臺灣博碩士論文加值系統

台灣為東南亞地區白點病毒最主要疫區之一，因白點病毒使蝦類養殖產生很大的危害，但卻沒有完全根治病毒的藥物或疫苗。現今的藥物相關研究常常仰賴實驗室大型儀器，雖然實驗結果準確，但太過耗時，且無法即時觀測結果，造成人力成本與資源的浪費。因此本研究希望可提供一靈敏度佳且即時觀測之檢測晶片，達到快速記錄白點病毒感染歷程之目的，並由此結果作為藥物篩選之依據，進而開發有效的抗病毒藥物及疫苗。
本研究整合半導體與微機電製程技術，利用CMOS壓阻式微懸臂樑晶片與微流道封裝技術，製作一微型生物晶片。將CMOS壓阻式微懸臂樑晶片黏著於PCB板中央，由於微流道無法直接與PCB板接合，所以在PCB板上黏著一環型矽結構，使微流道與PCB板結合為一微型生物晶片，其數根懸臂樑長寬約為150m×50m，而CMOS懸臂樑晶片大小為1.5mm×1.4mm，與PDMS微流道進行封裝後，整體晶片大小為3cm × 3cm。本研究利用注射幫浦將含細胞之培養液打入腔體使細胞貼附於懸臂樑上。續以白點病毒攻毒，並使用多功能數位電表紀錄其懸臂樑壓阻阻值變化量而得其感染歷程。
得到上述感染歷程後，可發現白點病毒有明顯的病毒入侵、複製、脫離時期，茲此量測結果，本研究進一步對病毒有效抗體(VP28)與無效抗體(山羊抗兔子之單株抗體)研究探討，經過分別與病毒混合後打入晶片內發現有效抗體，使白點病毒與抗體先接合，而使病毒無法侵入細胞。本研究為了驗證上述結果，利用不同抗體進行研究探討，可發現打入無效抗體時，細胞還是會受到白點病毒的入侵、複製，最後病毒死亡，藉此排除本研究之偽陽性。
本研究開發之生物晶片已成功量測病毒感染歷程與白點病毒抑制歷程，
茲此量測結果，可於數小時驗證抗體是否有治病之療效，不僅免除了傳統免疫分析之繁瑣與費時，且證實本系統可達到藥物篩選之目的，於生醫檢測可大幅縮短疫苗開發之進度，加速產業發展，若朝向商品開發，對未來市場發展潛力無窮。

Taiwan is one of the major area of White Spot Syndrome Virus(WSSV) epidemic Southeast Asia, but there are no useful drugs or vaccines to against WSSV. Nowadays, the studies of medicines usually have been done by laboratory scale instruments, the results of experiments are correct, but this process wastes too much time and resources, nay, the results are not able to be observed immediately. As a result, we hope that this research will provide a bio-chip with high sensitivity and real-time detection, and it could record infection process of WSSV immediately. Furthermore, according to the result of drug screening we could develop effective antiviral drugs and vaccines.
The study involved the integration of semiconductor and MEMS process. More specifically, the resultant bio-chip is a technological combination of Complementary Metal Oxide Semiconductor(CMOS) piezoresistive microcantilever chip and microfluidic packaging. The CMOS piezoresistive micro cantilever beam chip was bonded in the central of a ring-shape silicon structure adhered on a Printed Circuit Board(PCB) board and then the microfluidic was adhered onto the silicon structure to complete the bio-chip fabrication. The cantilever beams length and width are about 150µm*50µm, and the original dimensions of CMOS microcantilever chip is 1.5mm*1.4mm. Then the final package with poly-dimethysiloxane(PDMS) microfluidic size is 3cm*3cm. In this study, the fluid with SF9 cells was injected into the cavities of microfluidic to make the cells attach to the cantilevers. The experiment was then followed by infecting these cells by WSSV and using multimeter to record the resistance variations.
According to the consequence, we find that WSSV infection process in host cells comprises three phases: virus intrusion, replication, detachment. Based on the results, we had done more research on inhibition ability of effective antibody (VP28) and invalid antibody (Goat anti rabbit monoclonal antibody). The effective antibody can be linked with WSSV after they were injected into the chips, so the virus was not able to intrude into cells. In order to prove the results, there are some experiments has been done on different kinds of antibodies. It is found that the cells would still get infected when the antibodies were ineffective.
Moreover, all of the experiments mentioned above could be finish in 24 hours.
The bio-chip has been experimented and recorded the progresses of virus infection and virus inhibition. According to the consequence, the antibodies can be tested and proved that they are efficacious or not. It could speed up the development of effective vaccine or drug to against WSSV and promote research abilities of medicine industry.

摘要 I
Abstract II
目錄 III
表目錄 V
圖目錄 VI
第一章 緒論 1
1.1 前言 1
1.2 研究動機 2
1.3 文獻回顧 3
1.3.1 白點病毒檢測與抑制 3
1.3.2 微型懸臂樑生物感測器 4
1.3.3 CMOS生物晶片 6
1.4 研究目的 10
1.5 論文架構簡介 10
第二章 CMOS壓阻式微型懸臂樑生物晶片原理與設計製作 11
2.1 微懸臂樑工作原理 11
2.1.1 共振式微型懸臂樑感測器 11
2.1.2 彎曲式微型懸臂樑感測器 12
2.2 壓阻特性 13
2.3 CMOS壓阻式微型懸臂樑感測器 14
2.3.1 微型懸臂樑模擬 14
2.3.2 CMOS壓阻式微型懸臂樑佈局與製程 18
第三章 實驗材料與架設 23
3.1 實驗材料 23
3.1.1 細胞株 23
3.1.2 白點病毒 23
3.1.3 抗體 24
3.1.4 螢光染劑 25
3.2 CMOS壓阻式微型懸臂樑生物晶片封裝 25
3.2.1 微流道製作 25
3.2.2 微流道底座製作 26
3.2.3 CMOS微懸臂樑晶片封裝 27
3.2.4 晶片封裝與滅菌流程 28
3.3 實驗儀器 28
3.3.1 水循環式恆溫槽 28
3.3.2 多功能數位電表 28
3.4 實驗架構 29
3.5 實驗流程 30
第四章 實驗結果與討論 31
4.1 CMOS微型懸臂樑量測結果 31
4.1.1細胞貼附歷程紀錄 31
4.1.2 病毒感染歷程紀錄 33
4.1.3 抗體(VP28)抑制白點病毒歷程 35
4.1.4藥物篩選實驗結果 39
4.2 CMOS微型懸臂樑整合溫度感測晶片量測結果 40
4.2.1溫度晶片量測 40
4.2.2溫度晶片細胞貼附歷程紀錄 41
4.2.3溫度晶片病毒感染歷程紀錄 43
4.2.4溫度晶片抗體(VP28)抑制白點病毒歷程紀錄 47
第五章 結論與未來展望 51
5.1 結論 51
5.2 未來展望 52
參考文獻 53

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