人工關節感染 (PJI) 是關節置換手術後可能產生的嚴重併發症。然而，儘管人工關節感染具有其嚴重性及危險性，如何去診斷並管理此疾病在臨床上仍然是很大的挑戰。為了能更準確並客觀地檢測人工關節感染，本研究提出了一種新設計的整合式微流體晶片，能夠以關節液作為檢體樣品，同時檢測兩種在判別人工關節感染時採用的生物標誌物，α-防禦素-1（HNP-1）和 C-反應蛋白（CRP），來進行人工關節的檢測。為了同時檢測關節液中的兩種生物標誌物，整個檢測流程伴隨磁珠式酵素免疫分析法的步驟在晶片上自動化進行。預先將被人工適體包覆的磁珠滴至微混合器中，當關節液 (40 μL) 從存放槽被打入微混合器後，關節液中的生物標誌物（HNP-1 和 CRP）會被人工適體捕獲到磁珠上。之後在與檢測用的一抗以及與辣根過氧化物酶 (HRP) 偶聯的二抗接合後，隨後添加的呈色劑(冷光劑)會反應並產生光學訊號，透過檢測光學訊號大小進而可以在45分鐘內量化關節液中生物標誌物的濃度。而實驗結果表明，可得到的HNP-1標準曲線濃度範圍為0.01-50 mg/L而CRP標準曲線濃度範圍為1-100 mg/L，此兩條標準曲線展現了晶片對檢測關節液中的HNP-1和CRP具有足夠的靈敏度。我們也進行了特異性測試，確定HNP-1人工適體和CRP人工適體在面對目標蛋白與人類血清白蛋白(HAS)時具有對目標蛋白的高度專異性。此外，我們還進行了些許的臨床測試來驗證晶片與標準曲線的效能並得到不錯的結果。十二個臨床檢體通過類ELISA晶片進行檢測分析，所有檢體不僅可以用HNP-1和CRP正確地判斷陽性和陰性，還可以通過我們的方法進一步量化SF中的HNP-1濃度。結果指出此晶片對於檢測工關節感染來說是一項極具潛力的新工具。

Periprosthetic joint infection (PJI) is a serious complication after arthroplasty. However , diagnosis and management of PJI remains a major clinical challenge. In order to detect PJI more accurately and objectively, this study presents a new integrated microfluidic system capable of detecting two commonly used biomarkers based on pre-operative diagnostic criteria, including human neutrophil peptide 1 (HNP-1) and C-reactive protein (CRP) for periprosthetic joint infection from synovial fluid (SF) samples. The magnetic beads-based immunoassays were carried out automatically on a single microfluidic chip to detect these two biomarkers in SF simultaneously. The complete process of one-aptamer-one-antibody ELISA-like immunoassays was completed within 45 minutes. The new on-chip, one-aptamer-one-antibody ELISA-like assay demonstrated easy identification of negative and positive clinical samples for PJI along with the quantification of the target biomarkers. Magnetic beads coated with the primary aptamers against the target proteins were pre-loaded in a micromixer. When SF (40 μL) was injected into the micromixer, biomarkers in SF (HNP-1 and CRP) were captured separately onto the individual magnetic beads coated with specific aptamers for particular proteins. After mixing with the primary aptamer and secondary antibodies conjugated with horseradish peroxidase (HRP), addition of a substrate (lumino buffer, should be more specific) produced luminescence signals such that the concentrations of biomarkers in SF could be quantified within 45 minutes. Experimental results demonstrated that the standard curves with concentrations ranging from 0.01-50 mg/L for HNP-1 and 1-100 mg/L for CRP could be successfully achieved, indicating superior sensitivity for detecting HNP-1 and CRP in SF. The specificity tests were also conducted to determine the specificity of HNP-1 aptamer and CRP aptamer, showing that all HNP-1 and CRP aptamers have high specificity to target proteins towards human serum albumin (HAS). Additionally, clinical samples were used to testify the performance of the developed microfluidic chip. Twenty clinical samples were tested by a sandwich assay; not only were all samples determined positive and negative with HNP-1 and CRP correctly, also they could be quantified with HNP-1 concentrations in SF by the developed assay. It has been demonstrated that the developed microfluidic system can serve as a promising tool for detecting PJI.

Abstract I
中文摘要 III
誌謝 V
List of tables IX
List of figures X
Abbreviations and nomenclature XIV
Chapter 1: Introduction 1
1-1 Periprosthetic joint infection (PJI) 1
1-2 Current clinical methods and challenges for detection of PJI 3
1-3 Diagnostic criteria for PJI 4
1-3-1 Human Neutrophil Peptide 1 (HNP-1) 6
1-3-2 C-reactive protein (CRP) 7
1-4 Comparison of antibodies and aptamers 9
1-5 Bead-based enzyme-linked immunosorbent assay 10
1-6 Microfluidic chip for clinical detecting 12
1-7 Motivation and novelty 13
Chapter 2 Materials and methods 15
2-1 Enzyme-linked immunosorbent assay and ELISA-like assay 15
2-1-1 Enzyme-linked immunosorbent (ELISA) assay for HNP-1 15
2-1-2 ELISA-like assay for CRP and HNP-1 18
2-2 Chip design and microfabrication process 20
2-2-1 Design for ELISA–like chip 20
2-2-2 Microfabrication process of the ELISA-like chip 23
2-3 Preparation of magnetic beads coating 25
2-4 Specificity tests for HNP-1 and CRP aptamer 26
2-5 Preparation of samples for clinical tests 27
2-6 Experimental procedures of ELISA (ELISA-like) assay on chip 28
Chapter 3 Results and Discussion 32
3-1 Characterization of the integrated microfluidic system 32
3-1-1 Pumping volume of the micropumps 32
3-1-2 Mixing index of the micromixers 34
3-2 ELISA tests for HNP-1 on-chip 35
3-2-1 Standard curve of ELISA test for synovial fluid on-chip 35
3-2-2 Blinding tests for diagnosing PJI 37
3-3 ELISA-like tests for HNP-1 on-chip 39
3-4 ELISA-like tests for CRP on-chip 40
3-5 Specificity tests for HNP-1 and CRP 42
3-6 Quantitative tests for clinical samples 43
3-6-1 Gold standard for detecting HNP-1 in synovial fluid 44
3-6-2 Clinical tests from HNP-1 ELISA-like assay on-chip 46
3-6-3 Clinical tests from CRP ELISA-like assay on-chip 49
3-6-4 Cross-analysis of clinical samples with HNP-1 and CRP 52
Chapter 4 Conclusions and future works 53
4-1 Conclusions 53
4-2 Future works 54
4-2-1 Detect the HNP-1 in serum for PJI 54
4-2-2 Development of optical detection instruments 55
References 56
Publication list 63

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