【作者】 邹敏芬；

【导师】 张正奇；

【作者基本信息】 湖南大学 ， 分析化学， 2003， 硕士

【摘要】 本文在查阅大量有关生物传感器、植物DNA的提取与纯化以及DNA酶切等文献的基础上，研究了黄瓜DNA伏安传感器的制备及其应用。取得如下重要成果： 1．改进了植物DNA的提取方法．优化了用SDS法提取黄瓜DNA的条件。用4.0mL细胞提取液、0.6倍样液体积的酚／氯仿／异戊醇、0.6倍样液体积的氯仿／异戊醇、0.58倍样液体积的异丙醇及1.0 ml饱和KCl溶液，可从每克黄瓜中提取350 mg DNA。在此优化条件下提取黄瓜DNA产率高，纯度好；与已有提取方法相比较，本法有毒化学品用量显著降低。 2．采用电泳法纯化黄瓜DNA的优化条件：琼脂糖凝胶浓度为1.0％，凝胶厚度为4 mm，电泳缓冲溶液pH值为8.0，离子强度为0.050，电压为60 V。用琼脂糖包埋—小室电洗脱纯化DNA方法获取纯度更高的DNA。 3．采用Hind Ⅲ限制性内切酶降解所提黄瓜DNA，使之片断大小适合制备DNA传感器，显著改善了DNA传感器的重现性等性能。 4．以十八酸修饰的碳糊电极作基体传感器，制备了黄瓜DNA伏安传感器。优化了用所制备的传感器测定黄瓜DNA的条件，基本传感器的组成为：碳粉60％，十八酸5.0％，液体石蜡35％；光滑镜面抛光电极；ssDNA在基体传感器表面上固定条件为：表面活化液中含7.0mmol／LEDC，8.0mmol／LNHS，0.050mol／LNa₂HPO₄，pH为6.0，用量为100 μL；ssDNA固定液中含5.0 μg／mL变性DNA，0.010mol／LNa₂HPO₄，pH为6.9，用量为100 μL；DNA杂交和分析测定在含0.020mol／LNaCl5.0 mmol／L Tris，90 μmol／L Co（bpy）₃（ClO₄）₃配合物，pH为7.0的杂交液中反应30min。在优化条件下，黄瓜DNA伏安传感器的线性范围为0.010 μg／mL—0.090 μg／mL，检测下限为2.97ng／mL，相对标准偏差为1.80％。本类传感器可用于生物物质种属鉴定，在司法工作中有广泛应用前景。用所提出的方法测定了本室提取的黄瓜DNA样品，回收率在98％—104％之间。循环伏安法结果表明Co（bpy）₃（ClO₄）₃配合物嵌入双链DNA后，在伏安传感器上发生的电化学反应为可逆反应。更多还原

【Abstract】 On the basis of consulting a great amount of articles about biosensor, the extraction, the purification of plant DNA and the enzyme operation of DNA. The preparation and application of cucumber DNA voltammetric biosensor have been studied.1. The procedure of the extraction of plant DNA. The optimum conditions of the extraction of cucumber DNA using SDS method were as following ( for 1.0 g cucumber ) : 4.0 mL of cell extraction solution, 1.0 mL of saturated KC1 solution, the rate between the phenol / chloroform / isoamyl alcohol solution, chloroform / isoamyl alcohol solution or isopropanol and the sample solution being 0.6, 0.6, and 0.58, respectively. Under such optimum conditions, the yield of the cucumber DNA is high and the cucumber DNA is very pure. Comparing with the reported methods, the amounts of the poisonous chemicals in the proposed procedure were decreased greatly.2. The electrophoresis conditions for the purification of cucumber DNA were optimized. The optimum conditions are as following: the agarose concentration is 1.0%, and the thickness of gel, the pH, the ionic intention and the potential, 4.0 mm, 8.0, 0.050, and 60 V, respectively. We used the gel embedded - little chamber electrically washed method to obtain the purest cucumber DNA.3. The restriction enzyme Hind III was used to cut the cucumber DNA, and the fragment of the obtained DNA is suitable to prepare the cucumber DNA voltammetric biosensor. Thus the sensor possessed excellent analytical characteristics.4. The cucumber DNA voltammetric biosensor was prepared using the stearic acid-modified carbon paste electrode, and the conditions for the determination of cucumber DNA using the sensor were optimized. These conditions include the components of the basic sensor, the solution for the activation of the basic sensor, the solution for the immobilization of cucumber ssDNA and the solution for DNA hybridization reaction. The basic biosensor consists of 60% carbon powder, 5.0% stearic acid and 35% paraffin liquid, the surface of the sensor was polished on a smooth mirror. After the activation of the basic sensor in 100 μ L of solution (pH6.0) containing 7.0 mmol/L EDC, 8.0 mmol/L NHS and 0.050 mol/L Na2HPO4, theimmobilization of cucumber ssDNA on the activized surface was performed in 100 u L of immobilization solution (pH6.9) containing 5.0 μ g/mL denaturalized cucumber DNA, 0.010 mol/L Na2HPO4. The DNA hybridization reaction and the determination of DNA was carried out in a pH7.0 hybridization solution containing 0.020 mol/L NaCl, 5.0 mmol/L Tris and 90 u mol/L Co(bpy)3(ClO4)3. Under the optimum conditions, the linear range is from 0.010 μ g/mL to 0.090 μ g/mL with the detection limit of 2.97 ng/mL and the relative standard deviation 1.8%. This kind of biosensor can be used to identify the class of samples, which has a broad application in forensic science. The cucumber DNA samples extracted in our laboratory was determined by the proposed prdecdure and the recovery is between 98% - 104%. The cyclic voltammetric data showed that the electrochemical reaction of the Co(bpy).3(ClO4)3 complex insetted to the double-stranded DNA on the voltammetric biosensor is reversible.更多还原