Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35636
標題: Label-Free Nucleic Acid Sensors for Detection of Salmonella Based on Electrochemical Impedance Spectroscopy
以電化學阻抗法檢測沙門氏菌之免標定式核酸感測器的研發
作者: Wang, Yi-Ting
王怡婷
關鍵字: Salmonella
沙門氏桿菌
label-free
electrochemical impedence spectroscopy
cyclic voltammetry
nucleic acid sensor
electrode cleaning
免標定式
電阻抗分析法
循環伏安法
核酸感測器
電極清潔
出版社: 生物產業機電工程學系所
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摘要: In Taiwan, Salmonella is one of common sitotoxism-inducing microbes, causing diseases such as septicemia and gastroenteritis. Therefore, it is a very important issue to quickly detect the existence of Salmonella. This study attempts to develop a chip-type nucleic acid sensor for the detection of Salmonella by using electrochemical impedence spectroscopy (EIS) and cyclic voltammetry (CV) to measure the variation of impedence and current when performing the DNA hybridization. However, the initial cleanness states of chip-type electrodes would affect results such as the sensing reproducibility and the probe-DNA modification. Therefore, three kinds of cleaning procedures including (1) acetone, (2) oxygen plasma, and (3) piranha and aqua regia are compared for the electrode cleanness. The results show that the treatment of soaking in piranha for 2 min and in aqua regia for 1 min gives the best cleanness for the electrodes which obtains the smallest electron-transfer resistance and the best reproducibility. In addition, the effect of structures and charges of different buffering molecules on the sensing characteristics was compared. The results reveal that use of TES buffer can obtain the largest response of electron-transfer resistance to the MUA-modified or dsDNA- hybridized electrode-solution interfaces. The linear range and the detection limit for Target-DNA measured in TES buffer was 10-10~10-15 M and 10-15 M, respectively, which is better than the linear range (10-10~10-13 M) and the detection limit (10-13 M) measured in PBS buffer. Moreover, this nucleic acid sensor using EIS measurements can identity the signal difference between the single basepair mismatch and the 40-mer target-DNA and the complete complementary hybridization. The miniaturzied nucleic acid chip is beneficial for the integration of micro-fluidic system and has the advantages of portability and mass production.
沙門氏菌(Salmonella)為台灣常見導致食物性中毒病菌之一,易引發菌血症或急性腸胃炎,因此快速檢測出沙門氏菌是項重要議題。本研究利用電化學阻抗頻譜(electrochemical impedence spectroscopy, EIS)技術與循環伏安法(cyclic voltammetry, CV)量測DNA雜交前後電極介面電性響應的變化,開發一可檢測沙門氏菌特異性基因片斷的晶片式核酸感測晶片(chip)。 由於電極表面初始狀態的潔淨度,會影響電極的檢測再現性與DNA探針固定的結果,所以實驗初步先探討三種清潔方式(1) acetone, (2) oxygen plasma, (3) piranha /王水(aqua regia) 對電極表面清潔度的影響,結果顯示:piranha 2 min/aqua regia 1 min浸泡的清潔程序,可使電極表面具有最低的電子轉移阻抗與最佳的再現性。此外,也比較不同緩衝溶液分子的結構與電性對檢測特性的影響,結果顯示在相同MUA修飾或dsDNA雜交的介面,TES溶質分子可得最大的電子轉移阻抗量響應。在TES對target-DNA檢測的線性範圍為10-10~10-15 M,最低檢測極限可達10-15 M,皆較優於PBS中的線性範圍(10-10~10-13 M)與檢測極限值(10-13 M)。此外,此晶片藉由EIS量測可成功分辨出單鹼基配位錯誤和40 mer的target-DNA之檢測訊號與完全互補雜交後訊號間的差異。此微小化晶片的開發有利於微流體系統的整合並具有可攜式、大量生產等優勢。
URI: http://hdl.handle.net/11455/35636
其他識別: U0005-1908201014212400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1908201014212400
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