| Summary: | 碩士 === 國立成功大學 === 醫學工程研究所碩博士班 === 90 === 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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