- 學位論文
研發偵測人類去氧核醣核酸甲基轉移酶活性之免疫電化學感測系統
Development of a Highly Sensitive Electrochemical Immunosensor for the Detection of Human DNA Methyltransferase Activity
摘要
近來研究顯示，異常的DNA甲基轉移酶表現及活性，與癌症的發展及進程相關，因此偵測DNA甲基轉移酶活性將有助於了解及診斷癌症相關疾病。儘管許多測定DNA甲基轉移酶的方法已被發展，但大部分的方法皆較昂貴且靈敏度較低，許多團隊致力於發展新的方法。因此本研究利用電化學技術並搭配酵素進行訊號放大，發展出偵測DNA甲基轉移酶活性的新穎系統。將具有DNA甲基轉移酶及限制酶Sau3AI辨識序列的雙股DNA修飾在鍍有奈米金球的網版印刷碳電極上，DNA甲基轉移酶能將S-腺苷甲硫胺酸 (S-adenosylmethionine, SAM)的甲基轉移至雙股DNA分子上，而不被後續加入的限制酶Sau3AI剪切，保留完整序列的雙股DNA分子。接著透過尿素處理破壞雙股間氫鍵而成為單股;最後使用可辨識甲基 (5-methylcytosine)且修飾生物素 (biotin)的抗體進行辨識。由於生物素和卵白素 (avidin)或鏈黴親和素 (streptavidin)有很強之結合力，因此使用修飾卵白素的辣根過氧化物酶 (horseradish peroxidase, HRP)進行訊號放大。在分別進行限制酶剪切、DNA去雜合反應及抗體辨識驗證後，我們進一步模擬DNA甲基轉移酶作用情形，並獲得最適合修飾於電極上的甲基化DNA之比例，最後進行DNA甲基轉移酶活性分析。利用辣根過氧化物酶進行訊號放大的方式，能達到的最低偵測極限為3.03×10−2 U/ml，優於以往偵測DNA甲基轉移酶活性的方法，且與市售的人類DNA甲基轉移酶免疫分析試劑組相比，本研究之最低偵測極限低於市售試劑組377倍，因此相當有潛力作為癌症預後偵測系統。
並列摘要
Recent studies have shown that the aberrant expression and activity of DNA methyltransferase are associated with the development and progression of cancers. Therefore the assessment of the DNA methyltransferase activity may provide more information for the evaluation of cancer diagnosis and treatments. Despite the various methods currently available for the detection of DNA methyltransferase activity, most of them are expensive and not sensitive enough. We herein developed a super sensitive electrochemical immunosensor for the detection of DNA methyltransferase based on the catalysis of horseradish peroxidase (HRP-avidin). Gold-electrodeposited screen-printed electrodes were first modified with our thiol-sensitized DNA probes, it was followed by the formation of DNA duplex after an addition of their complementary DNA counterparts. The formed DNA duplexes on the electrode contained recognition sites for DNA methyltransferase and restriction enzyme Sau3AI. The methyl group from S-adenosylmethionine (SAM) was transferred to the DNA duplexes in the presence of DNA methyltransferase. The fully-methylated DNA duplexes inhibited the restriction enzyme from functioning. At last, DNA duplexes were dehybridized after treating with urea, and the immobile probes can therefore be recognized by biotin-tagged, anti-5 methylcytosine polyclonal antibodies. The signal current was subsequently measured by the catalysis of the avidin-HRP through a strong interaction between biotin and avidin. The electrochemical immunosensor using avidin-HRP for signal generation exhibited a detection limit of 3.03×10−2 U/ml for the analysis of DNA methyltransferase. Compared with most of the other DNA methyltransferase assays and commercially available kit, the sensitivity of our assay was greatly improved. We thus concluded that our sensor holds a great promise for being a practical tool for cancer prognosis.
參考文獻
延伸閱讀
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