WO2020228291A1 - 一种提高辣根过氧化物酶稳定性的固定化酶方法及应用 - Google Patents

一种提高辣根过氧化物酶稳定性的固定化酶方法及应用 Download PDF

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WO2020228291A1
WO2020228291A1 PCT/CN2019/119108 CN2019119108W WO2020228291A1 WO 2020228291 A1 WO2020228291 A1 WO 2020228291A1 CN 2019119108 W CN2019119108 W CN 2019119108W WO 2020228291 A1 WO2020228291 A1 WO 2020228291A1
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hrp
zif
horseradish peroxidase
stability
kan
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赵慧敏
谭冰
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Dalian University of Technology
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
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    • G—PHYSICS
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  • the invention belongs to the technical field of environmental monitoring, and relates to an immobilized enzyme method and application for improving the stability of horseradish peroxidase.
  • Natural enzymes are proteins or RNAs that are produced by living cells and are highly specific and highly catalytic for their substrates. Natural enzymes have the advantages of wide sources, various types, good substrate affinity, and high catalytic efficiency. They are widely used in various aspects of production and life such as catalytic analysis, disease diagnosis, and clinical treatment. However, as a typical biomolecule, natural enzymes have relatively poor stability, are easily affected by the external environment, and have strict requirements on storage conditions. For example, natural enzymes are easily damaged by extreme external conditions such as high temperature, strong acid, and strong alkali, and lose part of their functions or even their original activities.
  • Immobilized enzyme means that the enzyme is physically restricted or located in a certain spatial area, and its catalytic activity remains unchanged. Immobilized enzyme technology can more effectively control the reaction process, improve the storage capacity of the enzyme and its stability under extreme operating conditions. In addition, the hierarchical structure of the immobilized enzyme makes it easier to separate, minimize pollution, realize recycling, etc. to reduce production costs.
  • the commonly used methods for immobilizing enzymes on nanomaterials include sol-gel matrix, hydrogel, organic particles, mesoporous silica, metal organic framework (MOFs) materials, etc. These porous materials have large surface areas and voids. Volume is an ideal material for enzyme immobilization (Materials Horizons, 2017, 4(1): 55-63; Physical Chemistry Chemical Physics, 2018, 20(13): 8822-8831; Nature Catalysis, 2018, 1(9): 689-695). But on the other hand, these materials have poor catalytic performance.
  • the porous structure of zeolite imidazole ester framework materials (ZIFs) and the excellent stability of graphene (GO) materials are combined, and the zeolite imidazole ester framework-8/graphene composite material (ZIF-8/GO) is used to realize natural Fixation of horseradish peroxidase (HRP) (HRP@ZIF-8/GO).
  • Zeolite imidazole ester framework structure (ZIFs) materials as a type of MOFs materials, are a new type of porous material with zeolite topological structure formed by the connection of transition metal atoms with imidazole or imidazole derivatives, which provide for immobilization of natural enzymes An effective binding site.
  • ZIFs not only have the advantages of MOFs, but also have better thermal stability and aqueous solution stability than MOFs materials.
  • graphene material not only has abundant oxygen-containing functional groups and ⁇ -electron conjugated regions, but also has high environmental stability.
  • the abundant functional groups on its surface can provide covalent or non-covalent bonding reaction sites , Laid the foundation for building a multi-level structure.
  • the hierarchical structure built on the basis of the zeolite imidazole ester skeleton-8/graphene composite material has a cross-linked porous structure, which can provide more binding sites for natural enzymes, enhance the ability of natural enzymes to resist impact loads, and further enhance natural Environmental stability of enzymes.
  • DNA molecules to regulate the peroxidase catalytic activity of HRP@ZIF-8/GO biocomposite materials, a universal colorimetric analysis method was established for the detection of kanamycin (KAN).
  • the present invention solves the defects of insufficient stability of natural enzymes, and realizes the immobilization of natural horseradish peroxidase (HRP@ZIF-8) through the zeolite imidazole ester skeleton-8/graphene composite material (ZIF-8/GO) /GO), and applied it to the colorimetric detection of kanamycin (KAN).
  • HRP@ZIF-8 horseradish peroxidase
  • ZIF-8/GO zeolite imidazole ester skeleton-8/graphene composite material
  • KAN colorimetric detection of kanamycin
  • a biocomposite material of ZIF-8/GO nanocomposite material immobilized HRP is synthesized under normal temperature and pressure.
  • the technical means of ZIF-8/GO mineralization can retain the original peroxidase catalytic activity of HRP.
  • the ZIF-8/GO protective layer increases the resistance of HRP to extreme environments such as hydrolytic protease, high temperature and organic solvents, and can effectively improve the natural enzyme Storage stability and recycling performance.
  • This protective performance gradually increases with the increase of GO content in the composite material. It fully shows that the introduction of GO can compensate for the lack of stability of the ZIF-8 framework structure to a certain extent, and further enhance the natural enzymes. The stability.
  • An immobilized enzyme method for improving the stability of horseradish peroxidase the steps are as follows:
  • the aptamer sequence is
  • the concentration of aptamer is 2.14 ⁇ M
  • the concentration of HRP@ZIF-8/GO complex is 0.14mg/mL
  • the incubation and temperature are 20min and 25°C
  • the linear detection range of kanamycin for the reaction system of zeolite imidazole ester skeleton-8/graphene composite material immobilized horseradish peroxidase is 1-25 ⁇ g/L
  • the detection limit is 0.02 ⁇ g/L.
  • the method for immobilizing horseradish peroxidase on the zeolite imidazole ester skeleton-8/graphene composite material in the present invention is carried out by simple physical stirring under normal temperature and pressure, and does not require complex synthesis such as high pressure and high temperature condition.
  • the colorimetric detection system of zeolite imidazole ester skeleton-8/graphene composite material immobilized horseradish peroxidase can realize the detection of other targets by changing the type of aptamer, that is, it has universal detection Features.
  • the method for immobilizing horseradish peroxidase on the zeolite imidazole ester skeleton-8/graphene composite material of the present invention increases the resistance of HRP to extreme environments such as hydrolytic proteases, high temperatures and organic solvents, and is also effective Improve the storage stability and recycling performance of natural enzymes.
  • This protective performance gradually increases with the increase of GO content in the composite material, which fully shows that the introduction of GO can compensate for the lack of stability of the ZIF-8 framework structure to a certain extent. , Further enhance the stability of natural enzymes.
  • Figure 1 is a schematic diagram of the preparation process and detection mechanism of the immobilized horseradish peroxidase based on the zeolite imidazole ester skeleton-8/graphene composite material of the present invention.
  • Figure 2 shows the relative catalytic activity of the immobilized horseradish peroxidase on the zeolite imidazole ester skeleton-8/graphene composite material obtained by the present invention after extreme conditions (protein hydrolyzing agent, denaturant, high temperature, organic solvent).
  • Fig. 3 shows the relative catalytic activity of the immobilized horseradish peroxidase on the zeolite imidazole ester skeleton-8/graphene composite material obtained in the present invention after different storage days.
  • Figure 4 shows the relative catalytic activity of the zeolite imidazole ester skeleton-8/graphene composite material immobilized horseradish peroxidase after centrifugal collection and recycling.
  • Fig. 5 is a standard working curve of the application of the zeolite imidazole ester skeleton-8/graphene composite material immobilized horseradish peroxidase in the detection of kanamycin.
  • Fig. 6 is a standard linear curve of the immobilized horseradish peroxidase of the zeolite imidazole ester skeleton-8/graphene composite material obtained in the present invention applied to the detection of kanamycin.
  • Graphene oxide is prepared using a modified Hummers chemical method. The specific steps are as follows: Weigh 1.0g graphite powder and slowly add it to 23mL concentrated sulfuric acid. After fully stirring, slowly add 3g KMnO 4 in an ice water bath at 0°C, add slowly while fully stirring, and then continuously sonicate for 7 hours to obtain a dark brown solution , Then slowly add 46mL of deionized water, heat and boil for 15min, and then add 140mL of high purity water and 10mL of hydrogen peroxide to stop the reaction, and obtain a bright yellow graphene oxide aqueous solution.
  • the obtained products with different GO contents are named HRP@ZIF-8/GO-x, where x represents the mass percentage of the added GO in the final product. Therefore, the product obtained is HRP@ZIF-8/GO-4.
  • Graphene oxide is prepared using a modified Hummers chemical method. The specific steps are as follows: Weigh 1.0g graphite powder and slowly add it to 23mL concentrated sulfuric acid. After fully stirring, slowly add 3g KMnO 4 in an ice water bath at 0°C, add slowly while fully stirring, and then continuously sonicate for 6 hours to obtain a dark brown solution , Then slowly add 46mL of deionized water, heat and boil for 15min, and then add 140mL of high purity water and 10mL of hydrogen peroxide to stop the reaction, and obtain a bright yellow graphene oxide aqueous solution.
  • the obtained products with different GO contents are named HRP@ZIF-8/GO-x, where x represents the mass percentage of the added GO in the final product. Therefore, the product obtained is HRP@ZIF-8/GO-4.
  • Graphene oxide is prepared using a modified Hummers chemical method. The specific steps are as follows: Weigh 1.0g graphite powder and slowly add it to 23mL concentrated sulfuric acid. After full stirring, slowly add 3g KMnO 4 in an ice water bath at 0°C, add slowly while fully stirring, and then continuously sonicate for 5 hours to obtain a dark brown solution , Then slowly add 46mL of deionized water, heat and boil for 15min, and then add 140mL of high purity water and 10mL of hydrogen peroxide to stop the reaction, and obtain a bright yellow graphene oxide aqueous solution.
  • the obtained products with different GO contents are named HRP@ZIF-8/GO-x, where x represents the mass percentage of the added GO in the final product. Therefore, the product obtained is HRP@ZIF-8/GO-4.
  • kanamycin solution prepared with the water sample of the tap water sample at a concentration of 0.25 ⁇ g/L.
  • the sample is used in step (4) for detection, the detection result is compared with the standard working curve obtained in step (6), and the concentration of kanamycin is calculated.
  • the experimental results showed that the content of kanamycin was 0.30 ⁇ g/L, and the recovery rate was 120%.

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Abstract

提供了一种提高辣根过氧化物酶稳定性的固定化酶方法及应用。在常温常压下，利用共沉淀的方法，在水相中合成沸石咪唑酯骨架-8/石墨烯固定化天然辣根过氧化物酶的生物复合材料。ZIF-8/GO复合材料的包埋保留HRP的过氧化物酶催化活性，增强HRP的稳定性。相对于单独的ZIF-8矿化HRP材料，GO的引入增加了HRP对抗高温、有机溶剂和变性剂等复杂环境的耐受性，同时增强了HRP的存储稳定性和回收利用能力。利用DNA分子调控HRP@ZIF-8/GO生物复合材料的过氧化物酶催化活性，建立无标记的比色传感方法，实现了卡那霉素的特异性检测。

Description

一种提高辣根过氧化物酶稳定性的固定化酶方法及应用 技术领域
本发明属于环境监测技术领域，涉及一种提高辣根过氧化物酶稳定性的固定化酶方法及应用。
背景技术
天然酶是由活细胞产生的、对其底物具有高度特异性和高度催化效能的蛋白质或RNA。天然酶具有来源广泛、种类繁多、底物亲和力好、催化效率高等优点，广泛地应用于催化分析、疾病诊断、临床治疗等生产生活的各个方面。但是作为一类典型的生物分子，天然酶的稳定性相对较差，易受外界环境的影响，而且对存储条件有严格的要求。例如天然酶极易受到高温、强酸、强碱等极端外界条件的破坏而丧失部分功能甚至失去原有活性。随着纳米技术的迅速发展，近些年，结合纳米材料的稳定性，利用纳米材料固定化天然酶等生物分子已经成为新的研究热点(Small,2017,13(32):1700880)。固定化酶是指酶在物理上被限制或定位于一定的空间区域内，其催化活性保持不变。固定化酶技术能更有效地控制反应过程，提高酶的存储能力和在极端操作条件下的稳定性。此外，固定化酶的多级结构使其更容易分离，最大限度地减少污染，实现回收利用等，以降低生产成本。目前，常用的纳米材料固定化酶手段有溶胶-凝胶基质、水凝胶、有机微粒、介孔二氧化硅、金属有机骨架(MOFs)材料等等，这些多孔材料具有较大的表面积和空隙体积，是酶固定化的理想材料(Materials Horizons,2017,4(1):55-63；Physical Chemistry Chemical Physics,2018,20(13):8822-8831；Nature Catalysis,2018,1(9):689-695)。但另一方面，这些材料自身的催化性能较差，在固定酶后，往往抑制了天然酶的部分活性(Journal of the American Chemical Society,2017,139(19):6530-6533；Journal of the American  Chemical Society,2015,137(13):4276-4279)。
本发明中结合了沸石咪唑酯骨架材料(ZIFs)的多孔结构和石墨烯(GO)材料优异的稳定性，利用沸石咪唑酯骨架-8/石墨烯复合材料(ZIF-8/GO)实现了天然辣根过氧化物酶(HRP)的固定(HRP@ZIF-8/GO)。沸石咪唑酯骨架结构(ZIFs)材料作为MOFs材料的一类，是由过渡金属原子与咪唑或咪唑衍生物连接而生成的一类新型的、具有沸石拓扑结构的多孔材料，为固定化天然酶提供了有效的结合位点。ZIFs不仅具有MOFs的优点，而且同MOFs材料相比，具有更优良的热稳定性和水溶液稳定性。石墨烯材料作为典型的碳材料，不仅具有丰富的含氧官能团和π电子共轭区域，还具有较高的环境稳定性，其表面丰富的官能团可以提供共价或非共价结合的反应位点，为构建多级结构奠定了基础。基于沸石咪唑酯骨架-8/石墨烯复合材料构建的多级结构内部具有相互交联的多孔结构，可以为天然酶提供更多的结合位点，增强天然酶抗冲击负荷的能力，进一步提升天然酶的环境稳定性。此外，通过利用DNA分子调控HRP@ZIF-8/GO生物复合材料的过氧化物酶催化活性，建立了一种通用型的比色分析方法用于卡那霉素(KAN)的检测。
发明内容
本发明解决了天然酶存在的稳定性不足等缺陷，通过沸石咪唑酯骨架-8/石墨烯复合材料(ZIF-8/GO)实现了天然辣根过氧化物酶的固定(HRP@ZIF-8/GO)，并将其应用于卡那霉素(KAN)的比色检测。
本发明中，在常温常压下合成了ZIF-8/GO纳米复合材料固定化HRP的生物复合材料。ZIF-8/GO矿化的技术手段能够保留了HRP原有的过氧化物酶催化活性。相对于ZIF-8矿化HRP或游离的HRP来说，ZIF-8/GO保护层增加了HRP对抗极端环境如水解蛋白酶、高温和有机溶剂等的耐受性，还可以有效地提高 天然酶的存储稳定性以及回收利用性能，这种保护性能随复合材料中GO含量的增加而逐渐增强，充分说明GO的引入可以在一定程度上弥补ZIF-8骨架结构稳定性不足的缺点，进一步增强天然酶的稳定性。此外，利用DNA分子可以调控HRP@ZIF-8/GO催化活性这一特征，建立了基于HRP@ZIF-8/GO生物复合材料的无标记比色传感方法，反应体系的吸光度变化在一定范围内与卡那霉素的浓度正相关，从而为抗生素的定量分析提供依据。
本发明的技术方案：
一种提高辣根过氧化物酶稳定性的固定化酶方法，步骤如下：
(1)制备氧化石墨烯：石墨烯氧化物采用改进的Hummers化学法制备。步骤如下：取一定量的浓硫酸(98％)缓慢加入到石墨粉(浓硫酸体积:石墨粉质量＝23:1)中，充分搅拌后，在0℃冰水浴中缓慢加入KMnO 4(KMnO 4与石墨粉质量比为3:1)，同时充分搅拌。然后将混合物连续超声5-7h后，得到深褐色溶液。向深褐色溶液中缓慢加入高纯水，加热煮沸5-15min后，依次加入高纯水和30％的过氧化氢(H 2O 2)终止反应，得到亮黄色的石墨烯氧化物水溶液。离心分离后，用稀盐酸在8000-10000r/min的条件下离心洗涤2-3次去除杂质，然后用高纯水在8000-10000r/min的条件下离心洗涤5-7次去除杂质。洗涤后取出纯化的石墨氧化物，装入透析袋(MW＝14000)透析5-7天以进一步去除杂质，最后冷冻干燥得到固体氧化石墨烯。
(2)制备沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶(HRP@ZIF-8/GO)：氧化石墨烯(GO)与硝酸锌(Zn(NO 3) 2)在水中充分超声混合，形成混合液A；HRP和2-甲基咪唑溶于水中，形成混合液B；之后，将混合液B快速倒入混合液A中，得到的混合液在室温下搅拌12-24h；其中GO和HRP的质量比为在0:50-1:1，HRP质量、Zn(NO 3) 2的摩尔和2-甲基咪唑的摩尔量三 者比例为5g:100mol:7000mol；将得到的混合物用高纯水离心清洗3-5次，冷冻干燥12-24h后得到粉末状物质，将其研磨并置于-4～-20℃的条件下冷冻保存；得到的不同GO含量的产物命名为HRP@ZIF-8/GO-x，其中x代表加入的GO在最终产物中的质量。
一种提高辣根过氧化物酶稳定性的固定化酶的应用，卡那霉素(KAN)的定量检测：在25-35℃温度条件下，将浓度为0-50μg/L的KAN溶液与KAN适配体溶液置于HEPES缓冲液(10-25mM，pH＝7-8)中孵化20-30min；随后向混合液中加入HRP@ZIF-8/GO-x复合物在25-35℃下继续孵化20-30min，其中HRP@ZIF-8/GO-x复合物的质量与KAN适配体摩尔量的比值为14×10 6g:214mol；之后，再加入TMB(3,3',5,5'-四甲基联苯胺)和H 2O 2，其中KAN适配体、TMB和H 2O 2三者的摩尔比214:50:11。混合物在25-37℃下反应10-30min后，转移到石英比色皿中，记录体系吸光度随吸收波长的变化曲线。
所述的适配体序列为
5'-TGG GGG TTG AGG CTA AGC CGA-3'。
本发明的有益效果：
(1)当加入的GO比例为最终产物质量的4％，适配体浓度为2.14μM，HRP@ZIF-8/GO复合物浓度为0.14mg/mL，孵化和温度分别为20min和25℃，反应时间和温度分别为10min和37℃时，该沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶的反应体系对卡那霉素的线性检测范围在1-25μg/L，检测限为0.02μg/L。
(2)本发明中沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶的方法是在常温常压下通过简单的物理搅拌进行的，不需要高压、高温等复杂的合成条件。
(3)该沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶的比色检测体系可以通过更换适配体的种类，实现其他目标物的检测，即具有通用性检测的功能。
(4)本发明中沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶的方法增加了HRP对抗极端环境如水解蛋白酶、高温和有机溶剂等的耐受性，还可以有效地提高天然酶的存储稳定性以及回收利用性能，这种保护性能随复合材料中GO含量的增加而逐渐增强，充分说明GO的引入可以在一定程度上弥补ZIF-8骨架结构稳定性不足的缺点，进一步增强天然酶的稳定性。
附图说明
图1是本发明所述的基于沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶的制备过程与检测机理示意图。
图2是本发明获得的沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶经极端条件(蛋白水解剂、变性剂、高温、有机溶剂)处理后的相对催化活性。
图3是本发明获得的沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶经不同存储天数处理后的相对催化活性。
图4是本发明获得的沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶经离心收集循环利用后的相对催化活性。
图5是本发明获得的沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶应用于卡那霉素检测的标准工作曲线。
图6是本发明获得的沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶应用于卡那霉素检测的标准线性曲线。
图中：
Figure PCTCN2019119108-appb-000001
具体实施方式
以下结合技术方案具体说明本发明的具体实施方式。
实施例1
配置水样中卡那霉素含量的测定：
(1)氧化石墨烯的制备：
石墨烯氧化物采用改进的Hummers化学法制备。具体步骤如下：称取1.0g石墨粉缓慢加入到23mL浓硫酸中，充分搅拌后，在0℃冰水浴中缓慢加入3g KMnO 4，缓慢加入同时充分搅拌，然后连续超声7h后，得到深褐色溶液，然后缓慢加入46mL去离子水，加热煮沸15min后，再依次加入140mL高纯水和10mL双氧水终止反应，得到亮黄色的石墨烯氧化物水溶液。离心分离后，用5％的稀盐酸8000r/min离心洗涤2次去除杂质，然后用高纯水8000r/min离心洗涤3次去除杂质。洗涤后取出纯化的石墨氧化物，装入透析袋(MW＝14000)透析一周以进一步去除杂质，最后冷冻干燥得到固体氧化石墨烯。
(2)制备沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶(HRP@ZIF-8/GO)：1mg/mL的GO水溶液(10mL)与10mL的Zn(NO 3) 2·6H 2O(0.10M)充分超声混合(混合液A)。取50mg HRP溶于20mL的2-甲基咪唑(3.46M)水溶液中(混合液B)。之后，将混合液B快速倒入混合液A中，得到的混合液在室温下搅拌12h。之后，混合物经高纯水离心清洗3次，冷冻干燥24h，将得到的粉末研磨，-20℃下冷冻保存。得到的不同GO含量的产物命名为HRP@ZIF-8/GO-x，其中x代表了加入的GO在最终产物中的质量百分比。因此得到的产物为HRP@ZIF-8/GO-4。
(3)沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶(HRP@ZIF-8/GO)的极端条件稳定性研究：分别取不同浓度的HRP@ZIF-8和 HRP@ZIF-8/GO-x复合物与等物质的量的HRP水溶液与1mg/mL的胰蛋白酶或蛋白酶K在37℃下孵化1h后，测定其抗蛋白水解酶的能力。为测定尿素或EDTA对酶活性的影响，分别取不同浓度的HRP@ZIF-8和HRP@ZIF-8/GO-x复合物(等量HRP)在室温下与1wt％的EDTA或6M尿素孵化1h。为测试酶复合物耐受高温和有机溶剂的能力，分别取不同浓度的HRP@ZIF-8和HRP@ZIF-8/GO-x复合物(等量HRP，HRP量0.14mg/mL)在沸水中孵化10-30min和50％有机溶剂(甲醇、丙酮和N,N-二甲基甲酰胺)在室温下孵化30分钟。辣根过氧化物酶及其酶复合物经过上述不同的极端条件处理后，利用PBS(10mM，pH＝7.40)离心(8000r/min)洗涤3次。之后，利用典型的TMB-H 2O 2反应体系测定辣根过氧化物酶及其酶复合物处理前后催化活性的变化，分别取HRP、HRP@ZIF-8和HRP@ZIF-8/GO-x复合材料水分散液(等量HRP)，与0.75mM的TMB以及14.28mM的H 2O 2在0.20M的乙酸缓冲液中(pH＝4.00)孵化反应10min后(37℃)，转移到石英比色皿中，室温下测量体系在652nm处的吸光度，并与其未处理前原始吸光度对比，计算得到相对催化活性(图2)。
(4)卡那霉素(KAN)的定量检测：
取不同浓度的KAN溶液(0-50μg/L)与KAN适配体(2.14μM)混合，室温下在25mM的HEPES缓冲液(100mM NaCl，2mM MgCl 2，pH＝7.60)中孵化30min。随后向混合液中加入0.14mg/mL的HRP@ZIF-8/GO-4复合物继续孵化20min。再加入0.50mM的TMB和0.11mM的H 2O 2。
(5)检测方法：步骤(4)中的混合物在37℃下反应10min后，转移到石英比色皿中，记录体系吸光度随吸收波长(500-800nm)的变化曲线(图5)。
(5)标准工作曲线的绘制
步骤(4)中随着样品中卡那霉素浓度的增加，反应体系在652nm处的吸光度 不断增加，在0-50μg/L范围内，反应体系的吸光度与卡那霉素浓度有良好的线性关系，线性相关系数R 2＝0.99(图6)。
(6)配置水样中卡那霉素含量的测定：
用HEPES缓冲溶液配置卡那霉素浓度为0.02μg/L的水样。将样品用于步骤(4)方法进行检测，检测结果与步骤(5)得到的标准工作曲线对比，计算出卡那霉素的浓度。实验结果测出卡那霉素含量0.0225μg/L，回收率为113％。相对标准偏差RSD为2.35％(n＝5)。
实施例2
配置水样中卡那霉素含量的测定：
(1)氧化石墨烯的制备：
石墨烯氧化物采用改进的Hummers化学法制备。具体步骤如下：称取1.0g石墨粉缓慢加入到23mL浓硫酸中，充分搅拌后，在0℃冰水浴中缓慢加入3g KMnO 4，缓慢加入同时充分搅拌，然后连续超声6h后，得到深褐色溶液，然后缓慢加入46mL去离子水，加热煮沸15min后，再依次加入140mL高纯水和10mL双氧水终止反应，得到亮黄色的石墨烯氧化物水溶液。离心分离后，用5％的稀盐酸10000r/min离心洗涤2次去除杂质，然后用高纯水10000r/min离心洗涤5次去除杂质。洗涤后取出纯化的石墨氧化物，装入透析袋(MW＝14000)透析一周以进一步去除杂质，最后冷冻干燥得到固体氧化石墨烯。
(2)制备沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶(HRP@ZIF-8/GO)：1mg/mL的GO水溶液(10mL)与10mL的Zn(NO 3) 2·6H 2O(0.10M)充分超声混合(混合液A)。取50mg HRP溶于20mL的2-甲基咪唑(3.46M)水溶液中(混合液B)。之后，将混合液B快速倒入混合液A中，得到的混合液在室温下搅拌12h。之后，混合物经高纯水离心清洗3次，冷冻干燥24h，将得 到的粉末研磨，-20℃下冷冻保存。得到的不同GO含量的产物命名为HRP@ZIF-8/GO-x，其中x代表了加入的GO在最终产物中的质量百分比。因此得到的产物为HRP@ZIF-8/GO-4。
(3)沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶(HRP@ZIF-8/GO)的存储稳定性研究：为测定酶复合物的储存稳定性，分别取不同浓度的HRP@ZIF-8和HRP@ZIF-8/GO-x(等量HRP)的PBS分散液(10mM，pH＝7.40)，在4℃下储存不同的时间后，利用典型的TMB-H 2O 2反应体系测定辣根过氧化物酶及其酶复合物不同存储时间内催化活性的变化，分别取HRP、HRP@ZIF-8和HRP@ZIF-8/GO-x复合材料水分散液(等量HRP，HRP量0.14mg/mL)，与0.75mM的TMB以及14.28mM的H 2O 2在0.20M的乙酸缓冲液中(pH＝4.00)孵化反应10min后(37℃)，转移到石英比色皿中，室温下测量体系在652nm处的吸光度，并与其原始吸光度对比，计算得到相对催化活性(图3)。
(4)卡那霉素(KAN)的定量检测：
取不同浓度的KAN溶液(0-50μg/L)与KAN适配体(2.14μM)混合，室温下在25mM的HEPES缓冲液(100mM NaCl，2mM MgCl 2，pH＝7.60)中孵化30min。随后向混合液中加入0.14mg/mL的HRP@ZIF-8/GO-4复合物继续孵化20min。再加入0.50mM的TMB和0.11mM的H 2O 2。
(5)检测方法：步骤(4)中的混合物在37℃下反应10min后，转移到石英比色皿中，记录体系吸光度随吸收波长(500-800nm)的变化曲线(图5)。
(6)标准工作曲线的绘制
步骤(5)中随着样品中卡那霉素浓度的增加，反应体系在652nm处的吸光度不断增加，在0.01-0.50μg/L范围内，反应体系的吸光度与卡那霉素浓度有良好的线性关系，线性相关系数R 2＝0.99(图6)。
(7)配置水样中卡那霉素含量的测定：
用HEPES缓冲溶液配置卡那霉素浓度为0.30μg/L的水样。将样品用于步骤(4)方法进行检测，检测结果与步骤(5)得到的标准工作曲线对比，计算出卡那霉素的浓度。实验结果测出卡那霉素含量0.35μg/L，回收率为117％。相对标准偏差RSD为1.35％(n＝5)。
实施例3
自来水样品中卡那霉素含量的测定：
(1)氧化石墨烯的制备：
石墨烯氧化物采用改进的Hummers化学法制备。具体步骤如下：称取1.0g石墨粉缓慢加入到23mL浓硫酸中，充分搅拌后，在0℃冰水浴中缓慢加入3g KMnO 4，缓慢加入同时充分搅拌，然后连续超声5h后，得到深褐色溶液，然后缓慢加入46mL去离子水，加热煮沸15min后，再依次加入140mL高纯水和10mL双氧水终止反应，得到亮黄色的石墨烯氧化物水溶液。离心分离后，用5％的稀盐酸10000r/min离心洗涤2次去除杂质，然后用高纯水10000r/min离心洗涤5次去除杂质。洗涤后取出纯化的石墨氧化物，装入透析袋(MW＝14000)透析一周以进一步去除杂质，最后冷冻干燥得到固体氧化石墨烯。
(2)制备沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶(HRP@ZIF-8/GO)：1mg/mL的GO水溶液(10mL)与10mL的Zn(NO 3) 2·6H 2O(0.10M)充分超声混合(混合液A)。取50mg HRP溶于20mL的2-甲基咪唑(3.46M)水溶液中(混合液B)。之后，将混合液B快速倒入混合液A中，得到的混合液在室温下搅拌12h。之后，混合物经高纯水离心清洗3次，冷冻干燥24h，将得到的粉末研磨，-20℃下冷冻保存。得到的不同GO含量的产物命名为HRP@ZIF-8/GO-x，其中x代表了加入的GO在最终产物中的质量百分比。因此 得到的产物为HRP@ZIF-8/GO-4。
(3)沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶(HRP@ZIF-8/GO)的循化利用稳定性研究：为测试制备的酶复合材料的再生能力，在含有0.75mM的TMB和14.28mM的H 2O 2的0.2M醋酸缓冲体系(pH＝4.00)中加入一定浓度的HRP、HRP@ZIF-8或HRP@ZIF-8/GO-x复合材料(等量HRP)。混合液在37℃反应10min后，测定上清液吸光度。混合物在13000rpm下离心10min，以收集沉淀物，进行下一次酶催化反应。该过程重复4次以测定酶催化剂的重复利用能力。利用典型的TMB-H 2O 2反应体系测定辣根过氧化物酶及其酶复合物循环利用前后催化活性的变化，分别取HRP、HRP@ZIF-8和HRP@ZIF-8/GO-x复合材料水分散液(等量HRP，HRP量0.14mg/mL)，与0.75mM的TMB以及14.28mM的H 2O 2在0.20M的乙酸缓冲液中(pH＝4.00)孵化反应10min后(37℃)，转移到石英比色皿中，室温下测量体系在652nm处的吸光度，并与其原始吸光度对比，计算得到相对催化活性(图4)。
(4)卡那霉素(KAN)的定量检测：
取不同浓度的KAN溶液(0-50μg/L)与KAN适配体(2.14μM)混合，室温下在25mM的HEPES缓冲液(100mM NaCl，2mM MgCl 2，pH＝7.60)中孵化30min。随后向混合液中加入0.14mg/mL的HRP@ZIF-8/GO-4复合物继续孵化20min。再加入0.50mM的TMB和0.11mM的H 2O 2。
(5)检测方法：步骤(4)中的混合物在37℃下反应10min后，转移到石英比色皿中，记录体系吸光度随吸收波长(500-800nm)的变化曲线(图5)。
(6)标准工作曲线的绘制
步骤(5)中随着样品中卡那霉素浓度的增加，反应体系在652nm处的吸光度不断增加，在0.01-0.50μg/L范围内，反应体系的吸光度与卡那霉素浓度有良好 的线性关系，线性相关系数R 2＝0.99(图6)。
(6)自来水样品中卡那霉素含量的测定：
由于市政自来水样品中不含卡那霉素，故采用加标回收实验。用自来水样品的水样配制卡那霉素溶液，浓度为0.25μg/L。将样品用于步骤(4)方法进行检测，检测结果与步骤(6)得到的标准工作曲线对比，计算出卡那霉素的浓度。实验结果测出卡那霉素含量0.30μg/L，回收率为120％。相对标准偏差RSD为2.50％(n＝5)。

Claims (2)

  1. 一种提高辣根过氧化物酶稳定性的固定化酶方法，其特征在于，步骤如下：
    (1)制备氧化石墨烯：石墨烯氧化物采用改进的Hummers化学法制备；
    (2)制备沸石咪唑酯骨架-8/石墨烯复合材料固定化辣根过氧化物酶HRP@ZIF-8/GO：GO与Zn(NO 3) 2在水中充分超声混合，形成混合液A；HRP和2-甲基咪唑溶于水中，形成混合液B；之后，将混合液B快速倒入混合液A中，得到的混合液在室温下搅拌12-24h；其中GO和HRP的质量比为0:50-1:1，HRP质量、Zn(NO 3) 2的摩尔和2-甲基咪唑的摩尔三者比例为5g:100mol:7000mol；将得到的混合物用高纯水离心清洗3-5次，冷冻干燥12-24h后得到粉末状物质，将其研磨并置于-4～-20℃的条件下冷冻保存；得到的不同GO含量的产物命名为HRP@ZIF-8/GO-x，其中x代表加入的GO在最终产物中的质量百分比。
  2. 一种提高辣根过氧化物酶稳定性的固定化酶的应用，其特征在于，卡那霉素KAN的定量检测：在25-35℃温度条件下，将浓度为0-50μg/L的KAN溶液与KAN适配体溶液置于HEPES缓冲液中孵化20-30min；其中，HEPES缓冲液的浓度为10-25mM，pH＝7-8；随后向混合液中加入HRP@ZIF-8/GO-x复合物在25-35℃下继续孵化20-30min，其中HRP@ZIF-8/GO-x复合物的质量与KAN适配体摩尔量的比值为14×10 6g:214mol；之后，再加入3,3',5,5'-四甲基联苯胺TMB和H 2O 2，其中KAN适配体、TMB和H 2O 2三者的摩尔比214:50:11；混合物在25-37℃下反应10-30min后，转移到石英比色皿中，记录体系吸光度随吸收波长的变化曲线；
    所述的适配体序列为5'-TGG GGG TTG AGG CTA AGC CGA-3'。
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Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN116809946B (zh) * 2023-05-12 2025-06-27 江苏科技大学 一种FeCe/NC-TMB比色传感器及其制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103290124A (zh) * 2013-05-29 2013-09-11 华南农业大学 一种卡那霉素a酶联适配体检测方法及应用
CN105734038A (zh) * 2016-01-22 2016-07-06 南京工业大学 一种酶-GO-MOFs纳米复合催化剂及其制备方法
CN110218720A (zh) * 2019-05-14 2019-09-10 大连理工大学 一种提高辣根过氧化物酶稳定性的固定化酶方法及应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107446915B (zh) * 2017-08-30 2020-08-28 江苏大学 磁性氧化石墨烯复合材料固定化辣根过氧化物酶及其制备方法和应用
CN107497377A (zh) * 2017-10-19 2017-12-22 山东大学 一种形貌均一金属有机骨架化合物/氧化石墨烯复合微球的制备方法
CN107803119B (zh) * 2017-11-03 2021-01-05 宁夏大学 一种复合膜层的制备方法以及利用其去除制药废水中残留四环素的方法
CN108008128B (zh) * 2017-12-21 2020-03-31 江南大学 核酸适配体识别和功能化磁性微球分离预处理的卡那霉素快速检测试纸及其制备和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103290124A (zh) * 2013-05-29 2013-09-11 华南农业大学 一种卡那霉素a酶联适配体检测方法及应用
CN105734038A (zh) * 2016-01-22 2016-07-06 南京工业大学 一种酶-GO-MOFs纳米复合催化剂及其制备方法
CN110218720A (zh) * 2019-05-14 2019-09-10 大连理工大学 一种提高辣根过氧化物酶稳定性的固定化酶方法及应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
FAN, Z.J. ET AL.: "Metal-Organic Frameworks/Graphene Oxide Composite: A New Enzymatic Immobilization Carrier for Hydrogen Peroxide Biosensors. Journal of The Electrochemical Society", JOURNAL OF THE ELECTROCHEMICAL SOCIETY, vol. 163, no. 3, 19 December 2015 (2015-12-19), XP055753311, DOI: 20200215105921Y *
FAN, Z.J. ET AL.: "Metal-Organic Frameworks/Graphene Oxide Composite: A New Enzymatic Immobilization Carrier for Hydrogen Peroxide Biosensors. Journal of The Electrochemical Society", JOURNAL OF THE ELECTROCHEMICAL SOCIETY, vol. 163, no. 3, 19 December 2015 (2015-12-19), XP055753311, DOI: 20200215111956X *
KANOKPHANDHARANGKUL, K.: "Aptamer Biosensors.", THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF IMPERIAL COLLEGE LONDON., 30 April 2015 (2015-04-30), DOI: 20200215111250Y *
LIU, J.W. ET AL.: "Tunable Composites Prepared from Graphene Oxide and Zeolitic Imidazolate Framework-8 for Improved Selective Isolation of Hemoglobin.", MICROCHIMICA ACTA., vol. 185, 5 July 2018 (2018-07-05), XP036570198, DOI: 20200215110537X *
LIU, JIAWEI ET AL.: "Tunable Composites Prepared from Graphene Oxide and Zeolitic Imidazolate Framework-8 for Improved Selective Isolation of Hemoglobin.", MICROCHIMICA ACTA., vol. 185, 5 July 2018 (2018-07-05), XP036570198, DOI: 20200215110544Y *
LIU, XINJIA ET AL.: "Development of Enzyme-linked Aptamer Assay for Detection of Kanamycin A", CHINESE JOURNAL OF ANALYTICAL CHEMISTRY, vol. 41, no. 9, 30 September 2013 (2013-09-30), DOI: 20200215111452Y *

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