基于酶催化的生物传感体系的构建及其分析应用

发布时间：2018-01-26 19:51

  本文关键词： 天然酶 纳米材料 模拟酶 酶的活化 比色法　出处：《江南大学》2016年硕士论文　论文类型：学位论文

【摘要】：天然酶由于其具有专一性强,催化效率高等优点,被广泛应用于生物医学和分析检测等各个领域。但是天然酶在实际应用中存在一些缺点,如催化活性易受环境影响,易失活,提纯和贮存成本较高等。因此科研工作者们为开发出新的类酶催化方法进行了深入研究。一方面,开发大量的纳米材料模拟酶,它们合成方法简单,易存储,且可通过修饰改性应用于不同的体系中;另一方面,为了提高催化效率,开发出可以活化天然酶的纳米材料,可在提高催化效率的同时,给酶催化反应提供了宽泛的条件。这两种类酶催化方式都弥补了单纯天然酶的缺点,因而具有很高的研究价值和广阔的市场前景。基于此,本文主要为了解决天然酶的缺点而开发出两种类酶催化方式的材料,并应用于分析检测领域。具体研究内容如下:(1)以天然酶辣根过氧化酶(HRP)为模板合成金簇,在光照的条件下其具有优越的模拟氧化酶活性。在严苛的反应条件下,可以保持很高的催化效率,且其与显色底物3,3’,5,5’-四甲基联苯胺(TMB)的亲和力大于单纯的天然酶HRP。当加入二价汞离子时,其模拟酶活性被明显抑制,这是因为二价汞离子与金簇表面的金离子形成Au-Hg合金,改变了金簇的表面态,从而失去了催化活性,而三聚氰胺因为与二价汞离子具有更强的结合力,可以解除这种抑制作用,使得金簇重获模拟酶活性。我们根据此原理,建立了一个三聚氰胺的恢复型检测方法,并成功应用于牛奶和奶粉样品中三聚氰胺的检测。(2)原位合成的硫化镉量子点(CdS QDs)具有光模拟酶活性,修饰剂的种类与硫离子的浓度会直接影响CdS QDs的模拟酶活性。我们通过实验探究,原位合成出模拟酶活性最强的PO43-@CdS QDs,并将其与葡萄糖氧化酶分解1-巯基-β-D-葡萄糖可产生硫离子这一性质相结合,建立一个比色法检测葡萄糖氧化酶活性的方法。实验表明原位合成法具有背景信号弱,催化反应用量少的特点,可提高检测灵敏度。(3)邻苯二酚(CA)与二氧化钛(TiO2)复合物在可见光照下产生的活性中间体可以活化天然酶HRP,使其在无过氧化氢(H2O2)存在的条件下催化氧化底物TMB和2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS),其中,CA的浓度与TiO2-CA活化HRP的活性有关,而CA可以由碱性磷酸酯酶(ALP)催化底物邻苯二酚磷酸酯(OPP)产生。基于此,我们建立了一个三倍信号放大策略检测ALP的方法。(4)改进Hummers法合成得到的氧化石墨烯(GO)在可见光照射下可以产生活性中间体空穴(h+)和超氧阴离子(O2?-),其可活化天然酶HRP,可在无H2O2条件下可以催化氧化底物TMB和ABTS,且HRP的量与体系的催化氧化效果相关。同时GO的生物兼容性好,活化效率高,我们将其应用于免疫分析实验中。通过DNA放大技术,使DNA接上大量HRP,建立一个超灵敏检测抗原甲胎蛋白(AFP)的检测方法。得到的检测限是0.0001 pg/m L,远远低于传统方法检测AFP的结果。
[Abstract]:Natural enzymes have been widely used in biomedicine, analytical detection and other fields because of their strong specificity and high catalytic efficiency. However, there are some shortcomings in the practical application of natural enzymes. For example, catalytic activity is easy to be affected by environment, easily deactivated, purification and storage costs are high. Therefore, researchers have carried out in-depth research for the development of a new enzymic catalytic method. On the one hand. A large number of nanomaterials mimic enzymes have been developed, which have the advantages of simple synthesis, easy storage, and can be used in different systems through modification. On the other hand, in order to improve the catalytic efficiency, the development of nanomaterials that can activate natural enzymes can improve the catalytic efficiency at the same time. These two kinds of enzymatic catalysis methods both make up for the shortcomings of pure natural enzymes, so they have high research value and broad market prospect. In order to solve the shortcomings of natural enzymes, two kinds of enzyme catalytic materials were developed in this paper. And applied in the field of analysis and detection. The specific research contents are as follows: 1) the natural horseradish peroxidase (HRP) is used as template to synthesize gold clusters. Under the condition of light, it has superior mimic oxidase activity. Under harsh reaction conditions, it can maintain a high catalytic efficiency, and it can react with the chromogenic substrate (3 ~ (3) O ~ (5)). The affinity of TMBs was higher than that of natural enzyme HRP.The mimic enzyme activity was obviously inhibited when the divalent mercury ion was added. This is due to the formation of Au-Hg alloy by divalent mercury ions and gold ions on the surface of gold clusters, which changes the surface state of gold clusters and thus loses their catalytic activity, while melamine has stronger binding force with divalent mercury ions. This inhibition can be removed, resulting in the gold cluster to regain the mimic enzyme activity. Based on this principle, we established a recovery detection method for melamine. It has been successfully applied to the detection of melamine in milk and milk powder samples. The in situ synthesis of cadmium sulfide quantum dot CDs QDshas photomimic enzyme activity. The type of modifier and the concentration of sulfur ions will directly affect the mimic enzyme activity of CdS QDs. Through the experiment, we synthesized PO43-@CdS QDs, which has the strongest mimic enzyme activity. It was combined with glucose oxidase to decompose 1-mercapto- 尾 -D- glucose to produce sulfur ions. A colorimetric method for the detection of glucose oxidase activity was established. The results showed that the in situ synthesis method had the characteristics of weak background signal and low dosage of catalytic reaction. The active intermediate produced by the complex of catechol and titanium dioxide under visible light can activate the natural enzyme HRP. In the presence of H _ 2O _ 2 (H _ 2O _ 2), the substrates TMB and 2o _ 2-diazo-3-ethyl-benzothiazole-6-sulfonic acid) were oxidized in the presence of H _ 2O _ 2 (H _ 2O _ 2) in the presence of H _ 2O _ 2 (H _ 2O _ 2). The concentration of CA is related to the activity of HRP activated by TiO2-CA, and CA can be produced by alkaline phosphatase (ALP) catalyzed by catechol phosphate. We have established a tripling signal amplification strategy for detecting ALP. (4) the improved Hummers synthesis of graphene oxide (GOO) can produce active intermediate holes under visible light irradiation. H) and superoxide anion O 2? It can activate the natural enzyme HRP and catalyze the oxidation of substrates TMB and ABTS without H _ 2O _ 2. And the amount of HRP is related to the catalytic oxidation effect of the system. At the same time, go has good biocompatibility and high activation efficiency. We applied it to the immunoassay experiment by DNA amplification technique. A method was established for the detection of antigenic alpha-fetoprotein (AFP) by using a large number of DNA. The detection limit was 0.0001 pg/m / L. It is far lower than the result of traditional method for AFP detection.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3;O629.8
，

本文编号：1466443