基于二茂鐵衍生物的復(fù)合材料過氧化物模擬酶特性及其分析應(yīng)用研究
發(fā)布時(shí)間：2018-08-24 17:11
【摘要】：天然酶因具有高效的催化活性和高度的專一性等特點(diǎn),在很多領(lǐng)域占據(jù)著十分重要的地位。然而天然酶固有的一些缺點(diǎn),如易失活、難提純等限制了它的廣泛使用。因此,尋求具有天然酶活性的模擬酶顯得尤為重要。相比于天然酶,模擬酶具有易制備與保存、穩(wěn)定性好和活性易調(diào)控等優(yōu)點(diǎn)。其中對(duì)過氧化物模擬酶的研究最為居多,因?yàn)檫^氧化氫是生命體中一種重要的活性氧形態(tài)。自2007年四氧化三鐵磁性納米粒子被報(bào)道具有過氧化物模擬酶催化活性以來,人工合成材料在過氧化物模擬酶領(lǐng)域得到了快速發(fā)展,并廣泛應(yīng)用于電化學(xué)傳感器、環(huán)境污染物降解和生物大分子檢測(cè)等方面。為此,本文構(gòu)建了基于二茂鐵衍生物得到的一系列新型過氧化物模擬酶材料的光分析方法及其應(yīng)用。首先,我們利用二茂鐵甲酸中的羧基與鈷離子的配位作用,合成了鈷-二茂鐵甲酸(Fc-COOH-Co)。通過紅外表征證明了該配合物的成功合成。我們發(fā)現(xiàn)Fc-COOH-Co具有過氧化物模擬酶活性,即Fc-COOH-Co能夠催化H_2O_2氧化TMB的反應(yīng),而氧化后的TMB為一種藍(lán)色產(chǎn)物�；诖�,我們構(gòu)建了一個(gè)簡(jiǎn)單的比色法來檢測(cè)H_2O_2。在最佳反應(yīng)條件下,該方法具有較寬的線性范圍(0.01μM-70μM)和較低的檢出限(0.01μM)。穩(wěn)態(tài)動(dòng)力學(xué)實(shí)驗(yàn)證明,Fc-COOH-Co相對(duì)于底物H_2O_2的米氏常數(shù)值為0.63 mM,明顯低于HRP(3.70 mM),說明Fc-COOH-Co與H_2O_2有很好的親和力。通過結(jié)合葡萄糖氧化酶催化葡萄糖和氧氣產(chǎn)生過氧化氫的反應(yīng),該方法還可用于檢測(cè)葡萄糖,線性范圍為0.01μM-70μM,檢出限為0.01μM。該方法能夠成功用于血清中葡萄糖的測(cè)定。此外,相對(duì)于HRP,Fc-COOH-Co具有優(yōu)異的pH(2-11)和溫度(10-80 ℃)耐受性。其次,我們通過水熱法制備了二茂鐵基-L-組氨酸席夫堿衍生物(L-Fc-CHO)并研究其過氧化物模擬酶活性。相對(duì)于L-組氨酸和二茂鐵甲醛,L-Fc-CHO表現(xiàn)出了協(xié)同的催化效果�；诖�,我們構(gòu)建了一種超靈敏的H_2O_2和葡萄糖比色法。該方法的檢出限低至8 nM。穩(wěn)態(tài)動(dòng)力學(xué)實(shí)驗(yàn)證明,L-Fc-CHO相對(duì)于底物H_2O_2的米氏常數(shù)值為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對(duì)H_2O_2優(yōu)異的親和力。通過用對(duì)苯二甲酸作為自由基捕獲劑的熒光實(shí)驗(yàn)證明,該催化反應(yīng)中產(chǎn)生了活性中間體·OH。最后,該方法能用于準(zhǔn)確測(cè)定血清中葡萄糖。最后,以二茂鐵及其衍生物為前體通過溶劑熱法分別合成了不含官能團(tuán)以及含不同含氧官能團(tuán)(羥基、醛基和羧基)的磁性碳材料。通過XPS、紅外等表征證明磁性碳中仍保留了原材料中的含氧官能團(tuán)。我們發(fā)現(xiàn),這四種磁性碳材料具有不同的過氧化物模擬酶催化活性(醛基碳≈羧基碳碳羥基碳)。它們催化活性的差異主要?dú)w因于其含氧官能團(tuán)的不同,因?yàn)檫@四種磁性碳的其他性質(zhì)如表面Fe元素含量、形貌與尺寸、比表面積基本沒有差異。穩(wěn)態(tài)動(dòng)力學(xué)實(shí)驗(yàn)證明,相比于不含官能團(tuán)的磁性碳,醛基碳具有更高的最大反應(yīng)速率,羧基碳具有更低的米氏常數(shù)即更好的底物親和力,而羥基碳則具有更低的最大反應(yīng)速率和更高的米氏常數(shù)。這說明,在碳材料中不同官能團(tuán)在催化反應(yīng)的作用:-C=O是作為催化位點(diǎn),-COOH是作為親和位點(diǎn),而-OH起抑制作用。通過本文的研究可以達(dá)到由不同含氧官能團(tuán)調(diào)控磁性碳的模擬酶活性的目的。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O627.81;O629.8
本文編號(hào)：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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O627.81;O629.8
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