革蘭氏陰性菌細胞壁外膜之脂多醣體 (lipopolysaccharide, LPS)又稱為內毒素 (endotoxin)，在細菌死亡解體時會釋放出來，可引起人體產生休克或內毒素血症，嚴重者甚至可導致死亡。目前LPS檢測以鱟試驗法(Limulus amebocyte lysate, LAL)最常被使用，其檢測極限可約達到2 ng/ml。但此法仍有靈敏度低和檢測費用昂貴，以及原料來源取得困難等問題，本研究擬以電極表面的修飾技術配合交流電阻抗分析法(ACIS)，發展高靈敏度、成本低廉以及檢測程序簡便之LPS檢測系統。
實驗中我們以DTBA及PmB為感測元件進行LPS之檢測，亦即以自主性吸附 (SAM)之修飾程序，先將金電極浸泡於4,4’-dithiodi-(n-butyric acid) (DTBA)溶液中，使金電極表面產生帶有COOH之官能基，而成為我們探討之第一種電極(DTBA/Au)。其原理乃藉DTBA與LPS間之親疏水性作用，使LPS能夠吸附在電極表面上，當LPS添加後所產生之阻抗曲線即以架設於常數相位元件(CPE)元件下之電路模型，模擬分析LPS的檢測結果，其可使電阻(Rt)與電容(CSAM)分別產生48.1 kΩ/ng/ml及32.4 nF/ng/ml之變化量與0~0.6 ng/ml的線性範圍。DTBA/Au電極若再經由1-ethyl-3-(3-dimethylamionpropyl) carbodiimide (EDC)與N-hydrosuccinimide (NHS)的活化後，將抗生素Polymyxin B (PmB)固定於金電極表面，即成為本研究探討之第二種PmB-DTBA/Au電極。此則透過抗生素PmB與LPS之間的正負電吸引力，進而達到檢測LPS之目的，且其Rt與CSAM也分別有81.6 kΩ/ng/ml及56.6 nF/ng/ml之高偵測靈敏度及0~0.6 ng/ml的線性範圍。以PmB-DTBA/Au電極經pH 2.5之glycine buffer酸洗後可達到重複使用之目的，然靈敏度會逐漸呈略微降低。
以PmB/DTBA/Au修飾之電極在飲用水及生理食鹽水中進行LPS之檢測，在含有1 ng/ml之LPS溶液中，可分別使CSAM產生66 nF/ng/ml及33.2 nF/ng/ml之電容效應。前者之電容效應為後者之二倍，可歸因於溶液中離子較低的飲用水，有較大之電容效應；反之在生理食鹽水中，因其有許多正負離子存在，就會有較小之電容值。食鹽水受限於電容值不明顯，難以有較大之電容值變化。故本研究處在離子成分較低之溶液中，會有較明顯之LPS檢測結果。最後我們也以石英晶體微天秤(quartz crystal microbalance, QCM)方式佐證LPS的檢測結果，以DTBA-PmB/Au修飾之晶片在10 ng/ml LPS的濃度下會有15 Hz的變化量。

Lipopolysaccharide (LPS) was also called endotoxin which existed in the outer membrane of cell wall of Gram negative bacteria, it could induce some diseases such as shock, endotoxin, or death. Limulus amebocyte lysate (LAL) assay was the most common method for detecting LPS, which has the detection limit of 2 ng/ml. However, this method still defeats in its low sensitivity, high cost and the rare resource. In this study, modified electrodes combined with A.C. impedance spectroscopy (ACIS) were designed to develop a simpler, economical, and sensitive detection system.
DTBA and Polymyxin B (PmB) were separately used as the sensing elements for detecting LPS. The DTBA/Au electrode was fabricated by immobilization of 4,4’-dithiodi(n-butyric acid) (DTBA) on the gold surface through self-assembled membrane (SAM) process, which was the first detective electrode in our study (DTBA/Au). As LPS was added, it caused to an interaction between DTBA and LPS. This electrical change was detected by impedance analysis with circuit model, which was combined with a constant phase element (CPE). As a result, the sensitivity of Rt and CSAM for detecting LPS could be obtained to be 48.1 kΩ/ng/ml and 32.4 nF/ng/ml, and the linear range was 0 to 0.6 ng/ml. Then, the DTBA/Au electrode was successively activated with 1-ethyl-3-(3-dimethylamionpropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS) and coupled with PmB, this electrode was to be our second electrode (PmB-DTBA/Au). The sensitivity of Rt and CSAM for detecting LPS was 81.6 kΩ/ng/ml and 56.6 nF/ng/ml, and the linear range was 0 to 0.8 ng/ml. The electrode could be reused by washing with glycine buffer (pH 2.5), however its sensitivity gradually showed to be not sensitive enough.
The system with water and normal saline was evaluated, and the sensitivity of CSAM for detecting LPS with PmB-DTBA/Au electrode was 66 nF/ng/ml and 33.2 nF/ng/ml, respectively. It could be attributed that normal saline solution with higher ions content induced lower variation of capacitance. Namely, the detection system in this study is high sensitive to LPS in solution with lower ions content. We evidenced the effect of detecting LPS by quartz crystal microbalance (QCM), and the sensitivity of DTBA-PmB/Au modified electrode was 15 Hz in 10 ng/ml LPS.

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