分離去氧核醣核酸結構的分子與簡易癌症檢測的組件之研發與設計

Abstract

3,6-雙(4-甲基化砒啶烯基)咔唑(BMVC)是一個有機螢光分子，在DNA研究和癌細胞檢測上有特殊的應用價值。實驗發現它不僅和DNA結合後會產生大量的螢光增強，並且對於不同結構的DNA具有不同的結合趨勢。另外BMVC分子具有選擇性進入癌細胞之細胞核的能力，並且在癌細胞核內與DNA作用後，產生高螢光強度。本篇研究的主旨便是結合BMVC分子與材料設計的概念，分別著重在二方面：首先在DNA結構的研究，其次應用在癌細胞的檢測，將BMVC分子的價值提升到更實際且廣泛的應用範疇。 由於BMVC具有DNA結構上的選擇性，因此可以將BMVC應用在DNA結構的分離上。將BMVC分子改造成大型粒子，即可讓材料同時具有DNA結構上的選擇性並且允許我們利用過濾或者離心的方式，藉由粒子上BMVC的選擇作用，將不同的DNA結構進行分離。實驗結果顯示不論將BMVC設計成油/水界面活性劑或是修飾在奈米材料表面，都能夠讓我們將雙股、平行四股與非平行四股的DNA結構分離，配合光譜和電泳的結果，有效分析DNA結構上的複雜度，此成果為全球首次成功直接將不同DNA結構分離的方法，擴大了BMVC對於DNA研究上的應用範圍。 在DNA研究之外，利用BMVC在癌細胞中特殊的螢光效果，結合管柱層析的概念，可以準確地將BMVC應用在癌細胞的檢測上。將BMVC染色後的癌細胞捕捉在小型矽膠管柱，在適當光源下用照相機觀察，並藉由影像分析將螢光訊號轉為數位輸出，即可篩檢臨床檢體中是否有癌細胞，並且可估計檢體內癌細胞數。此方式可設計成小型的檢測裝置，在癌症檢測診斷上提供即時資訊。此簡易癌細胞偵測方法應用在台大醫院臨床胸腹水之112個案例檢測，結果已達到90%以上的準確度，希望有機會在醫院提供更準確的診斷服務，減少醫療成本的消耗。 本篇論文首先確立「DNA結構分離」與「臨床癌細胞檢測」兩項議題的重要性，針對目前的關鍵問題，嘗試利用BMVC分子的特質，結合材質的選擇性與孔洞的過濾分離，成功的將不同的DNA結構分離，確認了部分DNA序列在不同環境下的結構轉換；另外經由研發細胞組件 (cell kit)，提升臨床上BMVC檢測癌細胞的準確度達90%。這些結果在科學研究或者醫療應用上都具有重要意義，同時提升BMVC的分子優勢到更實際的應用。

Two new methods are developed here: one for DNA structural separation and another for cancer cell detection. Both of these methods have adapted the special characteristics of a small, fluorescent molecule called 3,6-Bis(1-methyl-4-vinylpyridinium) carbazole (BMVC). As an effective G-quadruplex stabilizer, BMVC has different binding preferences to different DNA conformations such as parallel or anti-parallel DNA quadruplex. On the other hand, it also exhibits different fluorescence intensity upon incubation with cancer and normal cell. Base on these two distinctive properties of BMVC, we synthesized a new derivative of BMVC for structural separation and designed a simple cell kit for rapid cancer detection. First, a novel method based on emulsion/filtration is introduced for DNA structural separation. We synthesized a lipophilic derivative of BMVC, 3,6-Bis(1-methyl-4-vinylpyridinium)-9-(12’-bromododecyl) carbazole (BMVC-12C-Br), which can form an oil-in-water (o/w) phase emulsion (average diameter ~2 μm). With its unique binding property, the BMVC-12C-Br emulsion shows an ability of selectively capturing certain specific DNA structures. Further filtration was employed by using a 0.22 μm pore size MCE membrane to separate the bound and free DNAs, i.e., the bound DNA will stick to the emulsion which cannot pass the MCE filter, while the free DNA can be collected after filtration. This emulsion induced filtration (EIF) method is able to isolate the non-parallel G-quadruplexes from the parallel G-quadruplexes and the linear duplexes from both G-quadruplexes. Using EIF method, one can isolate the conformation which has lower binding preference to BMVC. However, the emulsion bound DNA is trapped in the MCE membrane and is difficult to be obtained. We thus introduced nanotechnology to solve this problem. Our data shows that it provides some advantages and is a better method for DNA separation. Second, a simple cell kit is designed to collect cells from clinical pleural effusion for a rapid cancer diagnosis at low cost based on the enormous fluorescence intensity difference between BMVC in cancer and in normal cells. We consider that a pre-process for column separation and cell collection from sample is most likely the major factor to validate BMVC test for cancer diagnosis. After the pretreatment of clinical sample, the imaging-J software is used to analyze the BMVC fluorescence intensity. Compared with the gold standard of pathology for cancer diagnosis, this simple method without human factors gives a good discrimination with a sensitivity of ~90% (45/50) and a specificity of ~92% (56/61) for cancer diagnosis of pleural effusion from outpatients. In conclusion, combined filtration with material design, a method called emulsion induced filtration (EIF) is established, which can successfully separate two different DNA structures. On the other hand, through the development of cell kit for clinical cancer detection, BMVC molecule can achieve 90% accuracy in clinical pleural effusion samples. These results play important roles in both science research and medical therapy fields, and also provide further application using BMVC.