聚苯胺/辣根过氧化酶或多酚氧化酶生物传感器的构筑

【作者】 王鹏；

【导师】 阚锦晴；

【作者基本信息】 扬州大学 ， 物理化学， 2009， 硕士

【摘要】 质子酸溶液中合成的聚苯胺在pH大于4时失去氧化还原活性和导电性。这一缺点限制了它在酶生物传感器中的应用。我们通过改变实验条件来提高聚苯胺的性能。在含有离子液体的溶液中合成的聚苯胺,当溶液pH大于4时仍是电活性的聚合物。结果表明:此种方法合成的聚苯胺是制作酶生物传感器的理想材料。在导电玻璃上,用交联剂戊二醛把辣根过氧化酶和聚苯胺进行交联制备过氧化氢生物传感器。掺氟导电玻璃良好的光透明性使得传感器的表征非常容易。酶的固定过程十分温和,并且与酶的等电点无关。当过氧化氢浓度低于20 mmol dm^-3时,响应电流随过氧化氢浓度增加线性增长。最大响应电流I_max和米氏常数k_m’分别是1.189μA和27.11 mmol dm^-3。低于40℃时,该生物传感器的响应电流随着温度的升高而增大。活化能Ea是39.1 kJ mol^-1。扫描电镜和交流阻抗被用于表征此生物传感器。聚苯胺的合适多孔结构可延长传感器的寿期。用交联剂戊二醛把多酚氧化酶和聚苯胺进行交联制备邻苯二酚生物传感器。聚苯胺的生物兼容微环境可以保护多酚氧化酶。邻苯二酚为底物时,生物传感器的线性范围高达80μmol dm-3。最大响应电流I_max和米氏常数k_m’分别是9.44μA和117μmol dm^-3。表观米氏常数值表明:固定化的多酚氧化酶和邻苯二酚间的亲和力强于自由多酚氧化酶和邻苯二酚间的亲和力。pH值和工作电位的影响被研究以便优化测量条件。在B-R缓冲溶液中,多酚氧化酶催化反应的活化能E_a是30.23 kJ mol^-1。阻抗、紫外和扫描电镜被用于表征这个传感器。此传感器有好的长期稳定性。基于苯甲酸对多酚氧化酶催化活性的抑制作用,我们研究了检测苯甲酸的多酚氧化酶传感器。该传感器在不同浓度抑制剂中的米氏常数k_m’和最大响应电流I_max也被检测。动力学分析表明:苯甲酸对多酚氧化酶的抑制作用是可逆的,竞争抑制常数为28.7μmol dm^-3。苯甲酸的检测下限为0.3μmol dm^-3。酶活性下降到原来一半时,苯甲酸浓度为35μmol dm^-3。该传感器比较稳定且灵敏性较好,因此可作为一种新的用于检测苯甲酸的手段。更多还原

【Abstract】 PANI synthesized in proton acid solution losts its redox activity and conductivity at pH over 4. The drawback restricts its application in enzyme biosensor. So we will improve the properties of PANI through changing the experiment conditions. The PANI synthesized in a solution containing ionic liquid, 1-ethyl-3-methylimidazolium ethyl sulfate （EMIES）, is an electroactive polymer at pH over 4. The following results indicate that PANI is an ideal material for the construction of biosensor.The hydrogen peroxide biosensor is constructed by cross-linking between horseradish peroxidase （HRP） and polyaniline （PANI） using glutaraldehyde as a cross linking agent on F-doped tin oxide （FTO） electrode. It is easy to characterize the biosensor due to the excellent optical transparency of FTO. The process of immobilized HRP is unrelated to isoelectric point of the enzyme and is particularly mild. The response current increases linearly with increasing hydrogen peroxide concentration up to 20 mmol dm^-3. The maximum response current (I_max) and the Michaelis-Menten constant （ k_m’） are 1.189μA and 27.11 mmol dm^-3 respectively. The response current of the biosensor increases with increasing temperature below 40℃. The activation energy （E_a） of the HRP catalytic reaction is 39.1 kJ mol^-1 in the B-R buffer. The biosensor is also characterized with SEM and CV.The catechol biosensor is constructed by cross-linking between polyphenol oxidase （PPO） and polyaniline （PANI） using glutaraldehyde as a cross-linking agent. The PANI can protect the immobilized PPO due to the biocompatible microenvironment of PANI film. In the presence of catechol as a substrate, the biosensor exhibits a linear range up to 80μmol dm^-3. The maximum response current (I_max) and the Michaelis-Menten constant （ k_m’） are 9.44μA and 117μmol dm^-3, respectively. The value of the Michaelis-Menton constant k_m’ indicates that the interaction between immobilized PPO and catechol is stronger than that between free PPO and catechol. The effects of pH and operating potential are also explored to optimize measurement conditions. The activation energy （E_a） of the PPO catalytic reaction is 30.23 kJ mol^-1 in the B-R buffer. Electrochemical impedance spectroscopy （EIS）, UV-vis and SEM are used to characterize the PANI-PPO biosensor. The biosensor exhibits good long-term stability.A PANI/PPO biosensor for the determination of benzoic acid is reported. The biosensor functioning is based on the inhibition effect of benzoic acid on the biocatalytic activity of the enzyme. The kinetic parameters Michaelis-Menten constant （k_m’） and maximum current (I_max) in the absence and in the presence of benzoic acid are also evaluated. The kinetic analyses show that the inhibition of benzoic acid on the PPO activity is reversible and competitive with the inhibition constant determined to be 28.7μmol dm^-3. A limit of detection of 0.3μmol dm^-3 benzoic acid is obtained. The I_0.5 value is estimated to be 35μmol dm^-3. The biosensor has good sensivity and high stability. So it can be used as a new approach for the determination of benzoic acid.更多还原