| 摘要: | 結核菌曾為台灣最普遍的法定傳染病，目前仍為衛生署嚴格管控的疾病之一。目前的檢驗方法有X光檢查、結核菌素檢查、與細胞培植；細胞培植的檢驗方法又可以抗酸性染色、PCR、RFLP、等方式檢驗。本研究中採用生物感測器中的壓電式DNA感測器作為偵測此菌的方法。
實驗中的探針為常用於偵測結核菌屬之專一性序列IS6110的outer primer-Tb294，經由biotin修飾後藉由avidin-biotin interaction固定實驗中探針於晶片表面。(1)首先比較兩種不同晶片清洗方式-Piranha solution與Ammonium peroxide mixture清洗過後可於晶片表面形成的SAM。實驗結果顯示以Piranha solution所得的清洗效果較佳，經Piranha solution清洗過後的晶片,可於金表面形成更多的SAM。 (2)接著比較兩種不同的含硫化合物－Cystamine或Mercaptohexadecanoic acid(MHDA)作為固定Avidin方式。 以MHDA所形成的單層膜上，所能固定之avidin有較佳的數據重複性。之後使用不同濃度avidin進行注流式分析系統的分析，高濃度的avidin不僅可減少sample注入次數，也可達到比低濃度更大的頻率ΔF。之後使用未固定探針的晶片確定目標序列訊號並非由非專一性反應產生，而使用非互補序列作雜交反應則確定了此感測器的專一性。
探針固定化的最適化研究顯示探針濃度為10 μg/ml，所得結果最佳，而與不同濃度(0~20 μｇ/ml )的目標序列進行雜交試驗顯示，在目標序列濃度為1~5 μg/ml 間有線性反應。而之後使用了不同濃度的HCL (0.001 M、0.05 M、0.5 M)與NaOH (0.05 M、0.1 M、0.2M)進行晶片再生試驗得知 使用0.5 M HCl可成功達到感測器的再生，而使用450 μl的0.5 M HCl則可達到感測器100%再生。
Traditional detection methods of Mycobacterium tuberculosis, include X-ray, Tuberculin test, and sputum bacteria incubation. New method for tuberculosis detection is using biomolecular technology, such as PCR, RFLP, biosensor. In this study a DNA-based piezoelectric biosensor was used for the detection of Tuberculosis complex. The probe used in the experiment is the outer primer-Tb294, which is specific for IS6110 of Tuberculosis complex, was often used in PCR. The probe was biotinated and immobilized onto the crystal surface via avidin-biotin interaction.
Firstly, the effect of two cleaning methods was compared. The piranha solution was a better method in term of the following formation of self-assembled monolayer. Subsequently, SAM of 16-mercapto- hexadecanoic acid was formed and avidin was immobilized onto the SAM.
The higher concentration of avidin was used for shortening reaction times. There was no non-specific probe immobilization occurred on the avidin-modified chip by target sequence injection. Frequency shift was observed as the non-complementary sequence was applied. This test confirmed the high specificity of the DNA sensor. The optimal condintion of probe immobilization showed that the optimal probe concentration was 10 μg/ml. In the hybridization test, various concentrations of target sequence( 0.5 ~ 20 μg/ml) were examined, it showed that there is a linear relationship in the range of 1 ~5 μg/ml and the maxium reaction was occurred in 10 μg/ml. a lower reaction of higher concentration of target sequence was observed. In the regeneration test, various concentrations of acid ( 0.001 M, 0.05 M and 0.5 M HCl) and base ( 0.05 M, 0.1 M, 0.2M NaOH) was examined, the best method of regeneration was 450μl of 0.5 M HCl. |
