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標題: | 基於影像表面電漿子共振生物感測器的連續監測腫瘤細胞系外泌體研究 Continuous monitoring of exosomes from cancer cell lines by automatic imaging surface plasmon resonance |
作者: | 許晉懷 Jin-Huai Xu |
指導教授: | 林啟萬 Chii-Wann Lin |
關鍵字: | 表面電漿子共振,細胞培養,外泌體,整合素Alpha6beta4, SPR,Cell Culture,Exosome,Integrin Alpha6beta4, |
出版年 : | 2022 |
學位: | 碩士 |
摘要: | 外泌體的特徵大小為40-160納米的細胞外囊泡,已被證明有很大潛力作為早期或復發癌症預後的生物標誌物。然而,外泌體研究的發展對準確性、有限的樣品量和檢測等成本提出了更高的要求。表面電漿子共振(SPR)具有高靈敏度、即時性和非標記性等優點。本研究應用了影像SPR系統,並將其與設計的細胞培養管相連。 然後應用IDA-B適體探針檢測外泌體表面的特定亞型整合素Alpha6beta4,主要來自4175-LuT細胞系(實驗組)與MDA-MB-231細胞系(對照組)。方法:首先,通過三維建模設計並製作細胞培養管連結SPR系統,並使用NTA、基因表達和細胞形態學分析初步討論細胞和Alpha6beta4外泌體在15和40微升/分鐘的流速所產生的剪切力下和預先設計好的細胞培養管中的生長代謝狀態。其次,用IDA-B適體測量對照組和實驗組之間通過納米顆粒追蹤分析(NTA)定量的相同濃度整合素Alpha6beta4外泌體之SPR信號的差異。再次,將一種新設計的具有不同結構且不能捕獲Alpha6beta4的適體IDA-GC與IDA-B進行Alpha6beta4外泌體捕獲,從而證明IDA-B特異性結合性能。最後,分別使用適體IDA-B和IDA-GC對細胞培養基中的外泌體進行連續檢測測試。結果:影像SPR可以穩定地測量出對照組和試驗組培養基中整合素Alpha6beta4外泌體的信號差異。同時,細胞代謝不會受到剪切力的影響初步驗證了連接表面電漿子共振系統與細胞培養管的進行連續檢測外泌體之可行性。討論:基於表面電漿子共振的特點,SPR可以即時測量比其他方法更低濃度的樣品。同時SPR系統是一種很有前途的可以與其他系統整合成新的生物感測器系統,例如可以與細胞培養系統和藥物或化合物篩選機合作。 Exosomes, with a feature size of 40 - 160 nm and one of the extracellular vesicles, have been shown to have great potential as biomarkers for early or relapse cancer prognosis. However, the development of exosome research has put forward higher requirements in accuracy, limited sample volumes, and cost. On the other hand, surface plasmon resonance (SPR) has advantages in high sensitivity, real-time, and non-labeling. This research applies an imaging SPR system and connects it to a cell culture tube. And then, the IDA-B aptamers are used to detect a specific sub-type integrin alpha6beta4 on the surface of exosomes, which are mainly from test group 4175-LuT cell lines vs. control group MDA-MB-231 cell lines. Methods: First, cell culture tubes were designed and fabricated by 3D modeling linked to the SPR system, and nanoparticle tracking analysis (NTA), gene expression, and cell morphology analyses were used to initially discuss the state of cells and alpha6beta4 exosomes under the shear forces of flow rates of 15 µl/min and 40 µl/min under the designed cell culture tube. Then, the difference between the control and test group of integrin alpha6beta4 exosome signals, which were also quantified by NTA, was measured by imaging SPR with IDA-B aptamers. After that, to verify the IDA-B performance of integrin alpha6beta4 exosomes binding ability, new aptamers, IDA-GC, with different structures which could not capture integrin alpha6beta4 exosomes, were designed to compare. Finally, the aptamer IDA-B and IDA-GC were applied to continuously detect the exosomes from the cell culture medium. Results: The imaging SPR system could stably measure the signal difference of integrin alpha6beta4 exosomes between the control and the test group. At the same time, the shear force did not affect cell metabolism in this experiment. This result initially verified the feasibility of the imaging SPR system connecting with the cell culture tube. Discussion: Compared with other detected methods, SPR allows real-time and sensitive measurement of exosomes under lower concentrations. Meanwhile, the SPR system is robust and can be integrated with other systems, such as cell culture systems and drug/compound screening machines, to form new biosensor systems. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87635 |
DOI: | 10.6342/NTU202210023 |
全文授權: | 未授權 |
顯示於系所單位: | 生醫電子與資訊學研究所 |
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