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  • 學位論文

雙噻吩分子修飾生物感測器表面的同步量測技術與驗證平台之研發 —丙型干擾素重組蛋白質及其抗體間交互作用模組的開發

Synchronous Measuring Technique and Verification For the Development of the Biosensor Platform by Chip Surface Modification with Bithiophene Biolinker — A Module Developed For the Interaction of Interferon-gamma Recombinant Protein and Its Antibody

指導教授 : 李世光

摘要


技術的創新應用不僅加速了科技知識的普及化,也增進了知識的再加值化。自2003年4月人類基因圖譜宣布完全解碼後,生醫領域的趨勢乃聚焦於蛋白質體的研究,而研究蛋白質體學所需的技術或設備,則以具有即時(real-time)、無標記(label-free)、可直接監測生物分子間相互作用情形的生物感測器最具競爭優勢。 本研究整合臺大奈米生醫微機電系統研究群累積多年之研發基礎-光生化形生醫晶片檢測儀(OBMorph),及電化學阻抗譜系統(EIS)技術於一新創檢測平台上,使用自製型感測晶片搭配本團隊自行合成之電誘導型雙噻吩生物連結分子作為鍵結蛋白質的橋接物,藉由表面電漿共振(SPR)技術反射光的相位變化及電化學氧化還原反應,同步量測丙型干擾素(interferon-gamma, IFN-γ)與其抗體間的結合作用,以相互驗證檢測平台的效能。此外,本研究亦驗證雙噻吩生物連結分子用於SPR商用儀器和晶片之適用性,平行開發檢測自體免疫型之抗丙型干擾素抗體(anti-IFN-γ Ab)的優化試驗程序。 實驗結果顯示,本研究團隊開發之檢測平台和生物連結分子皆具有優異的量測性能,同步以電化學量測系統整合於OBMorph平台,利用雙噻吩生物連接分子的導電特性可以同步量測丙型干擾素12.8 pM ~ 1000 nM的濃度,並具多功整合型生物感測平台獨特的靈敏度和靈活性。同時也成功證實雙噻吩生物分子可用於固定抗體或抗原,雙向檢測抗體-抗原或抗原-抗體,故為一有效的生物連結分子。雙噻吩基晶片固定IFN-γ的訊號響應為聚葡萄醣晶片的8.31倍,而分析物的訊號響應可再增為2倍,顯示出其高靈敏度的特性。所測得抗丙型干擾素抗體濃度為0.67 nM ~ 83.33 nM範圍,以3.33 nM的濃度進行六次再生循環測試之感測訊號耗損小於1%,再現性表現優良。本研究所開發之檢測程序模型簡化便捷,不僅具大量、 可快速篩檢潛伏性結核病或定量自體免疫病灶,襄助臨床診治與用藥判斷的潛能,且具有時間和成本的優勢。 聯合國防治結核病的重點工作之一,即為發展更適切且可用在第一線的診斷工具。此外,據市場分析,2015年至2020年全球免疫分析市場規模將增長70億美元,而生物感測器市場規模則將增長95.3億美元。因此,生物分子交互作用分析,尤其是抗體與抗原之間的親和作用力,在生命科學研究、新藥開發與醫學臨床檢驗等領域的應用益發重要。本研究結果證實了研究團隊所開發的即時、無標記的檢測方法與系統-雙噻吩基生物感測系統可應用於檢測潛伏型結核菌感染之生物標誌物IFN-γ和anti-IFN-γ Ab,即時監測與分析生物分子間的相互作用,所得結果更證明其為高靈敏度的生物感測器,同步的量測系統與數據則可加速臨床的診斷與處置,而anti-IFN-γ Ab檢測模組的開發,對於自體抗體的檢測程序及感測晶片的再生循環試驗,也提供了進一步臨床研究的應用及縮短檢測時間與降低成本的效益。希冀,本研究成果對於未來技術研發的運用、疾病的防治或多元的應用上,具有為本土性發展體外檢測器材帶來實質的助力,並得使其更臻完善的潛能。

並列摘要


Innovations in technological applications not only accelerate the spread of knowledge in Science and Technology (S&T) but it can also increase the value of S&T knowledge. The integration of the fields of Life Science and Engineering has been a major research focus in achieving cross-disciplinary, cross-field knowledge. Since the completion of the Human Genome Project in April 2003, one of the major focus of biotechnology research has been on proteome-related research. New technology or equipment which are real-time, are label-free, and can handle direct detection of biomolecular interactions have been found to be the most advantageous. It is estimated that the global market for immunoassay technologies will grow by US$7 billion from 2015 to 2020 and the biosensor market will increase by US$9.53 billion. Biomolecular interaction detections, especially those between antigens and antibodies, have become more important in Life Science research, new medical development, and clinical studies. According to the World Health Organization (WHO), tuberculosis (TB) is a top infectious disease killer worldwide. In 2014, it was estimated that 9.6 million people fell ill with TB and 1.5 million died from the disease. Data shows that latent tuberculosis infection (LTBI), affects about one-third of the world’s population and approximately 10% of people with LTBI develop into active TB later on. Methods which facilitate efficient detection and interpretation of biomarkers for screening LTBI have been an important research topic as it attempts to deal with an international public health problem. Interferon-gamma (IFN-γ) as a biomarker indicator for tuberculosis and with antiviral, immunomodulatory and anti-tumor properties can be used as a reference to assess inflammatory status associated with risk of the disease. In addition, since the body immune function abnormalities produce neutralizing anti-IFN-γ antibodies (anti-IFN-γ Ab), it has the potential to be used in the development of drugs for antagonistic auto-antibodies associated with autoimmune diseases. With the limitations of current detection methods and costs, latent tuberculosis infection has not yet undergone thorough, large-scale screening. To this end, the development of a more appropriate and more readily available first-line diagnostic tool is one of the major priorities of the United Nations in combating tuberculosis. The experimental approach in this investigation was based on creating a bio-sensing platform. This platform, called OBMorph, was developed by the National Taiwan University (NTU), Nano-BioMEMS group. It integrated a surface plasmon resonance (SPR) technology and a phase modulated ellipsometer to improve the performance of biomolecular measurements. Electrochemical impedance spectroscopy (EIS) was also integrated simultaneously onto this measurement platform. Furthermore, a bithiophene-based conductive biolinker was also developed with an attempt to improve the antibody-antigen interactions for biosensors and to verify the performance as well as potential for synchronous measurement of IFN-γ binding interaction by the OBMorph platform and EIS. In addition, the bithiophene biolinker was introduced into our newly developed biochip and adopted into a commercially available SPR instrument (Biacore T200). We wanted to test and verify the suitability of the bithiophene biolinker and to optimize the test procedures for detecting anti-IFN-γ Ab. Our experimental results show excellent measurement performance for the OBMorph as well as for the newly developed biolinker. The interactions of the IFN-γ with immobilized anti-IFN-γ Ab at various concentrations (12.8pM ~ 1000nM) were investigated both optically and electrochemically. It was also verified that the bithiophene biolinker used was an effective biolinker for immobilizing antibody-antigen/antigen-antibody bio-detection. More specifically, we compared the response and concentration of the anti-IFN-γ Ab on a bithiophene-coated and dextran-coated biochip as well as on different thickness-modified surfaces under SPR measurement conditions. Our results indicate that a response to the IFN-γ molecules immobilized on a sensor using a bithiophene biolinker improved more than 8-fold when compared to that of a sensor using a dextran biolinker. A higher sensitivity on the immobilization of the IFN-γ and specific binding of the anti-IFN-γ Abs were both found in the newly developed bithiophene biochips when compared to that of a commercially available dextran biochip SPR measurement tool. Furthermore, the detection range of the anti-IFN-γ Ab obtained was 0.67nM to 83.33nM using a biosensor method without resorting to the use of second molecules for signal amplification. The regeneration ability of the sensor surface showed good repeatability as less than a 1% decrease was found after repeating the experimental work over 6 cycles. In this dissertation, we found that the OBMorph offered us a good platform for rapid screening, real-time monitoring and the potential for quantitative concentrations of the autoimmune antibody activities. Proper protocols were also developed to demonstrate the detection sensitivity, measurement resolution, dynamic detection range, and chip regeneration capability of this newly developed biochip. Effects associated with using the bithiophene as a biolinker for the anti-IFN-γ Ab interaction with IFN-γ are examined in this dissertation. Moreover, our newly developed integrated bio-sensing system has the potential to provide new insight into various conjugate phenomena and interfacial processes for observing molecular conformation changes. It provides an advantageous platform for proteomic research which is cost effective and cost efficient. In summary, the results obtained from the integrated bio-detection OBMorph platform in this dissertation, can be a good starting point for advancing future technology research. It can be used to further work on disease prevention or extending to other applications which has the potential to boost the local development of in vitro detection devices.

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