免疫生醫感測技術應用於癌症早期診斷

Immuno-biosensors for Carcinoma Early Detection

陳榮治(Jung-Chih Chen)

指導教授 ： 林峯輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.01232

摘要

石英微天平(Quartz qurstal microbalance；QCM)之理論與技術皆已十分成熟，藉以開發成一免疫感測器(Immunosensor)。石英晶體微天平法常見於極微量生化物質感測技術上，尤其在微生物檢測部分甚是；然而，QCM法用途雖不少，但卻少有臨床檢體實例。
本團隊具備微量生化物質分析經驗與癌症臨床檢體感測技術，在結合石英晶體微天平法後，更是集效率、靈敏、省時、經濟於一身。
本研究試圖在石英晶體表面修飾上生物標記物(Biomarkers)，也就是固定生物標記物之抗體在石英晶體表面，利用抗體-抗原彼此間會產生專一性吸附(Specific binding)之原理，藉此能夠進行包含癌症腫瘤標記等生物標記物之感測功能。
在第一部份實驗中，選用非小細胞肺癌（西方國家最常見致死的癌症，它包含了鱗狀細胞癌、腺癌、大細胞癌以及支氣管肺泡肺癌等）腫瘤因子的標記物：EGFr Ag-Ab當作研究對象；並利用特殊界面改質材料--Thiosalicylic acid (TSA)來修飾石英晶體表面，且導入Protein G來避免非專一性吸附。
經實驗證實，有使用Protein G進行抗體Site specific orientation步驟的結果，比一般化學吸附(Chemical cross-linking)固定抗體之方法效果來得好。另外，由實驗結果發現，經特定部位(Site-specific)固定抗體後之電極可經由0.1 M的氫氧化鈉進行再生，且重複使用次數可達6次。
以該技術將anti-EGFr antibody接枝在修飾後石英晶體表面，用於癌症早期診斷的偵測範圍由10 ng/ml∼10 μg/mL、實際檢測極限可低於1 ng/ml，這檢測極限足以與現有ELISA檢測技術分庭抗禮，甚至遠遠超越，因此可用於癌症的早期診斷上。
再者，因QCM實驗架構可採Flow-injection reaction設計，除了避免實驗過程造成訊號干擾外，更能達到篩選、富集樣本及即時觀察作用機制...等一貫化優勢。
第二部份我們選用與新生兒腦膜炎(Neonatal meningitis)發生息息相關的一種蛋白--OmpA，由於至今尚無任何快速檢測OmpA的技術，所以本研究創新於此。且由實驗結果發現，以此技術可以測得濃度20 ng/ml∼1.20 μg/ml的OmpA抗原；在OmpA抗原偵測極限探討上，由檢量曲線推估可達2 ng/mL。OmpA雖非癌症標記物，但一樣深具臨床檢測價值與意義。

關鍵字

石英晶體微天平 ； 免疫感測器 ； 生物標記物 ； 腫瘤標記 ； 非小細胞肺癌 ； 上皮細胞生長因子受體 ； 新生兒腦膜炎

並列摘要

The quartz crystal microbalance (QCM) technology has reached a mature and commonly used in biomedical sensing technology. Due to this system can detect extremely low concentrations, so it can develop into an immune sensor (Immunosensor), the most commonly used for microbial detection. However, the QCM method been widely used, but few clinical examples. As our team has the experience and analysis of trace substances in biological samples of cancer clinical sensing technology, once combined with quartz crystal microbalance method, the set of efficient, sensitive, time-saving and economic ... in one.
This study attempts to modify the quartz crystal surface with biological markers (biomarkers), which is a fixed biological marker of antibody in quartz crystal surface, and using the phenomenon of antibody and antigen specific adsorption, to detect cancer tumor markers.
In the first part of the experiment, we proposes the construction of a molecular recognition layer composed of thiol molecules and further activation with EDC-NHS complexes on the surface of a quartz crystal microbalance (QCM). The use of tumor markers factor: EGFr Ag-Ab as the object of study. EGFr is usually highly expression in non-small cell lung cancer patients (the most common cancer in Western countries, which includes squamous cell carcinoma, adenocarcinoma, large cell carcinoma and bronchioloalveolar lung cancer, etc.). We use special materials: thiosalicylic acid (TSA) to modify the surface of quartz crystal, and Protein G to avoid the import of non-specific adsorption. Two different antibody immobilization patterns such as chemical cross-linking and site-specific via protein G were attempted. Accordingly, protein G mediated antibody immobilization controls the quantity and orientation of the antibody molecules on the electrode surface for its high affinity to antigens. Thus, a similar immobilization strategy was utilized for the flow-cell analysis of real-time binding of EGFr. A linear relationship was observed between the frequency shift and different EGFr concentrations (0.01–10 μg/mL), and the detection limition lower than 1 ng/mL. The stepwise assembly of the immunosensor was characterized by quartz crystal microbalance. It was also demonstrated that, the prepared sensor surface was stable enough to withstand the repeated surface regeneration with 0.1 M NaOH.
In the second part, the ability to produce indigenous diagnostic tests for neonatal meningitis, that are novel, specific yet cost-effective would now provide a huge impact on public health management in developing countries. Escherichia coli (K1) is the major cause of neonatal meningitis through the interaction with human brain microvascular endothelial cells (HBMEC). Outer membrane protein A (OmpA) is a major protein in the E. coli outer membrane. However, there are not any rapid detections for OmpA. Immobilization of antibodies onto the crystal will be allowed to detect OmpA antigens for nanomedicine applications. This study originally developed the aforementioned QCM method to rapidly detect OmpA antigen. The method utilizes the piezoelectric immunosensor onto whose thiol activated crystal surface the affinity-purified antibodies would be immobilized, and the main objective of this study is to optimize the surface monolayer conditions to provide efficient binding of anti-OmpA IgG antibody and to exploit sensing surface for the detection of OmpA antigen, as an alternative diagnostic test. Though, the frequency shift was lower for OmpA antibody, still it can able to detect lower than 20 ng/ml of OmpA antigen. The detection of OmpA protein by QCM method is comparatively simple and sensitive than other systems.

並列關鍵字

Quartz crystal microbalance (QCM) ； Immunosensor ； Biomarkers ； Tumor markers ； non-small cell lung cancer ； Epidermal growth factor receptor (EGFr) ； Flow-injection Assay (FIA) ； Neonatal meningitis ； Outer membrane protein A (OmpA)

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