為利後續電化學檢測，本實驗所使用的基材為孔徑大小25µm的可導電不鏽鋼網。接枝流程如下，首先將不鏽鋼網與多巴胺(Dopamine)單體放置在鹼性的緩衝溶液中，在不鏽鋼網上形成奈米級多巴胺顆粒，隨後利用EDC/NHS共價固定法將明膠(Gelatin)與多巴胺結合，接枝上不鏽鋼網。接著，同樣利用EDC/NHS共價固定法修飾Protein G，再透過Protein G與抗體Anti-EpCAM的專一性強親和力固定抗體於基材表面。最後，為防止非特異性沾黏，將基材泡入牛血清蛋白(Bovine Serum Albumin，BSA)，在基材表面形成抗沾黏層，完成基材的製作。完成基材後，結合自製流體裝置進行循環腫瘤細胞以及病人檢體中目標細胞的抓取，接著利用電化學系統作定量分析，此實驗有進行至後端細胞培養以及利用胰蛋白酶(Trypsin)進行細胞無損釋放。
實驗結果以下歸納，利用各儀器定性確認高分子完整包覆及接枝上基材，並在確認最佳流速為1hr/3mL後，抓取率高達7成以上近8成，接著利用電化學阻抗頻譜偵測10、25顆循環腫瘤細胞時，阻抗變化呈現高度正相關，完成檢量線以利後續臨床分析，也透過DLD-1及Hela cells確認抗體之專一性。此外我們將細胞培養於基材上，細胞活性在第三天即可達338%，優於傳統培養盤許多，細胞培養至7天，利用胰蛋白酶裂解明膠，使細胞達到約90%的高釋放效果，也利用細胞死活染劑染色確認釋放之細胞存活率高達95%以上，顯示此試片未來有極大潛力可將病患血液中循環腫瘤細胞抓取後培養。
臨床分析上，本試片成功捕獲且利用螢光染色以及電化學阻抗法綜合分析判斷病患檢體之腫瘤細胞數，並利用病患不同治療時期之檢體，追蹤病人血液中循環腫瘤細胞之顆數，預期未來可利用此體外檢測之方法輔助醫生診斷，有機會進一步取代侵入式診斷方法。
本研究成功製備出專一性抓取EpCAM表現循環腫瘤細胞之流式篩網裝置，本試片具良好生物相容性，以及良好細胞釋放效果，提供一個高效率、低成本且靈敏度佳的體外檢測系統。

To facilitate subsequent electrochemical detection, the substrate used in this experiment is a conductive stainless steel mesh with a pore size of 25 µm. In the process, the stainless steel mesh and polydopamine are placed in a Tris buffer at first to form nano-scale dopamine particles on the stainless steel mesh, and then we used EDC/NHS to graft gelatin on to the surface of the stainless steel mesh. Same as well, Protein G was modified on the surface through EDC/NHS. We also modified specific antibody Anti-EpCAM on the surface through strong affinity between Protein G and Anti-EpCAM. Finally, we use bovine serum albumin ( Bovine Serum Albumin, BSA) to form am anti-fouling layer on the surface to complete the preparation of the substrate. After completing the substrate, we combine the self-made fluid device to capture the circulating tumor cells and the target cells in substrate, using electrochemical system for quantitative analysis. We applied it to do the cell culture and trypsin to release without damage the cells.
Experimental results are summarized as follows, we use instruments to
make sure polymer completely coating and graft on specimen. After decide the best flow rate 1hr/3mL, we use electrochemical system to do the quantitative analysis to 10 and 25 circulating tumor cells, a high positive correlation was found in impedance changes, and we use it to complete the calibration line and facilitate subsequent clinical analysis. The specificity of the antibody was also confirmed through
DLD-1 and Hela cells. In addition, we culture the cells on the substrate, and the cell activity can reach 338% on the third day, which is much better than the traditional culture plate. When the cells are cultured to 7 days, we use trypsin to lyse gelatin, the release rate has achieved 90%, we also use dead and live cell stain to make sure it’s good release effect of the cells, which showing that this substrate has great potential in the future to capture and culture circulating tumor cells in the patient’s blood.
In the clinical results, we successfully captured and used fluorescent and electrochemical method to comprehensively analyze and determine the number of circulating tumor cells in the patient sample, and we use specimen from patients during different treatment periods to track the number of circulating tumor cells in the patient’s blood, expected that this in vitro method can be used to assist doctors in diagnosis in the future, which has the opportunity to replace invasive diagnostic methods.
In this study, a fluid device was used to specifically capture cells with EpCAM performance, and it has good biological activity and cell release effevt. It can provide a high efficiency, low cost and highly sensitive in vitro detection system.

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