海洋环境腐蚀与生物污损重点实验室知识库

海洋环境资源开发过程中，微生物腐蚀几乎可以在所有海洋工程材料表面发生，严重威胁着海洋装备和工程设施的安全服役。因此，对海洋环境中微生物腐蚀的研究至关重要。快速测定腐蚀微生物膜的活性不仅可以揭示生物膜的发展阶段，还可以预测微生物腐蚀的发展趋势。本研究以海洋腐蚀微生物膜为研究对象，针对不同的应用背景，构建了腐蚀微生物膜特征代谢活性分子的快速检测方法，包括基于ZIF-90响应裂解和模拟酶活性抑制特性的生物膜ATP双信号检测体系、基于流动扩散的生物膜ATP纸基传感器、基于磁驱抗菌肽的生物膜中细胞内外ATP区分鉴别检测体系、基于CRISPR/Cas12a协同ZIF-90识别平台的生物膜ADP和ATP差异化检测体系、基于pH和ATP连续响应的生物膜精确定位及清除体系、基于流动扩散的生物膜cyt c双读数粘度流量试纸条、基于靶标竞争结合和Exo-I酶驱动的生物膜ATP和cyt c同时检测体系。系统地研究了每种检测体系的构建及实现过程、信号反馈及优化原理、性能测试及评价体系，并于生物膜体系进行了应用验证。本研究的开展为揭示生物膜内腐蚀微生物的代谢状态提供了可靠工具，为揭示腐蚀微生物代谢活性与腐蚀行为之间的关联性提供了技术保障，对微生物腐蚀的机理研究和防护技术开发具有重要意义。主要研究结果如下：

（1）开发了基于ZIF-90抑制MoS₂模拟酶活性的生物膜ATP双信号检测体系，利用ZIF-90纳米颗粒对ATP的响应特性实现了MoS₂模拟酶活性的可控释放，通过光学和电化学方法检测，建立了H₂O₂-TMB反应体系的催化活性与ATP浓度之间的关联，该方法具有良好的灵敏度和选择性，光学模式和电化学模式可满足不同应用环境ATP检测的需求，可实现生物膜中ATP的双信号灵敏检测。

（2）设计了基于ATP响应ChoA@ZIF-90纳米颗粒裂解的纸基双信号ATP检测体系，利用ATP特异性裂解ZIF-90的特性释放其中的壳聚糖酶，进而裂解壳聚糖水凝胶产生溶液黏度变化，此外ZIF-90的裂解可以释放纳米酶的催化活性，进而产生颜色变化，使其可以用于便携、快速、无仪器的ATP现场检测。检测结果结合距离和比色读数直接显示在滤纸上，实现了生物膜内ATP现场定性检测和肉眼定量测量。

（3）构建了基于抗菌肽功能化磁性纳米颗粒（M-AMPs）的生物膜中细胞内和细胞外ATP鉴别检测的阻抗型适体传感体系。利用M-AMPs温和、快速的细胞裂解能力，通过M-AMPs释放前后电荷转移电阻差异得到细胞内外ATP含量。该体系为区分生物膜细胞内和细胞外ATP的检测提供了一种方便的方法，可实现各种海洋腐蚀微生物细胞内和细胞外ATP的鉴别检测。

（4）开发了基于dsDNA-ZIF-90@Ag₃AuS₂@Fe₃O₄纳米颗粒的ADP和ATP同时检测体系，该体系包含适配体识别和DNA激活CRISPR/Cas12a信号放大的ADP敏感模块，以及基于ZIF-90纳米颗粒裂解和PEI-碳量子点淬灭的ATP响应模块。首次成功实现了ADP和ATP的同时、高灵敏度定量，排除了ATP测定时ADP分子对测试信号的干扰，实现了海洋腐蚀微生物SRB生长过程中细胞内ADP和ATP的精确定量，为SRB生物膜体系中ADP和ATP超灵敏双模定量提供了一种有前景的方法。

（5）设计了基于pH和ATP连续响应型生物膜靶向纳米容器的生物膜精确定位及清除体系，用于精确定位生物膜并同时杀灭细菌。针对生物膜微酸性和富ATP环境，分别以无定形碳酸钙/聚丙烯酸（ACC/PAA）为识别外壳， ZIF-90为响应内核，提高生物膜识别和去除精度。此外，为满足不同生物膜场景的应用需求，可通过改变ACC/PAA外壳结构中掺杂的金属离子调节纳米容器的pH响应性，可通过直接喷涂或嵌入智能防卫涂层功实现海洋腐蚀微生物SRB生物膜的精确定位和同步清除。

（6）构建了基于响应透明质酸酶释放的双读出粘度流量cyt c传感体系，以负载透明质酸酶的UiO-67-NH₂作为纳米探针，双链DNA为智能门控，利用cyt c识别释放透明质酸酶裂解水凝胶，进而可以通过pH纸条上扩散距离和颜色信号变化确定生物靶标cyt c浓度。整个检测流程避免了昂贵的仪器和复杂的操作程序，完成了不同基底上腐蚀微生物SRB生长过程中生物膜cyt c浓度的便捷、准确检测。

（7）开发了基于靶标调节适体竞争结合和核酸外切酶I（Exo I）加速信号分子释放的多靶点同时检测体系，设计了具有高信号分子负载特性的UiO-67纳米载体，利用靶标分子的特异性识别过程释放信号分子，并引入Exo I以加速信号分子的释放。该体系具有响应快、选择性强和灵敏度高的优点，实现了ATP和cyt c同时、快速、准确检测，可用于包括海洋腐蚀微生物SRB在内的多种细菌ATP和cyt c同时检测，实现了腐蚀微生物生长过程中生物膜ATP和cyt c的同时检测。

Microbial corrosion can occur on the surface of almost all marine engineering materials during resource exploitation in the marine environment, which seriously threatens the safe service of marine equipment and engineering facilities. Therefore, the study of microbially influenced corrosion in the marine environment is crucial. Rapid determination of the activity of corrosive microbial biofilms can not only reveal the development stage of biofilm, but also predict the development trend of microbial corrosion. In this study, the rapid detection methods of characteristic metabolic active molecules of corrosive microbial biofilms were constructed for different application backgrounds, including a dual-signal ATP detection system based on artificial nanozymes inhibited by ZIF-90 nanoparticles, an instrument-free paper-based system for ATP detection, a intracellular and extracellular ATP discriminative detection system based on magnetically modified antimicrobial peptide, a simultaneous ultrasensitive ADP and ATP quantification system based on CRISPR/Cas12a integrated ZIF-90 nanocomposites, a precise localization and simultaneous bacterial eradication system based on successive responsive towards pH and ATP, an instrument-free dual-readout viscosity flow system for cyt c detection, and a target-modulated competitive binding and exonuclease I-powered system for the simultaneous detection of ATP and cyt c. The construction and implementation process, signal feedback and optimization principle, and performance testing and evaluation system of each detection system were systematically investigated and validated in biofilms system. This study provides reliable tools to reveal the metabolic state of corrosive microorganisms in biofilms, and provides a technical guarantee to reveal the correlation between the metabolic activity of corrosive microorganisms and corrosion behavior, which is of great significance to reveal the microbial corrosion mechanism and develop protection technology. The main research results are as follows:

(1) A dual-signal detection system for biofilm ATP based on ZIF-90 inhibition of MoS₂ nanoenzyme activity was developed The controlled release of MoS₂ mimic enzyme activity was achieved by using the response property of ZIF-90 nanoparticles to ATP, and the correlation between catalytic activity of H₂O₂-TMB reaction system and ATP concentration was established by optical and electrochemical methods. The proposed detection method holds good sensitivity and selectivity towards ATP, and the optical and electrochemical dual modes can meet the demand of ATP detection in different application environments. The prepared system enabled dual-signal detection of ATP in SRB biofilms.

(2) A paper-based dual-signal ATP detection system based on ATP responsive cleavage of ChoA@ZIF-90 nanoparticles was designed, using the property of ATP-specific cleavage of ZIF-90 to release the encapsulated chitosanase, which cleaves chitosan hydrogels to produce solution viscosity changes. In addition, the cleavage of ZIF-90 releases the catalytic activity of the nanoenzyme, which is used to produce color changes. The proposed method is portable, rapid, and instrument-free for detection of ATP, and the detection are displayed directly on the filter paper results with distance and colorimetric readings, enabling qualitative on-site detection and quantitative visual measurement of ATP within microorganisms and biofilms.

(3) A novel impedimetric aptasensor was designed for the discriminative detection of intracellular and extracellular ATP using antimicrobial peptide-functionalized magnetic particles (M-AMPs). For discriminative intracellular and extracellular ATP detection, magnetically controlled M-AMPs were introduced into the detection system for mild and rapid bacterial disintegration. The intracellular and intracellular ATP levels were obtained via the difference of charge transfer resistance before and after M-AMPs release. The proposed aptasensor exhibited distinguished sensitivity and selectivity, which enabled the discriminative intracellular and extracellular ATP detection in various corrosive microorganism.

(4) A nanoplatform based on dsDNA-ZIF-90@Ag₃AuS₂@Fe₃O₄ nanocomposites was established for the simultaneous detection of ADP and ATP, which was integrated with an ADP sensitive module based on aptamer recognition and DNA-activated CRISPR/Cas12a amplification, and an ATP responsive module based on ZIF-90 nanocarrier fragmentation and quencher loading release. The absolute concentrations of nucleotides rather than the ADP/ATP ratio were successfully determined using the developed biosensor, which was helpful to exclude the interference of ADP during ATP detection. The proposed biosensor enabled accuracy detection of ADP and ATP, thus the nanoplatform provides a promising method for ultrasensitive dual-mode quantification of ADP and ATP in SRB biofilms.

(5) A novel biofilm-targeted nanocontainer with successive responsive property towards pH and ATP for precise localization and simultaneous bacterial eradication. The biofilm-targeted nanocontainer was composed of a ATP-responsive zeolitic imidazolate framework-90 (ZIF-90) core encapsulated in the pH-responsive amorphous calcium carbonate/poly(acrylic acid) (ACC/PAA) shell. Moreover, to meet the application requirements of different biofilm scenarios, the pH response ability of the nanocontainers could be adjusted by changing the metallic ions doped into the structure of the ACC/PAA shell. The precise localization and simultaneous bacterial eradication were successfully realized via a simple spray process and embedding in self-defensive coatings. The nanocontainer endowed the nanocontainers high precision for localization and simultaneous eradication of SRB biofilms.

(6) A dual-readout viscosity flow sensor based on cyt c-triggered hyaluronidase (HAase) release was designed for on-site detection. The probe was fabricated using UiO-67-NH₂ loaded with HAase as a nanocarrier, and the double-stranded DNA (dsDNA) as a gatekeeper. Release of HAase to lyse hyaluronic acid hydrogels was triggered by cyt c recognition,,and the changes in the diffusion distance and the color signal were used to quantify the biotarget concentration. The proposed sensor avoids expensive instruments and sophisticated operation procedures, making it practical for on-site cyt c detection. This sensor exhibited excellent specificity and reproducibility for detection of cyt c, enabling accurate detection of cyt c concentration in SRB biofilm.

(7) A novel strategy based on target-modulated competitive binding and exonuclease I (Exo I)-powered signal molecule release was established for simultaneous multiple-target detection. UiO-67 nanocarriers with high signal molecule loading characteristics were designed to release signal molecules using the specific recognition process of target molecules, and Exo I was introduced to accelerate the release of signal molecules. With the advantages of rapid response and high selectivity and sensitivity, this strategy provides guidance for simultaneous detection of ATP and cyt c in a variety of bacteria, including SRB.

第1章 绪论 1

1.1 海洋微生物腐蚀概述 1

1.1.1 微生物腐蚀概述 1

1.1.2 海洋腐蚀微生物 5

1.2 硫酸盐还原菌种群浓度的检测方法 8

1.2.1 传统培养法 9

1.2.2 聚合酶链式反应法 9

1.2.3 荧光法 10

1.2.4 代谢物检测法 11

1.2.5 生物传感器法 12

1.3 硫酸盐还原菌代谢活性的检测方法 15

1.3.1 培养法 15

1.3.2 活性微电极法 15

1.3.3 活性指示分子检测法 15

1.4 活性指示分子的检测方法 18

1.4.1 活性指示分子ATP的检测 18

1.4.2 活性指示分子cyt c的检测 23

1.5 选题依据 27

1.5.1 选题依据 27

1.5.2 研究内容 28

第2章 基于ZIF-90响应裂解和模拟酶活性抑制特性的生物膜ATP双信号检测体系 31

2.1 前言 31

2.2 实验部分 31

2.2.1 材料与仪器 31

2.2.2 纳米颗粒制备 32

2.2.3 抑制性能测定 32

2.2.4 ATP双信号检测 32

2.2.5 生物膜ATP双信号检测 33

2.3 结果与讨论 33

2.3.1 检测原理 33

2.3.2 纳米颗粒表征 34

2.3.3 抑制性能测定 34

2.3.4 ATP双信号检测性能 37

2.3.5 生物膜ATP双信号检测 40

2.4 本章小结 43

第3章 基于流动扩散的生物膜ATP纸基传感器 45

3.1 前言 45

3.2 实验部分 46

3.2.1 材料与仪器 46

3.2.2 纳米颗粒制备 47

3.2.3 水凝胶浓度选择 47

3.2.4 ATP检测 47

3.2.5 纸基传感器的应用 47

3.3 结果与讨论 48

3.3.1 纳米颗粒表征 48

3.3.2 ATP检测可行性 49

3.3.3 ATP检测性能 51

3.3.4 选择性、重现性和稳定性 54

3.3.5 纸基试剂盒 54

3.4 本章小结 56

第4章 基于磁驱抗菌肽的生物膜中细胞内外ATP区分鉴别检测体系 57

4.1 前言 57

4.2 实验部分 58

4.2.1 材料与仪器 58

4.2.2 适配体传感器制备 58

4.2.3 细菌培养 58

4.2.4 ATP检测 58

4.2.5 细胞内和细胞外ATP鉴别检测 59

4.3 结果与讨论 59

4.3.1 检测原理 59

4.3.2 适体传感器表征 60

4.3.3 纳米颗粒表征 61

4.3.4 ATP检测性能 63

4.3.5 细胞内和细胞外ATP鉴别检测 64

4.4 本章小结 66

第5章 基于CRISPR/Cas12a协同ZIF-90识别平台的生物膜ADP和ATP差异化检测体系 67

5.1 前言 67

5.2 实验部分 68

5.2.1 材料与仪器 68

5.2.2 纳米颗粒制备 69

5.2.3 ADP检测 69

5.2.4 ATP检测 69

5.2.5 生物膜ADP和ATP差异化检测 70

5.3 结果与讨论 70

5.3.1 检测原理 70

5.3.2 纳米颗粒表征 71

5.3.3 ADP和ATP检测可行性 76

5.3.4 ADP和ATP检测性能 78

5.3.5 ADP和ATP差异化检测 81

5.3.6 生物膜ADP和ATP差异化检测 83

5.4 本章小结 84

第6章 基于pH和ATP连续响应的生物膜精确定位及清除体系 85

6.1 前言 85

6.2 实验部分 86

6.2.1 材料与仪器 86

6.2.2 纳米颗粒制备 86

6.2.3 纳米颗粒的响应释放 87

6.2.4 细菌培养 87

6.2.5 纳米颗粒的抗菌性能 88

6.2.6 纳米颗粒的生物膜精确定位及清除 88

6.3 结果与讨论 89

6.3.1 检测原理 89

6.3.2 纳米颗粒表征 89

6.3.3 纳米颗粒的响应释放 93

6.3.4 纳米颗粒的抗菌性能 98

6.3.5 纳米颗粒的生物膜精确定位及清除 99

6.4 本章小结 102

第7章 基于流动扩散的生物膜cyt c双读数粘度流量试纸条 103

7.1 前言 103

7.2 实验部分 104

7.2.1 材料与仪器 104

7.2.2 纳米颗粒制备 105

7.2.3 Cyt c检测 105

7.2.4 生物膜cyt c检测 105

7.2.5 数据分析 106

7.3 结果与讨论 106

7.3.1 纳米颗粒表征 106

7.3.2 Cyt c检测可行性 107

7.3.3 Cyt c检测性能 110

7.3.4 选择性、重现性和稳定性 112

7.3.5 生物膜cyt c检测 113

7.4 本章小结 114

第8章 基于靶标竞争结合和Exo-I酶驱动的生物膜ATP和cyt c同时检测体系 117

8.1 前言 117

8.2 实验部分 118

8.2.1 材料与仪器 118

8.2.2 纳米颗粒制备 118

8.2.3 ATP和cyt c检测可行性 119

8.2.4 ATP和cyt c同时检测 119

8.2.5 生物膜ATP和cyt c同时检测 119

8.3 结果与讨论 119

8.3.1 检测原理 119

8.3.2 纳米颗粒表征 120

8.3.3 ATP和cyt c检测可行性 121

8.3.4 ATP和cyt c检测性能 124

8.3.5 ATP和cyt c同时检测 127

8.3.6 生物膜ATP和cyt c同时检测 129

8.4 本章小结 130

第9章 总结与展望 131

9.1 结论 131

9.2 创新点 132

9.3 展望 132

参考文献 135

致 谢 155

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 157