雙功能核酸適體辨識元件設計於表面電漿子共振生物感測器之應用： 以丙型干擾素檢驗於結核感染診斷為例

Design of Bifunctional Aptamer-Based Recognition Elements for Surface Plasmon Resonance Biosensors Applications: Using Interferon-r Detection for Tuberculosis Infection Diagnosis as an Example

李忠翰(Chung-Han Lee)

指導教授 ： 林啟萬

國立臺灣大學/工學院/醫學工程學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10925

摘要

近年來具分子辨識功能的核酸適體（Aptamers）逐漸用於設計成為取代傳統抗體免疫檢測的分子檢測平台。核酸適體是由單股寡核苷酸構成（ssDNA或ssRNA），其功能類似抗體，能與特定的目標分子具有高度的專一性辨識，但其製造卻可以經由化學合成因此品質容易控制而且核酸本身穩定度高於抗體，此外，核酸適體所能辨識的目標分子尺度從小分子如：胺基酸、抗體、維生素等；至較大分子如：蛋白質、生長因子；甚至大到整個細胞或微生物如:細菌都有其核酸適體可做應用，使得核酸適體在光學、電化學等感測技術中，已被廣泛應用作為分子辨識元件。其中，在這些不同的感測技術裡，利用光學感測原理的表面電漿子共振（surface plasmon resonance, SPR）生物感測器因為其具有可定量（quantitative）、高靈敏（high sensitive）、即時性（real-time）與不須標記（label-free）的特性而日漸受到重視。對於免標定的生物感測器而言，其檢測後得到的訊號通常來自於目標分子吸附於感測表面所造成的物理性或化學性變化，但是，當待測物在檢測環境中的濃度極低時，檢測極限 （limit of detection，LOD） 往往將受限於感測器本身之最小訊雜比而無法有效檢測，因此，如何將探針辨識到低濃度目標分子後的微小訊號變化進一步放大，便成了生物感測器發展過程中一個評估其應用價值的重要指標。基於上述原因，本研究試圖發展一個兼具分子辨識與訊號放大的雙功能核酸適體探針，希望藉由探針具有此雙功能的特性，期望使得生物感測器能同時具備專一性與靈敏度的目的。研究中，辨識探針的設計概念是將具有特定專一性的核酸適體之DNA序列設計成具有自發形成hairpin （stem-loop）結構的辨識探針，並且為了達到放大SPR生物感測訊號的功能，我們利用在探針stem部分的末端修飾上生物素（biotin），目的是藉由當探針與目標分子結合後打開探針原有的hairpin結構使得stem末端的biotin能夠外露，接著利用加入放大分子鏈黴卵白素共軛金奈米粒子（streptavidin conjugated gold nanoparticles，SA-AuNP）與生物素結合以達到放大SPR訊號而進一步提升SPR感測之靈敏度。為了驗證我們設計的探針結構能否具有雙功能的特性並能成功應用於發展SPR生物感測器上，本研究我們以檢測丙型干擾素（Interferon-r）於潛伏性結核感染診斷之臨床需求為例驗證我們的設計概念。從實驗結果顯示，在探針的固定化上，我們成功利用在探針5’端修飾上硫醇官能基將IFN-r aptamers探針固定於金表面；在雙功能辨識探針設計上，藉由反應動力學與熱力學的分析我們成功設計了具有開關功能（turn on/off）的hairpin 探針結構；最後，我們在緩衝溶液中實際檢測低濃度25 nM IFN-r 的實驗結果顯示，本研究所設計之雙功能丙型干擾素核酸適體探針的確可以成功專一地辨識丙型干擾素並進一步放大SPR訊號達10倍，得到明顯的SPR反應訊號，不僅如此，放大分子的使用更可以作為報導探針（reporter probe）以再次確認探針是否有與目標分子結合以減少實際檢驗上偽陽性的發生率。整體來說，本研究所提出之雙功能核酸適體辨識分子的設計，兼具對於目標分子的專一性，也進一步提升了SPR感測的靈敏度，有助於SPR感測應用於低濃度或是小分子的檢測上，因此於未來將能夠廣泛應用於醫學診斷、環境與食品安全等檢測上。

關鍵字

生物感測器 ； 核酸適體 ； 表面電漿子共振 ； 潛伏性結核感染 ； 丙型干擾素

並列摘要

Recently, aptamers are considered to be ideal recognition elements for biosensing applications. Aptamers are oligonucleotides （ssDNA or ssRNA） which can specifically bind to targets. The characteristics of aptamers are similar with antibodies. Moreover, aptamers can be produced by chemical synthesis appoarches, so we can easily control the quality of aptamers. To compare with antibodies, aptamers are more stable and have a wide range of targets recognition such like amino acids, proteins, and even cells. Based on tha advantages of aptamers, aptamers are applied for being recognition elements in optical, electric, and others senseing platforms. Although there are many different sensing approaches to develop aptamer-based biosensors, surface plasmon resonance （SPR） biosensors are becoming more and more important because of their several unique advantanges such as quantitative, high sensitivity, real-time detection and label-free. However, for SPR and other label-free biosensors, the signal of detection is usually caused by target adsorption on the sensing surface. Therefore, the limit of detection （LOD） is very important for lebel-free biosensors expecially on detecting low concentration of targets. As the result, how to amplify the signal change after binding is an essential requirement for developing higher sensitive SPR biosensors. In this thesis, we want to design a bifunctional aptamer-based recognition element for SPR biosensors to not only specifically recognize targets but also amplify SPR response after binding. To achieve this purpose, we constructed an aptamer-based hairpin （stem-loop） probe configuration and modified a biotin at the end of stem. We expect that the hairpin probes we proposed can selectively bind to our targets and open the hairpin conformation. Then, we can further add the amplifier, streptavidin-AuNP to enhance SPR response and improve sensitivity. To demonstrate our appoarch, we utilize Interferon-r (IFN-r) as our target in this sutdty for latent tuberculosis infection diagnosis. The results showed that we successfully immobilized the 5’ end of hairpin on the gold sensing surface of SPR by Au-S covalent bond. Moreover, the immobilized hairpin structure might not influence the specificity between IFN-r aptamer and IFN-r. According to these unique properties of hairpin probe, we integrated the principle of kinetics and thermodynamics to design a suitable “aptamer beacon” which spontaneously folded to hairpin structure. In the detection of 25 nM IFN-r, the aptamer beacons specifically bound to IFN-r and amplified SPR response upto 10 times by using streptavidin-AuNP as amplifiers. Besides, the use of amplifier might also be a reporter to avoid false-positive in practical applications. Consequently, this research efforts provid a novel design strategy of bifuctional aptamer-based recognition elelmets to selectively bind to IFN-r and improve the sensitivity of SPR biosensors, which is of great benefit to a wide range of applications especially on detection of low concentration of targets or small molecules for medical diagnosis, environmental monitoring, and food safety.

並列關鍵字

biosensors ； aptamers ； surface plasmon resonance ； latent tuberculosis infection diagnosis ； interferon-r

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