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基于光学纳米材料的海洋腐蚀微生物快速检测方法
郑来宝
Subtype博士
Thesis Advisor张盾
2019-05-15
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Keyword海洋腐蚀微生物 快速检测 光学纳米材料 识别分子
Abstract

海洋腐蚀微生物的快速检测对微生物腐蚀机理及早期微生物腐蚀防护的研究具有重要的指导意义。本研究以海洋腐蚀微生物-硫酸盐还原菌(SRB)为主要检测目标,通过制备先进的光学纳米材料,结合合适的生物识别分子,分别构建了基于苯硼酸识别的银纳米颗粒的比色传感器、基于脱氧核酶识别的银纳米簇荧光传感器和基于GSH-Au(Ⅰ)-Pb(Ⅱ)配合物的荧光传感器和基于多重识别的碳点荧光阵列传感器,建立了对海洋腐蚀微生物的高灵敏度的快速检测方法。该研究的开展对海洋腐蚀微生物的检测及海洋微生物腐蚀的机理研究具有重要意义。主要研究结果如下:

1)构建了一种基于苯硼酸识别的银纳米颗粒的简单、快速、价格低廉的比色微生物检测方法。该检测方法利用细菌表面存在的邻二羟基基团可与苯硼酸功能化银纳米颗粒共价结合,从而抑制由过量巯基苯硼酸诱导的苯硼酸功能化银纳米颗粒的聚集,从而使其产生不同的颜色变化。通过肉眼观察、智能手机拍照或者紫外可见吸收光谱仪可实现对细菌的快速检测。结果证明,苯硼酸功能化银纳米颗粒在400 nm处的吸光度与SRB的浓度在5.0×106 cfu·mL-15.0×107 cfu·mL-1之间存在良好的线性关系。同时,整个检测过程可在20 min内完成。

2)构建了一种基于脱氧核酶识别的银纳米簇荧光传感器用于微生物的高灵敏度检测。该检测体系,以磁性微球为载体,以脱氧核酶为识别分子,引入乙酰胆碱酯酶,构建了一种MNP-DNAzyme-AChEMDA)复合物。在目标细菌裂解液存在下,脱氧核酶可与其特异性结合,并发生催化裂解,使连接其上的乙酰胆碱酯酶,从MDA复合物中脱离进入溶液。利用乙酰胆碱酯酶的催化产物,使DNA银纳米簇的荧光信号发生增强。该荧光传感平台表现出了高特异性和高灵敏度,其最低检测限为60 cfu·mL-1,线性检测范围为1.0×102-1.0×107 cfu·mL-1。整个检测过程可在2 h内完成。通过变换该检测体系中的脱氧核酶,可实现对特定海洋腐蚀微生物的高特异性、高灵敏度检测,为海洋腐蚀微生物的快速检测提供了新的策略方法。

3)构建了一种基于GSH-Au(Ⅰ)-Pb(Ⅱ)配合物的荧光传感器,其可对SRB代谢产物S2-产生特异性响应,在S2-存在下其可发生荧光猝灭。本研究基于Pb2+诱导的GSH-Au(Ⅰ)聚集诱导发光,以及S2-导致荧光猝灭现象,开发了一种用于检测SRB的荧光传感器。该方法显示出良好的线性检测范围和极好的选择性。鉴于所提出的荧光平台的快速和简便性,该方法在SRB的快速检测中具有潜在的应用价值。

4)构建了一种基于苯硼酸、多粘菌素和万古霉素功能化碳点的荧光阵列传感器用于海洋腐蚀微生物的识别、鉴定。由于不同细菌表面的物理化学性质的多样性,不同碳点对细菌具有不同的结合能力,因此该荧光阵列传感器对不同细菌产生不同程度的荧光信号响应。该荧光阵列传感器可以简单、快速、有效地实现对包括SRB和铜绿假单胞菌等海洋腐蚀微生物在内的6种细菌的准确识别。

Other Abstract

The detection of corrosive microorganisms is of great significance for studying the microbial corrosion mechanism and timely taking microbial corrosion protection measures. In this work, a phenylboronic acid functionalized silver nanoparticles based colorimetric sensor, a DNAzyme-based fluorescence sensor and a GSH-Au(Ⅰ) complex based fluorescence sensor and a three receptors-functionalized carbon dots based fluorescence sensor array have been established for sensitive and rapid detection of marine corrosive microorganisms. Main research work is as follows:

(1) A simple, rapid and cost-effective colorimetric bacterial detection strategy based on MPBA-AgNPs has been demonstrated. The as-prepared MPBA-AgNPs would be easily aggregated with the addition of excess MPBA. However, the bacteria cells containing cis-diol groups could absorb MPBA-AgNPs onto their surfaces to keep the MPBA-AgNPs dispersed and anti-aggregated, resulting in the various color changes which could be monitored by the naked eye and UV-vis spectrometer. There was a good linear relationship between the absorbance intensity at 400 nm and SRB concentration from 5.0×106 cfu·mL-1 to 5.0×107 cfu·mL-1. And the total detection procedure could be finished within 20 minutes. Though semi-quantitative analysis could be realized by the color change which could be distinguished with the naked eye, smartphone based measuring mean makes it suitable for routine laboratory applications. Moreover, the concept of the MPBA-AgNPs based colorimetric strategy can be expanded to other Gram-negative bacteria including corrosive microorganisms suggesting a universal platform for corrosive microorganisms assay.

(2) A novel DNA-templated fluorescent silver nanoclusters based sensing system was developed for pathogenic bacterial detection integrated with DNAzyme as the recognition molecule. In the sensing platform, a MNP-DNAzyme-AChE (MDA) complex was constructed, where magnetic nanoparticles plays the role of a carrier, DNAzyme acts as the recognition molecule and a bridge to linke to the acetylcholinesterase. In the presence of the target bacterial lysate, the MDA-target complex was formed through the reaction between DNAzyme and the target molecules lysed by bacteria. And thereby the DNAzyme substrate could be cleaved into two pieces to release AChE into the solution to catalyze the hydrolysis of ATCh to produce TCh, which could significantly enhance the fluorescence of DNA-AgNCs. The detection method has an ultrasensitive detection limit of 60 cfu·mL-1 with a broad linear range from 1.0×102 to 1.0×107 cfu·mL-1, and good specificity to the target bacteria. The total detection procedure could be finished within 2 days. The proposed fluorescence detection method can be expanded to the specific detection of corrosive microorganisms by simply replacing with the DNAzyme specific to the certain corrosive microorganisms.

(3) Au(Ⅰ)-thiolate complexes exhibit AIE property with the presence of Pb2+ and its fluorescence could be quenched by S2-. Herein, for the first time, the S2- responsive fluorescence turn off phenomenon based on Pb2+ induced aggregation of AIE-active GSH-Au(Ⅰ) complexes was used to develop a fluorescence sensor for detection of SRB, which could generate S2- through reducing SO42-. The proposed method shows good liner detection ranges and satisfactory selectivity. Given the simplicity and timesaving of the proposed fluorescence platform, we expect it could be used for the rapid detection of SRB in the environmental analysis.

(4) A novel fluorescent sensor array was designed for identification of bacteria based on three kinds of facile synthesized CDs functionalized with boronic acid, polymixin and vancomycin. The CDs displayed different binding abilities to bacteria due to the variety of physicochemical nature of different bacteria surfaces. The generated fluorescence pattern allowed for the good discrimination of the six kinds of bacteria. The proposed sensor array also show satisfactory discrimination ability in bacteria mixture and real samples. Thus, our proposed sensor array provides a new strategy for simple, rapid, and effective identification of bacteria including SRB and P. aeruginosa.

Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156788
Collection海洋环境腐蚀与与生物污损重点实验室
Recommended Citation
GB/T 7714
郑来宝. 基于光学纳米材料的海洋腐蚀微生物快速检测方法[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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