使用奈米探針裝置進行細胞外囊泡的全自動化晶片抓取與檢測

Abstract

癌症是造成現代人死亡的重要疾病之一，由於其偵測困難以至難以早期診斷。傳統的腫瘤活體檢測風險高又可能因為採取部位差異導致診斷誤差，這些問題難以突破，而液態活體檢測技術提供了癌症診斷及監控一個全新的方向，技術的方便、即時性高且低侵入性的優點，比起傳統侵入式檢測，更能加入伴隨性檢測中，近年相關研究與應用越發廣泛。 外泌體(Exosomes)是液態活體檢測重要的檢測標的物，其中含有與原細胞相關且豐富的遺傳物質mRNA, miRNA,蛋白質等訊息，有細胞與細胞間傳遞訊息的功能，接受到這些外泌體的細胞也會有所改變。近年來，研究顯示液態活體檢測技術的高度發展，也有許多外泌體在癌症早期診斷的相關應用，使得外泌體越來越受醫學界重視。然而，因於血液中外泌體體積百分比低，再加上粒徑微小，約為30~150 nm，使得抓取或純化一直都是一項艱鉅的任務。 本研究中，使用了奈米探針裝置，藉由其親和力來抓取微小的外泌體，此技術的價格相對抗體抓取與超高速離心等現今較常使用的抓取技術而言都相對低廉，且實驗過程都是由軟體操控的完整的機械化過程，除了減少操作者的誤差外，也降低環境的汙染及干擾。實驗方面，使用的是AU565的細胞株(1.13×10^10±1.7×10^9particles/ml)，設計一系列的實驗，藉由白金漢π定理分析後獲得的無因次參數進行優化，選擇出固定時間中最高且穩定的光強度參數，設計為擾動優化下最佳的樣品孵化過程，進而進行不同濃度的定量實驗，最後加入脂蛋白，進行干擾物實驗，證實加入粒徑較小的特定濃度(5×10^9particles/ml)干擾物後擾動仍具有優化的效果。另外，也利用COMSOL軟體進行動態分析，解釋well的幾何設計，以及剪切力與well粗糙度對樣品接合的影響。 對液態活體檢測技術而言，現今機械化的設備並不普及，結合奈米探針系統的抓取機制更是不普遍，綜合研究中討論到的優點，希望在經過不同種類的癌症測試後，可以在往後應用於臨床上。

Liquid biopsy provides cancer a new direction of diagnosis and monitoring. The advantages of this technology are minimally invasive, real time monitoring and cost effective. Exosome is an important detection target for liquid biopsy, containing abundant genetic material mRNA, miRNA, protein and other information related to the original cells. In recent years, studies have substantially advanced the development of exosomes in liquid biopsy, for example in early diagnosis of cancer. However, the clinical usage of exosomes remains technically challenging, particularly in developing technology for exosomes isolation. In this thesis, the nanoprobe technology was used to apply in an automatic process of exosomes capture. Sample loading, antibodies and reagents reaction, washing and releasing the residual particles away from the well and the detection of the chemiluminescence signal were controlled in order to reduce operator error and environmental contamination. Exosomes from a breast cancer cell line AU565 (1.13×10^10±1.7×10^9particles/ml) was used to test non-dimensional parameters analyzed by the Buckingham π theorem. Moreover, selection of the parameter with the highest and most stable intensity in a fixed time was used to optimize the incubation protocol. Then, different concentration of exosomes was used and lipoproteins were added to conduct interference experiments. In addition, COMSOL Multiphysics was also utilized for dynamic analysis to explain the geometric design of the well and the effect of shear force related to well roughness on the sample conjugation. In conclusion, the platform for capturing exosomes has completed preliminary tests. Further effort is needed to apply the technology in clinical applications.