假體周圍關節感染是關節置換手術術後最嚴重的併發症之一，其被公認為災難性的併發症，一旦假體周圍受到感染，再感染的機率亦會提升，進而使整個治療程序變得更加棘手。隨著高齡化社會的到來，人類關節上的老化伴隨而來，關節置換手術的比例也隨之增加，據文獻估計在2030年將會有42萬人在關節置換手術術後受到感染，此將會消耗非常大的醫療資源。
　　目前臨床於假體周圍關節感染診斷技術上有時間耗費太長、人力成本過高、具偽陽性與偽陰性的結果及複雜的檢驗程序等缺點，而另一方面若臨床檢測方式是使用分子診斷技術，常因無法辨認結果為死菌或活菌的DNA所造成的核酸增幅結果，而得到偽陽性的結論，進而拖延了治療時間。
　　於本篇研究中，我們發展了一檢測假體周圍關節感染的整合型微流體系統，利用恆溫環型核酸增幅法與溴化乙錠於微流體晶片，在整合型控制系統中做即時偵測人類關節液樣本，此研究不僅可讓所有流程包括細菌分離、細胞裂解、核酸增幅、光學檢測等都被整合化外，還能夠全自動處理，如此便能減少一般大型系統在人力資源上的耗費。與我們團隊先前的研究相比，本篇研究使用恆溫環型核酸增幅法使核酸增幅技術變得更加有效率(30-55分鐘內能完成核酸增幅反應)，在偵測極限上則是又比聚合酶連鎖反應要來得低一個等級，並且是由4個引子對應目標基因的6個區域，因此敏感性和特異性也相對變得更好，另又因其為恆溫反應，故亦使增幅過程變得更加穩定。除此之外，本研究的新穎性還包括在整合型控制系統機台上的改良，不僅將新機台的體積做得比舊機台小至四分之三的大小，重量則是少了三分之一，同時本研究亦整合小型化的氣壓源系統，進而大幅度地提升了整套檢測系統的便利性與接受度。總結來說，我們發展了一整合型微流體定點醫療照護系統，此複合式的恆溫環型核酸增幅檢測系統可在短時間內獲得高精確度的診斷結果，它偵測極限可低至5 fg。我們希望藉由這套檢測系統，在未來能夠讓臨床醫生及早了解假體周圍關節感染病人的病情，並且予以適當的治療。

關鍵詞：恆溫環型核酸增幅法、溴化乙錠、微流體、假體周圍關節感染

　　Periprosthetic joint infection (PJI) is one of the severe and acute/chronic complications of prosthetic joint replacement. The necessity for joint replacement is increasing in the older generation. Therefore the subsequent complications also become serious. By 2030, it is expected to have 420,000 patients in the USA who may suffer from the infection in their artificial joints, and the cost for medication would consume one of the largest amounts of hospital resources.
　　There are several disadvantages such as time consumption, human error, false-positive and false-negative results and the complicated process in recent diagnosis methods of PJI. Another important issue is how to distinguish the live bacteria from dead bacteria in PJI. As the deoxyribonucleic acid (DNA) of dead bacteria remains intact during the analysis procedure of molecular diagnostic methods, it would cause inaccurate treatment results from false-positive conclusion.
　　An integrated microfluidic chip has been developed in this study to perform the overall process for PJI diagnosis in a single chip. It is the first time that loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) was executed in microfluidic systems to diagnose PJI automatically. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic chip by using a compact custom-made control system. Compared to our previous works, this molecular diagnosis for multiple pathogens could be done faster (30 to 55 min) and simultaneously. It is more stable with higher specificity (four primers complementary to six regions of the target gene) and sensitivity (i.e. the detection limit for LAMP is one order of magnitude higher than PCR in this work), the limit of detection (LOD) in this multiple LAMP assay could be as low as 5 fg to 50 pg for different bacteria. And the new custom-made control system is improved to be smaller and lighter with the miniaturized pneumatic supply. Thus, this system for PJI diagnosis has great potential to become a point-of-care device. In conclusion, a novel integrated microfluidic system with a point-of-care device for rapid, stable, high specificity and sensitivity detection of live bacteria for PJI has been developed.

Keyword: Loop-mediated isothermal amplification, Ethidium monoazide, Microfluidics, Periprosthetic joint infection

Abstract I
摘要 III
誌謝 V
Table of contents VII
List of Tables IX
List of Figures X
Abbreviations and nomenclature XVI
Chapter 1 Introduction 1
1.1 Background and literature survey 1
1.1.1 Total joint arthroplasty (TJA) 1
1.1.2 Periprosthetic joint infection (PJI) 2
1.1.3 Current diagnosis for PJI 5
1.2 Ethidium monoazide (EMA) 7
1.3 Loop-mediated isothermal amplification (LAMP) techniques 9
1.4 MEMS-based microfluidic technology 11
1.5 Motivation and novelty 13
Chapter 2 Materials and methods 16
2.1 System design and fabrication 16
2.1.1 Fabrication of microfluidic chip 16
2.1.2 Copper sheet for thermal management 20
2.1.3 Miniaturized pneumatic supply 22
2.2 The working principle of the integrated microfluidic chip 25
2.3 Compact custom-made control system 29
2.4 Preparation of the experimental materials 32
2.4.1. EMA 32
2.4.2. LAMP reagent design and reaction 32
2.4.3. Clinical samples 36
2.4.4. Magnetic beads conjugated with vancomycin 36
2.4.5. Gel electrophoresis 38
2.5 Experimental process 40
2.6 Experimental setup 43
Chapter 3 Results and discussion 45
3.1 Characterization of microfluidic chip 45
3.1.1 Chip structure 45
3.1.2 The pumping rate of micropump 47
3.2 Characterization of miniaturized system 49
3.2.1 Performance of miniaturized pneumatic supply 49
3.2.2 Copper sheet for thermal management 51
3.2.3 Optimization of the operation conditions 53
3.3 Limit of detection (LOD) of the proposed system 55
3.3.1 Sensitivity 55
3.3.2 Time of LAMP process 61
3.4 Specificity 64
3.5 Spiked sample tests 71
Chapter 4 Conclusions and future perspectives 73
4.1 Conclusions 73
4.2 Future perspectives 75
References 77

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