IOCAS-IR  > 海洋环境腐蚀与生物污损重点实验室
基于MOF材料的非生物基荧光探针的构建及其在生物膜测试中的应用研究
辛悦
Subtype硕士
Thesis Advisor戚鹏
2023-05-19
Degree Grantor中国科学院海洋研究所
Place of Conferral中国科学院海洋研究所
Degree Name生物与医药硕士
Keyword生物膜 非生物基 荧光探针 三磷酸腺苷 微生物腐蚀
Abstract

海洋工程装备和船舶在服役过程中不可避免的要遇到各种腐蚀问题,其中微生物腐蚀(MIC)几乎可以在所有常用工程材料表面发生,微生物腐蚀约占整个腐蚀的20%。微生物腐蚀不仅影响着海洋工程设施的安全服役,还威胁着从业人员的人身安全,为国民经济带来巨大损失。采取有效手段现场、原位评估生物膜内微生物的代谢状态,研究微生物膜发展规律,能为深入揭示微生物在腐蚀过程中的作用机理,开发微生物腐蚀状态的快速监测技术提供重要依据。三磷酸腺苷(ATP)是生物膜活性的重要标志性物质,传统ATP的检测方法流程复杂、仪器昂贵、费时长、无法进行原位检测。荧光检测技术具有灵敏性高、选择性好、可实现原位检测等优点,在生物膜内微生物活性分析中的应用潜力巨大。锌基沸石咪唑框架(ZIF-90)是一种合成路线简单、孔隙率高、醛基丰富、生物相容性好、环境友好的材料,被广泛应用于生物系统中。本研究以金属有机框架结构(MOFs)为基础,构建了三种不同类型的非生物基荧光探针,并且将其应用于生物膜内ATP的检测。主要的研究结果如下:

1)合成了ZIF-90特异性识别的材料,并构建了非生物基荧光增强探针CDs@ZIF-90。研究以细菌培养基的主要成分酵母粉和胰蛋白胨为原料,合成了一种具有聚集荧光抑制效应的蓝色荧光碳量子点(CDs),通过ZIF-90框架结构将ATP浓度和碳点荧光强度联系起来,实现了ATP浓度的快速、高稳定性荧光检测。实验表明,ZIF-90能够与ATP的特异性结合并发生裂解,进而使CDs@ZIF-90可对ATP进行灵敏度高、选择性好的检测,该非生物基荧光探针具有较好的光稳定性和化学稳定性,在固体基质表面生物膜内ATP的检测中展现出良好的可行性。

2)构建了一种基于ZIF-90识别的双发射比率型荧光探针Cu NCs-Al@ZIF-90。研究合成了具有聚集诱导荧光发射(AIE)效应的铜纳米簇(Cu NCs),在铝离子的存在下,Cu NCs被诱导聚合从而荧光显著增强。Cu NCs-Al@ZIF-90探针可以在ATP存在时裂解,释放出发蓝色荧光的咪唑-2-甲醛(2-ICA),和发橙色荧光的Cu NCs,二者可分别在450 nm620 nm处观察到荧光发射,两种荧光变化趋势相反。这种双发射比率荧光探针可以实现检测信号的自校准,具有更高的准确性,进而实现ATP的高效快速测定。研究进一步将Cu NCs-Al@ZIF-90探针应用于生物膜内ATP的检测,取得良好效果,测试了Cu NCs-Al@ZIF-90探针应用于四种不同金属材料表面生物膜的荧光成像分析,观察到Cu NCs-Al@ZIF-90双发射探针能够指示生物膜ATP的分布。

3)构建了一种基于ZIF-90识别的检测-抑菌双功能荧光探针DNA-FAM-CeO2@ZIF-90。探针中的CeO2纳米颗粒具有抑制细菌附着的功能,将ZIF-90ATP识别特性与吸附DNA-FAM的特性结合,可以实现对ATP的特异性、高灵敏度检测。因此,DNA-FAM-CeO2@ZIF-90探针具有ATP荧光特异性检测和抑制细菌生长的双重功能,初步测试其在生物膜样品中检测的性能。

Other Abstract

Marine engineering equipment and ships inevitably encounter various corrosion problems during service. Among which, microbially influenced corrosion can occur on all commonly used engineering materials, accounting for about 20% of corrosion loss. Microbiologically influenced corrosion not only affects the safe service of offshore engineering facilities, but also threatens the personal safety of employees, bringing huge losses to the national economy. Taking effective means to assess the metabolic state of microorganisms in biofilms on-site and in situ, and to study the development of biofilms can provide important foundationsfor revealing the mechanism of microorganisms in the corrosion process and developing rapid monitoring techniques towards microbiologically influenced corrosion. Adenosine triphosphate (ATP) is an important marker of biofilm activity, but traditional ATP detection methods are complex, expensive, time-consuming, and unable to perform on insitu detection. Fluorescence detection technology has the advantages of high sensitivity, good selectivity, and in situ, which are great helpful for applying fluorescence detection to the analysis of microbial metabolic activity in biofilms. Zinc-based zeolite imidazole framework (ZIF-90) is a material with simple synthesis routes, high porosity, abundant aldehyde groups, good biocompatibility, and it’s environmental-friendly, which was widely used in biological systems. In this study, based on metal-organic framework (MOFs), three different types of abiotic fluorescence probes based on metal-organic frameworks (MOFs), and applied to the detection of ATP in microbial biofilms. The main research results are as follows:

(1) Synthesized ZIF-90 specific recognition material and developed an abiotic fluorescence enhancement probe CDs@ZIF-90. Using yeast powder and tryptone, the main components of bacterial culture medium, as raw materials, blue-fluorescent emission carbon quantum dots (CDs) with aggregation fluorescence inhibition effect were synthesized. Then the ATP concentration and CDs fluorescence intensity were linked through the degree of cleavage of ZIF-90, realizingfast and stable fluorescence detection of ATP concentration. Experiments have proved that ZIF-90 can be cleaved with the specific binding of ATP, thereby causing ATP can be detected by CDs@ZIF-90 with high sensitivity and selectivity. This non-biological fluorescence probe has good photostability and chemical stability, demonstrating good feasibility in the detection of ATP in biofilms on solid substrates.

(2) A dual-emission ratiometric fluorescent probe Cu NCs-Al@ZIF-90 based on ZIF-90 recognition was constructed. Copper nanoclusters (Cu NCs) with aggregation induced fluorescence emission (AIE) effects were synthesized. In the presence of aluminum ions, Cu NCs were induced to aggregate, resulting in significant fluorescence enhancement. While, Cu NCs-Al@ZIF-90 was cleaved in the presence of ATP, releasing imidazole-2-formaldehyde (2-ICA) with blue fluorescence emission, and Cu NCs with orange fluorescence emission. Two reverse fluorescence changes were observed at 450 nm and 620 nm, respectively. This dual-emission ratiometric fluorescent probe couldself calibrate the detection signals and thus exhibited a higher accuracy, thereby realizing efficient and rapid ATP detection. Cu NCs-Al@ZIF-90 was further applied to the detection of ATP in biofilms with good performances. Lastly, the Cu NCs-Al@ZIF-90 probe was applied to fluorescence imaging of biofilms on the surface of four different materials, and it was confirmed that Cu NCs-Al@ZIF-90 could indicate the distribution of ATP in biofilms.

(3) A dual functional detection and bacteria inhibition fluorescent probe DNA-FAM-CeO2@ZIF-90 based on ZIF-90 recognition was constructed. The CeO2 nanoparticles in the probe exhibited the function of inhibiting bacterial adhesion, and the combination of the ATP recognition property of ZIF-90 and the adsorption characteristics of DNA-FAM made DNA-FAM-CeO2@ZIF-90 could achieve a high-specific and high-sensitive detection of ATP. Therefore, the DNA-FAM-CeO2@ZIF-90 probe possessed dual functions of ATP fluorescence detection and inhibition of bacterial growth, and its application performance in biofilm samples was preliminarily tested.

MOST Discipline Catalogue生物与医药
Funding ProjectYoung Elite Scientists Sponsorship Program by CAST[20180276] ; National Natural Science Foundation of China[41876101] ; National Natural Science Foundation of China[41506094]
Language中文
Table of Contents

1  绪论......................................................................................... 1

1.1  海洋腐蚀........................................................................................................... 1

1.2  微生物腐蚀....................................................................................................... 1

1.3  ATP荧光探针的研究进展.................................................................................. 4

1.3.1  打开型荧光探针...................................................................................... 5

1.3.2  关闭型荧光探针...................................................................................... 6

1.3.3  比率型荧光探针...................................................................................... 8

1.4  ZIF-90框架结构.............................................................................................. 10

1.5  选题依据及研究思路....................................................................................... 11

1.5.1  选题依据............................................................................................... 11

1.5.2  研究思路............................................................................................... 12

2  快速、可靠检测ATP的非生物CDs@ZIF-90荧光探针的构建与应用............................................................................................... 15

2.1  引言................................................................................................................ 15

2.2  实验部分......................................................................................................... 16

2.2.1  实验试剂............................................................................................... 16

2.2.2  CDs@ZIF-90合成.................................................................................. 16

2.2.3  CDs性能优化........................................................................................ 17

2.2.4  CDs@ZIF-90荧光探针性能优化............................................................. 17

2.2.5  CDs@ZIF-90检测ATP........................................................................... 17

2.2.6  CDs@ZIF-90探针选择性检测................................................................ 17

2.2.7  血清样品检测和生物膜内ATP含量检测................................................. 18

2.2.8  CDsCDs@ZIF-90ZIF-90表征......................................................... 18

2.3  研究结果与分析.............................................................................................. 18

2.3.1  CDsCDs@ZIF-90ZIF-90表征分析.................................................. 18

2.3.2  CDs性能优化分析................................................................................. 23

2.3.3  CDs@ZIF-90荧光探针性能优化分析...................................................... 23

2.3.4  ATP检测标准曲线................................................................................. 24

2.3.5  血清检测及生物膜内ATP含量检测分析................................................. 26

2.4  小结................................................................................................................ 26

3 快速检测ATPCu NCs-Al@ZIF-90双光路比率型荧光传感系统的构建与应用............................................................................... 29

3.1  引言................................................................................................................ 29

3.2  实验部分......................................................................................................... 30

3.2.1  实验试剂............................................................................................... 30

3.2.2  Cu NCs-Al@ZIF-90合成........................................................................ 31

3.2.3  Cu NCs-Al@ZIF-90 荧光探针性能优化.................................................. 31

3.2.4  Cu NCs-Al@ZIF-90检测ATP................................................................. 31

3.2.5  Cu NCs-Al@ZIF-90 荧光探针选择性检测............................................... 31

3.2.6  血清样品检测和生物膜内ATP含量检测................................................. 32

3.2.7  生物膜双光路荧光成像.......................................................................... 32

3.2.8  Cu NCsCu NCs-Al@ZIF-90ZIF-90表征.......................................... 32

3.3  研究结果与分析.............................................................................................. 33

3.3.1  Cu NCsCu NCs-Al@ZIF-90ZIF-90表征分析................................... 33

3.3.2  Cu NCs-Al@ZIF-90 荧光探针性能优化分析........................................... 37

3.3.3  ATP检测标准曲线................................................................................. 38

3.3.4  Cu NCs-Al@ZIF-90探针选择性分析....................................................... 39

3.3.5  Cu NCs-Al@ZIF-90探针性能分析.......................................................... 40

3.3.6  血清检测及生物膜内ATP含量检测分析................................................. 41

3.3.7  生物膜双光路荧光成像分析................................................................... 42

3.4  小结................................................................................................................ 46

4  检测-抑菌双功能DNA-FAM-CeO2@ZIF-90荧光探针的构建与应用................................................................................................... 47

4.1  引言................................................................................................................ 47

4.2  实验部分......................................................................................................... 48

4.2.1  实验试剂............................................................................................... 48

4.2.2  DNA-FAM-CeO2@ZIF-90合成............................................................... 48

4.2.3  DNA-FAM-CeO2@ZIF-90荧光探针性能优化.......................................... 49

4.2.4  DNA-FAM-CeO2@ZIF-90检测ATP........................................................ 49

4.2.5  生物膜内ATP含量检测......................................................................... 49

4.2.6  CeO2@ZIF-90抑菌效果检测.................................................................. 49

4.2.7  CeO2纳米颗粒、CeO2@ZIF-90DNA-FAM-CeO2@ZIF-90ZIF-90表征........................................................................................................................ 50

4.3  研究结果与分析.............................................................................................. 50

4.3.1  CeO2纳米颗粒、CeO2@ZIF-90DNA-FAM-CeO2@ZIF-90ZIF-90表征分析.................................................................................................................... 50

4.3.2  DNA-FAM-CeO2@ZIF-90荧光探针性能优化分析................................... 53

4.3.3  ATP检测标准曲线和探针选择性分析..................................................... 55

4.3.4 生物膜内ATP含量检测分析.................................................................... 56

4.3.5  CeO2@ZIF-90抑菌效果分析.................................................................. 57

4.4  小结................................................................................................................ 59

5  结论与展望........................................................................... 61

5.1  结论................................................................................................................ 61

5.2  创新点............................................................................................................ 62

5.3  展望................................................................................................................ 62

参考文献............................................................................................... 63

  ................................................................................................... 73

作者简历及攻读学位期间发表的学术论文与研究成果..................... 75

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/181168
Collection海洋环境腐蚀与生物污损重点实验室
中国科学院海洋研究所
Recommended Citation
GB/T 7714
辛悦. 基于MOF材料的非生物基荧光探针的构建及其在生物膜测试中的应用研究[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,2023.
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