基于二茂铁衍生物的复合材料过氧化物模拟酶特性及其分析应用研究
发布时间：2018-08-24 17:11
【摘要】：天然酶因具有高效的催化活性和高度的专一性等特点,在很多领域占据着十分重要的地位。然而天然酶固有的一些缺点,如易失活、难提纯等限制了它的广泛使用。因此,寻求具有天然酶活性的模拟酶显得尤为重要。相比于天然酶,模拟酶具有易制备与保存、稳定性好和活性易调控等优点。其中对过氧化物模拟酶的研究最为居多,因为过氧化氢是生命体中一种重要的活性氧形态。自2007年四氧化三铁磁性纳米粒子被报道具有过氧化物模拟酶催化活性以来,人工合成材料在过氧化物模拟酶领域得到了快速发展,并广泛应用于电化学传感器、环境污染物降解和生物大分子检测等方面。为此,本文构建了基于二茂铁衍生物得到的一系列新型过氧化物模拟酶材料的光分析方法及其应用。首先,我们利用二茂铁甲酸中的羧基与钴离子的配位作用,合成了钴-二茂铁甲酸(Fc-COOH-Co)。通过红外表征证明了该配合物的成功合成。我们发现Fc-COOH-Co具有过氧化物模拟酶活性,即Fc-COOH-Co能够催化H_2O_2氧化TMB的反应,而氧化后的TMB为一种蓝色产物。基于此,我们构建了一个简单的比色法来检测H_2O_2。在最佳反应条件下,该方法具有较宽的线性范围(0.01μM-70μM)和较低的检出限(0.01μM)。稳态动力学实验证明,Fc-COOH-Co相对于底物H_2O_2的米氏常数值为0.63 mM,明显低于HRP(3.70 mM),说明Fc-COOH-Co与H_2O_2有很好的亲和力。通过结合葡萄糖氧化酶催化葡萄糖和氧气产生过氧化氢的反应,该方法还可用于检测葡萄糖,线性范围为0.01μM-70μM,检出限为0.01μM。该方法能够成功用于血清中葡萄糖的测定。此外,相对于HRP,Fc-COOH-Co具有优异的pH(2-11)和温度(10-80 ℃)耐受性。其次,我们通过水热法制备了二茂铁基-L-组氨酸席夫碱衍生物(L-Fc-CHO)并研究其过氧化物模拟酶活性。相对于L-组氨酸和二茂铁甲醛,L-Fc-CHO表现出了协同的催化效果。基于此,我们构建了一种超灵敏的H_2O_2和葡萄糖比色法。该方法的检出限低至8 nM。稳态动力学实验证明,L-Fc-CHO相对于底物H_2O_2的米氏常数值为0.22 m M,比HRP的低了近17倍,表明L-Fc-CHO的高效催化活性是源于对H_2O_2优异的亲和力。通过用对苯二甲酸作为自由基捕获剂的荧光实验证明,该催化反应中产生了活性中间体·OH。最后,该方法能用于准确测定血清中葡萄糖。最后,以二茂铁及其衍生物为前体通过溶剂热法分别合成了不含官能团以及含不同含氧官能团(羟基、醛基和羧基)的磁性碳材料。通过XPS、红外等表征证明磁性碳中仍保留了原材料中的含氧官能团。我们发现,这四种磁性碳材料具有不同的过氧化物模拟酶催化活性(醛基碳≈羧基碳碳羟基碳)。它们催化活性的差异主要归因于其含氧官能团的不同,因为这四种磁性碳的其他性质如表面Fe元素含量、形貌与尺寸、比表面积基本没有差异。稳态动力学实验证明,相比于不含官能团的磁性碳,醛基碳具有更高的最大反应速率,羧基碳具有更低的米氏常数即更好的底物亲和力,而羟基碳则具有更低的最大反应速率和更高的米氏常数。这说明,在碳材料中不同官能团在催化反应的作用:-C=O是作为催化位点,-COOH是作为亲和位点,而-OH起抑制作用。通过本文的研究可以达到由不同含氧官能团调控磁性碳的模拟酶活性的目的。
[Abstract]:Natural enzymes play an important role in many fields because of their high catalytic activity and specificity. However, some inherent shortcomings of natural enzymes, such as inactivation and difficulty in purification, limit their widespread use. Therefore, it is particularly important to seek mimetic enzymes with natural enzymes. Among them, peroxidase mimetic enzymes have been studied mostly because hydrogen peroxide is an important active oxygen species in living organisms. Since ferrous oxide magnetic nanoparticles were reported to have peroxidase mimetic catalytic activity in 2007, synthetic materials have been synthesized. The field of peroxidase mimetic enzymes has been rapidly developed and widely used in electrochemical sensors, degradation of environmental pollutants and detection of bio-macromolecules. In this paper, a series of new methods for the optical analysis of peroxidase mimetic enzymes based on Ferrocene Derivatives and their applications have been developed. Cobalt-ferrocene formic acid (Fc-COOH-Co) was synthesized by the coordination of carboxyl group in ferric acid with cobalt ion. The successful synthesis of the complex was proved by IR characterization. We found that Fc-COOH-Co had peroxidase mimic enzyme activity, that is, Fc-COOH-Co could catalyze the oxidation of TMB by H_2O_2, and the oxidized TMB was a blue product. A simple colorimetric method has been developed to detect H_2O_2. Under the optimum reaction conditions, the method has a wide linear range (0.01_ M-70_ M) and a low detection limit (0.01_ M). The steady-state kinetic experiments show that the Michaelis constant of Fc-COOH-Co relative to substrate H_2O_2 is 0.63 mM, significantly lower than that of HRP (3.70 mM), indicating that Fc-COOH-Co and H_2O_2 have a lower detection limit. The method can also be used for the determination of glucose in serum with a linear range of 0.01-70 mu M and a detection limit of 0.01 mu M. In addition, Fc-COOH-Co has excellent pH (2) relative to HRP. Secondly, we prepared ferrocenyl-L-histidine Schiff Base Derivatives (L-Fc-CHO) by hydrothermal method and studied their peroxidase mimic enzyme activity. Compared with L-histidine and ferrocene formaldehyde, L-Fc-CHO showed synergistic catalytic effect. Based on this, we constructed a highly sensitive H_2O_2 and Portuguese. The detection limit of L-Fc-CHO was as low as 8 nM. The steady-state kinetic experiment showed that the Michaelis constant of L-Fc-CHO relative to substrate H_2O_2 was 0.22 m M, which was nearly 17 times lower than that of HRP, indicating that the high catalytic activity of L-Fc-CHO was due to its excellent affinity for H_2O_2. Finally, ferrocene and its derivatives were used as precursors to synthesize magnetic carbon materials without functional groups and with different oxygen-containing functional groups (hydroxyl, aldehyde and carboxyl) by solvothermal method. Oxygen-containing functional groups in the raw materials are still retained in the magnetic carbon. We found that these four magnetic carbon materials have different catalytic activities for peroxidase mimics (aldehyde-based carbon carboxyl-based carbon hydroxyl carbon). Their catalytic activities are mainly attributed to the difference of their oxygen-containing functional groups because of the other properties of the magnetic carbon such as surface Fe. The results of steady-state kinetic experiments show that aldehyde-based carbon has higher maximum reaction rate, carboxyl-based carbon has lower Michaelis constant, i.e. better substrate affinity, and hydroxyl-carbon has lower maximum reaction rate and higher Michaelis ratio than magnetic carbon without functional groups. This indicates that different functional groups play a catalytic role in the catalytic reaction of carbon materials: -C=O is the catalytic site, -COOH is the affinity site, and-OH inhibits the reaction.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O627.81;O629.8
本文编号：2201475
[Abstract]:Natural enzymes play an important role in many fields because of their high catalytic activity and specificity. However, some inherent shortcomings of natural enzymes, such as inactivation and difficulty in purification, limit their widespread use. Therefore, it is particularly important to seek mimetic enzymes with natural enzymes. Among them, peroxidase mimetic enzymes have been studied mostly because hydrogen peroxide is an important active oxygen species in living organisms. Since ferrous oxide magnetic nanoparticles were reported to have peroxidase mimetic catalytic activity in 2007, synthetic materials have been synthesized. The field of peroxidase mimetic enzymes has been rapidly developed and widely used in electrochemical sensors, degradation of environmental pollutants and detection of bio-macromolecules. In this paper, a series of new methods for the optical analysis of peroxidase mimetic enzymes based on Ferrocene Derivatives and their applications have been developed. Cobalt-ferrocene formic acid (Fc-COOH-Co) was synthesized by the coordination of carboxyl group in ferric acid with cobalt ion. The successful synthesis of the complex was proved by IR characterization. We found that Fc-COOH-Co had peroxidase mimic enzyme activity, that is, Fc-COOH-Co could catalyze the oxidation of TMB by H_2O_2, and the oxidized TMB was a blue product. A simple colorimetric method has been developed to detect H_2O_2. Under the optimum reaction conditions, the method has a wide linear range (0.01_ M-70_ M) and a low detection limit (0.01_ M). The steady-state kinetic experiments show that the Michaelis constant of Fc-COOH-Co relative to substrate H_2O_2 is 0.63 mM, significantly lower than that of HRP (3.70 mM), indicating that Fc-COOH-Co and H_2O_2 have a lower detection limit. The method can also be used for the determination of glucose in serum with a linear range of 0.01-70 mu M and a detection limit of 0.01 mu M. In addition, Fc-COOH-Co has excellent pH (2) relative to HRP. Secondly, we prepared ferrocenyl-L-histidine Schiff Base Derivatives (L-Fc-CHO) by hydrothermal method and studied their peroxidase mimic enzyme activity. Compared with L-histidine and ferrocene formaldehyde, L-Fc-CHO showed synergistic catalytic effect. Based on this, we constructed a highly sensitive H_2O_2 and Portuguese. The detection limit of L-Fc-CHO was as low as 8 nM. The steady-state kinetic experiment showed that the Michaelis constant of L-Fc-CHO relative to substrate H_2O_2 was 0.22 m M, which was nearly 17 times lower than that of HRP, indicating that the high catalytic activity of L-Fc-CHO was due to its excellent affinity for H_2O_2. Finally, ferrocene and its derivatives were used as precursors to synthesize magnetic carbon materials without functional groups and with different oxygen-containing functional groups (hydroxyl, aldehyde and carboxyl) by solvothermal method. Oxygen-containing functional groups in the raw materials are still retained in the magnetic carbon. We found that these four magnetic carbon materials have different catalytic activities for peroxidase mimics (aldehyde-based carbon carboxyl-based carbon hydroxyl carbon). Their catalytic activities are mainly attributed to the difference of their oxygen-containing functional groups because of the other properties of the magnetic carbon such as surface Fe. The results of steady-state kinetic experiments show that aldehyde-based carbon has higher maximum reaction rate, carboxyl-based carbon has lower Michaelis constant, i.e. better substrate affinity, and hydroxyl-carbon has lower maximum reaction rate and higher Michaelis ratio than magnetic carbon without functional groups. This indicates that different functional groups play a catalytic role in the catalytic reaction of carbon materials: -C=O is the catalytic site, -COOH is the affinity site, and-OH inhibits the reaction.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O627.81;O629.8
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