidium mosaic virus, which is of similar sensitivity of existing, delicate laborious methods. Therefore, by using the integrated microfluidic system with buried optical fibers, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65 minutes. Furthermore, the integrated system is compact in size and is relatively easy to operate. The power　and reagent consumption are greatly reduced. Therefore, it would be promising for in-field study.

Abstract i
摘要 iii
誌謝 v
Table of Contents vii
List of Figures x
Abbreviations xvii

Chapter 1 Introduction 1
1.1 Virus in Phalaenopsis orchid and its diagnostic methods 1
1.2 MEMS and bio-MEMS 5
1.3 Motivation and objectives 7

Chapter 2 Design and Fabrication 10
2.1 Integrated microfluidic chip design 10
2.2 The fabrication processes of the microfluidic chip 12
2.3 The working principle of the integrated microfluidic chip 14
2.4 The working principle of stirring device and optical detection 15

Chapter 3 Materials and Methods 21
3.1 Experimental procedures 21
3.2 Preparation of primer, nucleotide probe and nucleotide-probe-coated magnetic beads 24
3.3 Gel electrophoresis 26
3.4 Custom-made microfluidic control system 27
3.5 Optical fiber and its working principles 28
3.6 Fluorescent dye and its optimization tests 30
3.6.1 Optimization test of concentration 30
3.6.2 Optimization test of temperature 32

Chapter 4 Results and Discussion 37
4.1 Surface modification and the pumping performance of the integrated microfluidic chip 37
4.2 Performance of the optical detection of the buried optical fibers 39
4.3 The results of the real-time turbidity detection and the effect of the stirring membrane 42
4.4 The limit of detection of the optical detection system 44
4.4.1 The limit of capsicum chlorosis virus (CaCV) detection 44
4.4.2 The limit of cymbidium mosaic virus (CymMV) detection 45
4.5 Virus detection for field samples by using the turbidity and fluorescent detection 48
4.5.1 The detection of field samples infected with capsicum chlorosis virus (CaCV) 48
4.5.2 The detection of field samples infected with cymbidium mosaic virus (CymMV) 49

Chapter 5 Conclusions 64

References 66

Publication list 78

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