心血管疾病(CVD)每年導致數百萬以上的死亡，因此我們迫切地需要快速且具靈敏度的心血管疾病診斷技術。 對於未來的醫療保健方案和及時檢測來說，適體(aptamers) 由於其優於傳統的基於抗體的CVD生物標誌物檢測篩選，近年來備受關注。然而，適體的篩選經由 Systematic Evolution of Ligands by EXponential enrichment (SELEX) ，為 一複雜且耗時的技術，需要數個篩選循環才能得到具專一性和親和性的適體。為簡化及自動化整個SELEX流程, 我們開發一自動微型化裝置以篩選出與CVD生物標誌具專一性之適體：心臟肌鈣蛋白I(cTnI), B型鼻尿肽 (NT-proBNP) 的N端激素, 以及fibrinogen。已建立的微流體平臺配有數個微裝置以達到自動化的樣本混和與傳輸，晶片上亦配有一結合核酸擴增模組。此高效的裝置可以在 8 小時內連續完成整個篩選過程。微流體平臺適體篩選與CVDs相關的蛋白質生物標誌物提供了有利、精確且經濟高效的平台。通過篩選過程獲得的適體可用於進一步實驗中的捕獲探針, 並搭配使用不同的感應器進行敏感的生物感應器. 為了證明這一想法,考慮了兩種獨特的方法。首先, 為自動流體輸送和新型的貼合三明治測定應用了獨特的集成氣動驅動微流體平臺, 該平臺包含高效的微設備(微泵、微混合器和微閥) 在25分鐘內從多達6個臨床樣本中同時檢測NT-proBNP。計算出的檢測極限(LOD)低至1.53皮克/mL。值得注意的是與預置作品相比，晶片的緊湊性增加了 64%, 所需的樣品輸入僅為 5 μL。這可能有助於作為早期診斷的一種強大手段。如果使用合適的捕獲探針, 該平臺也可用於檢測其他生物標誌物。其次，新設計的多層微流體平臺上固定了目標特定的適體探針於場效應電晶體 (FET)的感應器陣列上，可從臨床樣本(~4 μL)中快速檢測(5 分鐘),。 根據當前結果，這四種CVD生物標誌物(C-反應蛋白(CRP)、NT-proBNP、cTnI和纖維蛋白原)的FET感應器陣列的標準曲線顯示他們的濃度依賴性分別為CRP (0.1-50毫克/升)、NT-proBNP (50-10,000 皮克/升)、cTnI (1-10,000 皮克/升)和纖維蛋白原 (0.1-5 毫克/升)。這種全自動可擕式設備的原型機可在最少的試劑、輸入步驟顯示出未來POC設備從患者臨床樣本中檢測與CVD相關的大量生物標誌物。
總括來說，在心血管生物標誌物檢測領域，已經證明了所開發的微流體系統對於晶片系統的未來POC/實驗室的潛力。本篇的微流體平臺用於適體篩選，以及後續使用三明治測定法同時檢測多個樣品，並在在FET嵌入式微流體晶片上進行多個生物標記定量，可視為在心血管疾病診斷領域做出的巨大貢獻。

Cardiovascular diseases (CVDs) causes more than millions of deaths every year. In the present scenario, there is an urgent need for fast and sensitive diagnosis of CVDs. For future health care scenario and point-of-care (POC) perspectives, aptamers screened by Systematic Evolution of Ligands by EXponential enrichment (SELEX) have obtained considerable attention due to its superiority over traditional antibody-based detection of CVD biomarkers. Nonetheless, SELEX being an intricate, prolonged technique, demands several cycles of selection to screen the aptamers. To avoid such complications, an automatic, miniaturized setup for SELEX to screen aptamers against protein-based biomarkers linked to CVDs: cardiac troponin I (cTnI), N-terminal prohormone of B-type natriuretic peptide (NT-proBNP), and fibrinogen has been demonstrated. The established microfluidic platform was furnished with several microdevices proficient in pumping mixing and transportation of required regents and a module for on-chip nucleic acid amplification was incorporated. This efficient setup could accomplish the entire screening process continuously including 5 rounds (35 L of reagents in each cycle) of selection in 8 hrs. This microfluidic platform served as a favorable, precise, and cost-effective layout for aptamers screening specific to protein biomarkers related to CVDs. The aptamers obtained through the screening process could be used as capture probe for further experiments leading to a sensitive biosensor using different transducers. In order to demonstrate this idea, two unique approaches have been considered. Firstly, a unique integrated pneumatically driven microfluidic platform containing efficient microdevices (micropumps, micromixers and microvalves) were implemented for the automatic fluidic transportation and a novel aptamer-based sandwich assay for the simultaneous detection of NT-proBNP from up to six clinical samples in 25 min. The calculated limit of detection (LOD) was as low as 1.53 pg/mL. It was important to note that the deigned chip was 64% more compact compared to our pre-demonstrated works and the required sample input was only 5 µL. This may assist as a capable means for early diagnosis of HF. This platform could be applied for the detection of other biomarkers as well, if suitable capture probes are used. Secondly, a newly designed multilayered microfluidic platform with target specific aptamer probes immobilized on field-effect transistor (FET)-based sensor arrays were used to rapid detection (5 minutes) up to four protein biomarkers related to CVDs from clinical samples (~4 µL). As per current process demonstration, the standard curve from the FET sensor arrays for those four CVDs biomarkers (C-reactive protein (CRP), NT-proBNP, cTnI, and fibrinogen) showed concentration dependency in the wide concentration assortments for CRP (0.1-50 mg/L), NT-proBNP (50-10,000 pg/mL), cTnI (1-10,000 pg/mL), and fibrinogen (0.1-5 mg/mL). The established prototype of this fully automated portable device necessitates minimal quantity of reagents, input steps and showed great potential for future POC devices for examining numerous biomarkers related to CVDs from clinical samples from patients.
To sum up, the potential of the developed microfluidic systems for future POC/lab on a chip system has been demonstrated in the field of cardiovascular biomarker detection. The respective microfluidic platforms and their application for screening aptamers, simultaneous detection of multiple samples using a sandwich assay and multiple biomarker quantification on an FET embedded microfluidic chip could be considered as a prodigious contribution in the field of CVD diagnosis.

Table of contents

Acknowledgments II
Abstract IV
Abstract in Mandarin VI
Table of contents VIII
List of tables XIV
List of equations XV
List of figures XVI
Abbreviations XXIX
Chapter 1: Introduction 1
1.1. MEMS and microfluidics devices 1
1.1.1. Introduction to MEMS and microfluidic devices 1
1.1.2. Introduction to soft lithography and PDMS microfluidics 3
1.1.3. Microfluidic microdevices 5
1.2. Biosensors 7
1.2.1. Introduction to biosensing 7
1.2.2. Role of microfluidics in biosensing 8
1.2.3. Point-of-care devices and role of microfluidics 8
1.2.4. Aptasensors: the next generation biosensors and POCT 9
1.3. Biomarkers 10
1.3.1. CVDs and introduction to cardiovascular biomarkers 11
1.4. Aim of this study, final goals and purpose 12
Chapter 2: Screening of aptamers specific to cardiovascular biomarkers 18
2.1. Introductions and motivations 18
2.1.1. Comparison of aptamers and antibodies 20
2.1.2. Types of SELEX 21
2.1.3. Selection of aptamer for CVDs 22
2.1.4. Magnetic bead-based selection 23
2.1.5. Aptamer-protein interaction study 25
2.1.6. Microfluidic SELEX 26
2.2. Materials and Methods 27
2.2.1. Design and fabrication of the microfluidic chip 27
2.2.2. Reagents, ssDNA library, and proteins 32
2.2.3. Magnetic beads and their coating with probes 33
2.2.4. Mixing and pumping units 34
2.2.5. Experimental procedure and on-chip PCR 35
2.2.6. Monitoring the enrichment of the ssDNA pool 39
2.2.7. Competitive assay, cloning, sequencing, and synthesis of screened aptamers 39
2.2.8. Analysis of the aptamer equilibrium dissociation constants by surface plasmon resonance. 41
2.2.9. Evaluation of the specificity of the selected aptamers by PCR 41
2.2.9. Aptamer binding force calculations 42
2.3. Results and Discussion 44
2.3.1. Characterization of the chip 44
2.3.2. On-chip SELEX 46
2.3.3. Monitoring the enrichment of the ssDNA pool 49
2.3.4. Affinity of selected aptamers to CVDs biomarkers 51
2.3.5. Analysis of the equilibrium dissociation constant of aptamers by SPR 51
2.3.6. Evaluation of the specificity of the selected aptamers by qPCR 53
2.3.7. Exploring the binding force between aptamer and protein 55
2.4. Conclusions 56
Chapter 3: An integrated microfluidic system for simultaneous detection of cardiovascular biomarkers in an aptamer sandwiched assay 58
3.1. Introduction and motivation 58
3.1.1. Enzyme-linked immunosorbent assay (ELISA) 59
3.1.2. Aptamer sandwich assay 60
3.1.3. Exploring an aptamer-based sandwiched assay in a microfluidic system 61
3.2. Materials and methods 62
3.2.1. Required reagents 62
3.2.2. Magnetic breads 63
3.2.3. Working process of the aptamer sandwiched assay 64
3.2.4. The design of the microfluidic system 66
3.2.5. Characterizing the mixing and pumping microdevices 75
3.2.6. Aptamer-based sandwich assay on-chip 76
3.2.7. Specificity tests 79
3.2.8. Detection of unknown serum concentration and recovery analysis 79
3.3. Results and discussion 80
3.3.1. Characterization of the microdevices 80
3.3.2. Optimization of the aptamer-based sandwich assay 82
3.3.3. Calibration curve of the aptamer-based sandwich assay 83
3.3.4. Specificity tests of the developed assay and detection in serum 85
3.3.5. Recovery percentage analysis 86
3.4. Conclusions 88
Chapter 4: Detection of multiple protein biomarkers in an integrated microfluidic system equipped with FET-based detection system 89
4.1. Introduction and motivation 89
4.2. Materials and methods 91
4.2.1. Microfluidic FET biosensor 91
4.2.2. Design and fabrication of the EDL-gated HEMT sensor 91
4.2.3. Design and fabrication of microfluidic chip 93
4.2.4. Characterization of mixing and pumping devices 98
4.2.5. Reagents and materials 98
4.2.6. Aptamer functionalized sensor preparation 99
4.2.7. Optimization of analytical conditions 100
4.2.8. Analytical performance of the microfluidic aptamer-FET sensor 101
4.2.9. Design of the portable control system 103
4.2.10. Portable control system: 104
4.3. Results and discussion 105
4.3.1. Optimization of the micro-devices on the chip 105
4.3.2. Characterization of the sensors. 107
4.3.3. Characterization of the mixing and pumping devices on the microfluidic chip 109
4.3.4. Analytical performance of the microfluidic aptamer-based FET biosensor 111
4.3.5. LOD determination: 113
4.3.6. Detection of proteins from clinical samples and recovery rate measurements 113
4.3.7. Portable control system 115
4.4. Conclusions and future potential of the developed system 117
Chapter 5: Conclusion and future works 119
References 123
Publication list: 135

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