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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林啟萬(Chii-Wann Lin) | |
| dc.contributor.author | Tsung-Liang Chuang | en |
| dc.contributor.author | 莊琮亮 | zh_TW |
| dc.date.accessioned | 2021-06-16T04:12:33Z | - |
| dc.date.available | 2017-09-05 | |
| dc.date.copyright | 2014-09-05 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-20 | |
| dc.identifier.citation | Reference
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55611 | - |
| dc.description.abstract | 目前影像式表面電將共振(SPR)生物檢測系統已經有許多實驗室型的機種商品化，並已成為生物分子檢測上廣泛使用的工具。然而因其操作的複雜性，系統的體積，操作環境需求與價格等因素，在面臨未來預防性醫學的使用需求，包含重點照護測試(POCT)，田野動植物檢測，食品與環境監測等，SPR生物檢測系統仍須持續改善才能達到應用目的。在物理上，SPR感測器只能靈敏地反應出共振平面附近的折射率變化而非造成變化的因素; 因此為了讓檢測數據具有生物學上的分析意義，會據檢體種類(基因、蛋白質體或細胞)，輔以適當生化反應，例如核酸聚合反應與免疫結合反應等，以達成有效辨識出待測物的目的。因此對於SPR生物感測系統的設計而言有以下重點;包括傳感器，光學機構，流體操作與其它輔助系統(機械結構、溫控與資料紀錄與分析)皆很重要, 然而考慮現場檢測的推廣，本研究將著重在流體操作系統的簡化、傳感器多步驟生物操作流程的整合與可拋棄式的設計。據此前提因應基因與蛋白質體的可拋棄式晶片的研發，配合不同典型的生化反應為偵測機制，建構LAMPSPR與薄膜式的微流體晶片，此外基因與蛋白質體層級的檢測為細胞生理表現的重要指標，因此須以此研究為基礎，亦能推廣其應用範疇。
為了證明基因快速檢測的可行性，提出以聚合物材質的SPR感測器，針對恆溫核酸放大反應與蛋白質體-適體結合反應，提出因應的設計，作為不同型生化反應偵測機制的結構基礎。首先，為確保晶片功能的可行性，因此利用目前常使用的Kretschmann設計架構作為晶片量測平台， 用以量測聚合物材質的SPR感測器之光學訊號。接著，為了檢測環化恆溫放大反應(loop-mediated isothermal amplification LAMP)，發展了SPRLAMP 系統，包括了整合了聚甲基丙烯酸甲酯(polymethyl methacrylate, PMMA)微反應槽與聚碳酸酯(polycarbonate, PC) 微小稜鏡的SPRLAMP感測晶片。為實現環化恆溫放大反應來檢測B型肝炎病毒(hepatitis B virus, HBV)的片段基因。首先，我們驗證試劑溶液的體折射率在反應前後的變化量約0.0011。PC 稜鏡的線性度與熱反應與玻璃稜鏡比較結果，顯示PC 稜鏡可用來作為SPR的量測。最後發現到，HBV模板濃度，即使在低濃度2fg/ml的情況下，亦可在17分鐘檢測到。我們亦做各種DNA模板起始濃度與反應時間的相關係數分析，以建立最佳的反應終點時間25分鐘(R2=0.98)。由此可知，利用SPRLAMP感測晶片與 系統可以用來直接偵測溶液體折射率變化來檢測LAMP反應。 酶聯免疫吸附試驗(ELISA) 與酶聯免疫吸附斑點試驗(ELISPOT)被用來作為丙型干擾素釋放分析用以偵測從T細胞分泌出的的丙型干擾素。然而此一堆步驟的分析操作流程仍只適用在實驗室裡的設備並且由訓練過的人來操作。於此，我們提出薄膜式的微流體晶片整合了表面電漿共振感測器以實現方便操作與具經濟效益的多步驟定量分析。為了處理表面電漿共振的量測，我們提供了薄膜式SPR感測器，其中螺縈薄膜位於吸收片下300微米處。此運作機制以Darcy 定理來表示。另外，從蔗糖處理過的玻璃纖維膜釋出的鍊黴親合素之濃度，亦可限縮在一小範圍(0.81 μM ± 6%)。最後未反應的分子可被預先儲存在晶片儲存槽中的清洗液移除。利用雙功能髮夾形的適體作為感測探針，可以偵測丙型干擾素同時經由鍊黴親合素放大其訊號。在0.01nM至100 nM的範圍內，具有高相關係數(R2=0.98)。在30分鐘的反應時間內，可獲得10 pM的檢測極限。因此以表面電漿共振偵測丙型干擾素的分析程序，可在無外加幫浦系統下用此簡單的裝置實現 | zh_TW |
| dc.description.abstract | At present, many surface potential resonance (SPR) detection systems based on laboratory models has been commercialized and have become a power tools widely used in the detection of biological molecules. However, because of the complexity of operating procedure, system size, and limitation of operating environment, SPR biological detection system shall continue to improve to achieve the application purpose, including the demands of preventive medicine in the future, including point of care testing (POCT), flora and animal testing in field, food and environmental monitoring. Physically, SPR sensor can only response sensitively to the refractive index change near the plane of the resonance but the reason of refractive index change; Therefore, in order to make sense of the biological test result, people will chose appropriate biochemical reactions, such as nucleic acid polymerization reaction and immuno reaction according to the type of the specimen (genes, proteins, or cell), to recognized the analytes. Therefore, for the design of SPR biosensing systems, all of the following priorities; including sensors, optical construction, fluid operation system and other auxiliary systems (mechanical structure, temperature and analysis of the data records) are important. Considering the expression of on-site application in near future, the study will focus on the simplification of fluid operating systems, the design of multi-step and disposable sensor to promote the performance and practicability of SPR biosensing system. Therefore, the LAMPSPR chip and membrane based processing chip was provided based on different reaction mechanism to develop the disposable chip for genetic and proteomic detection. In addition, the detection of gene and protein expression is critical for determination of cell behavior and thus the relative technique could be used as the fundamental to extend the application scope to cell level detection.
In this work, a simple, low-cost surface plasmon resonance (SPR)-sensing cartridge based on a loop-mediated isothermal amplification (LAMP) method was provide for the on-site detection of the hepatitis B virus (HBV). To detect LAMP reaction, a SPR based LAMP sensing system (SPRLAMP) was constructed, including a novel SPRLAMP sensing cartridge integrating a polymethyl methacrylate (PMMA) micro-reactor with a polycarbonate (PC)-based prism coated with a 50 nm Au film. First, we found that the change of refractive index of the bulk solution was approximately 0.0011 refractive index (RI) units after LAMP reaction. The PC-based prism’s linearity and thermal responses were compared to those of a traditional glass prism to show that a PC-based prism can be used for SPR measurement. Finally, the HBV template mixed in the 10 μl LAMP solution could be detected by SPRLAMP system in 17 minutes even at the detection-limited concentration of 2 fg/ml. We also analyzed the correlation coefficients between the initial concentrations of HBV DNA templates and the system response (ΔRU) at varying amplification times to establish an optimal amplification time endpoint of 25 minutes (R2= 0.98). In conclusion, the LAMP reaction could be detected with the SPRLAMP sensing cartridge based on direct sensing of the bulk refractive index. ELISA and ELISPOT methods are utilized for interferon-gamma (IFN-γ) release assays (IGRAs) to detect the IFN-γ secreted by T lymphocytes. However, the multi-step protocols of the assays are still performed with laboratory instruments and operated by well-trained people. Here, we report a membrane-based microfluidic device integrated with a surface plasmon resonance (SPR) sensor to realize an easy-to-use and cost effective multi-step quantitative analysis. To conduct the SPR measurements, we utilized a membrane-based SPR sensing device in which a rayon membrane was located 300 μm under the absorbent pad. The basic equation covering this type of transport is based on Darcy's law. Furthermore, the concentration of streptavidin delivered from a sucrose-treated glass pad placed alongside the rayon membrane was controlled in a narrow range (0.81 μM ± 6%). Finally, the unbound molecules were removed by a washing buffer that was pre-packed in the reservoir of the chip. Using a bi-functional, hairpin-shaped aptamer as the sensing probe, we specifically detected the IFN-γ and amplified the signal by binding the streptavidin. A high correlation coefficient (R2 = 0.995) was obtained, in the range from 0.01 to 100 nM. A detection limit of 10 pM was achieved within 30 min. Thus, the SPR assay protocols for IFN-γ detection could be performed using this simple device without an additional pumping system. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T04:12:33Z (GMT). No. of bitstreams: 1 ntu-103-D96548001-1.pdf: 2276389 bytes, checksum: 76d842118d22ed593596566fd4fcbc91 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 目 錄
國立台灣大學(碩) 博士學位論文口試委員會審定書 i 中文摘要 viii Abstract x 1 Introduction - 1 - 1.1 Requirements of life sciences - 1 - 1.2 Scheme of SPR system for bio-sensing - 2 - 1.3 Characteristics of Surface plasmon resonance - 3 - 1.4 SPR coupling schemes - 4 - 1.5 Recognition of bio-molecules - 4 - 2 NIR surface instrument design and fabrication - 6 - 2.1 Introduction - 6 - 2.2 Materials and methods - 7 - 2.2.1 Design concept - 7 - 2.2.2 Light source apparatus design and construction - 7 - 2.2.3 Prism and detector - 8 - 2.2.4 Temperature control - 8 - 2.2.5 Evaluation of light source uniformity - 9 - 2.2.6 Noise test - 9 - 2.2.7 Refractometry test - 10 - 2.3 Results and Discussion - 10 - 2.3.1 Light source uniformity and Imaging resolution - 10 - 2.3.2 Instrument performance - 10 - 2.4 Summary - 11 - 3. A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loop-mediated isothermal amplification - 12 - 3.1 Introduction - 12 - 3.2 Materials and methods - 14 - 3.2.1. Preparation of LAMP assay - 14 - 3.2.2 Measurement of refractive index - 15 - 3.2.3 The SPRLAMP sensing cartridge - 16 - 3.2.4 SPRLAMP instrument - 16 - 3.2.5 Polycarbonate (PC) prism test - 18 - 3.3 Results and discussion - 19 - 3.3.1 HBV LAMP assay - 19 - 3.3.1.1 HBV LAMP validation - 19 - 3.3.1.2 Measurement of bulk refractive index of LAMP mixture - 19 - 3.3.2 Isothermal SPRLAMP system - 20 - 3.3.2.1 Isothermal SPRLAMP instrument - 20 - 3.3.2.2 Polycarbonate (PC) prism - 21 - 3.3.2.3 PMMA cartridge - 22 - 3.3.3 HBV LAMP analysis using SPR isothermal system - 23 - 3.4 Summary - 25 - 4. Disposable surface plasmon resonance aptasensor with membrane-based sample handling design for quantitative interferon-gamma detection - 26 - 4.1 Introduction - 26 - 4.2 Materials and methods - 29 - 4.2.1 Preparation of hairpin DNA for an IFN-γ aptamer and a streptavidin aptamer - 29 - 4.2.2 Process designs of the membrane-based microfluidic device - 30 - 4.2.3 Aptamer Immobilization of the COC prism modified with DNA - 30 - 4.2.4 Construction of a membrane-based quantitative SPR chip - 31 - 4.2.5 SPR imaging instrument - 32 - 4.2.6 Visualization of the test of the chip processes - 32 - 4.2.7 Estimation of the variation in the streptavidin concentration - 33 - 4.2.8 Interferon-gamma measurement - 33 - 4.3 Results and Discussion - 34 - 4.3.1 Surface plasmon resonance instrument - 34 - 4.3.2 Configuration of the autonomous process sensing cartridge - 35 - 4.3.3. Functional verification of autonomous processing for SPR detection - 36 - 4,3,4 Stability of the streptavidin concentration with different sample addition volumes - 37 - 4.3.5 SPR-based analysis for the multi-step process sensing cartridge - 39 - 4.4 Summary - 41 - 5. Conclusion - 43 - 圖目錄 Fig. 1.1 - 45 - Fig. 1.2 - 46 - Fig.1.3 - 47 - Fig. 2.1 - 48 - Fig. 2.2 - 49 - Fig. 2.3 - 50 - Fig. 2.4 - 51 - Fig. 2.6 - 53 - Fig. 3.1 - 54 - Fig. 3.2 - 55 - Fig. 3.3 - 56 - Fig. 3.4 - 57 - Fig. 3.5 - 58 - Fig. 4.1 - 59 - Fig. 4.2 - 60 - Fig. 4.4 - 62 - Fig. 4.6 - 64 - 表目錄 Table 1.1 - 65 - Table 2.1 - 66 - Table 2.2 - 67 - Table 3.1 - 68 - Table 3.2 - 69 - | |
| dc.language.iso | zh-TW | |
| dc.subject | 重點照護 | zh_TW |
| dc.subject | B肝病毒 | zh_TW |
| dc.subject | SPRLAMP檢測匣 | zh_TW |
| dc.subject | 孔隙薄膜微流體裝置 | zh_TW |
| dc.subject | 丙型干擾素 | zh_TW |
| dc.subject | 影像式表面電漿共振儀 | zh_TW |
| dc.subject | interferon-gamma | en |
| dc.subject | SPR image Hepatitis B virus | en |
| dc.subject | point of care | en |
| dc.subject | SPRLAMP sensing cartridge membrane-based microfluidic device | en |
| dc.title | 拋棄式表面電漿共振快速篩檢晶片之設計與應用 | zh_TW |
| dc.title | Design and Application of Surface Plasmon Resonance Based Disposable Rapid Screen Chip | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 王安邦(An-Bang Wang),薛博仁(Po-Ren Hsueh),賴信志(Hsin-Chih Lai),李世元(Shih-Yuan Lee) | |
| dc.subject.keyword | 影像式表面電漿共振儀,B肝病毒,重點照護,SPRLAMP檢測匣,孔隙薄膜微流體裝置,丙型干擾素, | zh_TW |
| dc.subject.keyword | SPR image Hepatitis B virus,point of care,SPRLAMP sensing cartridge membrane-based microfluidic device,interferon-gamma, | en |
| dc.relation.page | 77 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-20 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| 顯示於系所單位： | 醫學工程學研究所 | |
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