微流道系統結合免疫磁珠偵測淋巴癌細胞

Lymphatic cancer cells detection utilizing an immunomagnetic bead-based microfluidic system

董正霖(Cheng-Lin Dong)

指導教授 ： 吳見明 ； 賴麗珍

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2012年)

摘要

現今臨床醫學中的應用上，即時檢測扮演極重要的角色。以一般疾病檢測來說，細胞培養是非常需要的，然而早期疾病顯現的細胞數量非常少，因此需要大量培養細胞以達到偵測的數量。常用來偵測細胞的方法，有酶聯免疫斑點法(Enzyme- linked Immunospot Assay, ELISPOT)，和流式細胞儀（Flow Cytometry，）。ELISPOT雖可得到較好的偵測靈敏度，卻需要數天的反應時間；而FCM雖偵測快速，但靈敏度大約僅有0.01%，且價錢昂貴。因此本研究將以高靈敏度的檢測概念結合於生醫晶片中，進行癌細胞的偵測。生醫晶片透過大量製造的方式，不僅可以減少成本，晶片一次性的拋棄使用，也可避免生物感染及藥品交叉汙染。本實驗，將以免疫磁珠與螢光結合於微流道系統，利用磁珠分離特定細胞，再利用螢光訊號偵測癌細胞。流道層包含兩個反應區，其中第一混合反應區，是以圓形結構作為設計，配合平行錯位的微管道引入，以及蠕動幫浦驅動達到藥品均勻混和的效果；而第二反應區為偵測區，使用外接488 nm波長雷射激發偵測區樣品，並利用光纖接收樣品的螢光訊號。在本實驗中，癌細胞將與螢光藥物先行反應，再與免疫磁珠進行反應，並於第二反應區底部，外加磁鐵進行吸引，最後進行螢光訊號量測。本實驗結果僅需約5秒的偵測時間即可達到即時檢測目的，並達到0.001%的靈敏度；此外，本實驗系統可精準的傳輸於不同的反應區域，效率可達95%以上；最後，相較於傳統臨床流式細胞儀的檢測極限0.01%，本系統不但可以達成，甚至可達0.001%的靈敏度。

關鍵字

流式細胞儀 ； 酶聯免疫斑點法 ； 免疫磁珠 ； 微機電系統

並列摘要

It is an important issue to have real-time and highly sensitive detection of the disease. For a tumor in the early stage, the concentration of disseminated cells circulatinge in the blood is extremely low, making the detection of low concentration cancer cells difficult. Conventional methods for detecting small amount of cells include culture-based techniques and enzyme-linked immunospots (ELISPOT). However, it is labor-intensive and time-consuming to increas the number of cells by culture or ELISPOT to a number that suffices for measurement. Another approach, flow cytometry (FCM) has been established for detecting low concentration of cancer cells. However, the detection sensitivity of FCM is approximately 0.01%.
In this thesis, combining the magnetic bead and fluorescence in the microfluid system was applied to obtain rapid and sensitive detection of cancer cells. Microfluidic system has a number of advantages, including rapid detection of small amounts of samples, low-cost, and disposable.
The detection of cancer cells is made by measuring the fluorescence which is conjugated with the first marker of the specific cells in the micro-reaction chamber. Additionally, the magnetic bead is adopted to conjugated with the second marker of the specific cells. The magnet is applied in the second reaction zone of the microfluid system to isolate the specific cells. The results show the detection sensitivity of the cancer cells is around 0.001% within 5 seconds period of the exposure time. And the percentage of biological cells can be transported into another reaction zone of microfluid system for above 95%. As a result, the immune magnetic bead-based microfluidic system successfully integrates rapid and high sensitivity on cancer cell detection .

並列關鍵字

FCM ； ELISPOT ； Immunomagnetic bead ； MEMS

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