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新型光学生物传感方法的构建及其在环境检测中的应用
殷堃
Subtype博士
2016-05-30
Degree Grantor中国科学院大学
Place of Conferral北京
Keyword生物材料 光学传感 比色检测 荧光检测 环境分析
Abstract光学生物传感方法因其出色的特异性以及优良的生物相容性等诸多优点,广泛应用于环境污染分析和生物分析等领域。在本文中,我们专注于开发具有优良光学响应的智能生物材料以及基于生物材料的光学化学反应来构建新颖的光学生物传感方法用于典型环境污染物的分析。这些生物传感方法拥有出色的光学响应性能,可以把对目标物的识别过程转化为简单易读的光学信号,进而实现对目标物的检测。本文的详细内容如下:
1.      基于荧光铁载体特异性检测铜离子。在铜离子的存在下,荧光铁载体的荧光会因为电荷转移以及铜离子的重金属效应而发生猝灭。在0.2-10 μM的铜离子的浓度范围内,荧光铁载体的荧光猝灭率和铜离子的浓度有着良好的线性关系(R = 0.997),检出限为50 nM。该光学生物传感方法成功地用于饮用水、海水和生物加标样品中铜离子的检测,并与传统的电感耦合等离子体质谱方法获得的数据相吻合。因此,构建的光学生物传感方法可以简单、特异性地检测实际样品中的铜离子。
2.      基于荧光铁载体检测呋喃唑酮。呋喃唑酮上的硝基官能团是一个强吸电子基团,可诱导电子转移而作为一种荧光猝灭剂。呋喃唑酮分子可能比较容易进入到荧光铁载体分子结构的空腔中,进而高效猝灭荧光铁载体的荧光。在EDTA的掩蔽下,该体系可以有效避免铜离子的干扰。基于此,构建了检测呋喃唑酮的光学生物传感方法,该生物传感方法可以在1分钟内完成对呋喃唑酮的检测。在2-160 μM的呋喃唑酮的浓度范围内,荧光铁载体的荧光猝灭率和呋喃唑酮的浓度有着良好的线性关系(R = 0.997),检出限为0.5 µM。该工作开发了第一个检测呋喃唑酮的荧光生物传感方法。该荧光生物传感方法可以快速灵敏的检测加标环境水体样品中的呋喃唑酮。
3.      基于催化氧化半胱氨酸特异性检测二价铜离子。在特定条件下,半胱氨酸可以与2,4-二硝基氯苯发生亲核反应并生成黄色产物。在氧气的存在下,二价铜离子可以特异性地催化氧化半胱氨酸生成胱氨酸,进而阻止了上述反应的发生。因此,黄色产物吸光度的降低可以反映溶液中二价铜离子的浓度。基于此,构建了一个检测二价铜离子的光学生物传感方法。在0.8-10 nM二价铜离子的浓度范围内,黄色产物吸光度的降低和二价铜离子的浓度有着良好的线性关系(R = 0.996)。检出限为0.5 nM。该生物传感方法成功地用于饮用水、海水和生物样品中铜离子的检测,并与传统的电感耦合等离子体质谱获得的数据相吻合。因此,构建的光学生物传感方法可以高灵敏、特异性地检测二价铜离子。
4.      基于羧酸酯酶E2的表面展示体系特异性吸附和检测汞离子。铜绿假单胞菌PA1作为汞离子耐受菌具有良好的汞离子吸附能力。研究发现该菌株分泌的羧酸酯酶E2也可以有效的吸附汞离子,而且在吸附汞离子后酯酶E2的酶活性会被抑制。因此我们将羧酸酯酶E2表达在大肠杆菌的表面来吸附和检测汞离子。实验结果表明,该表面展示体系可以有效地吸附环境中的汞离子,并可以通过调节pH使体系再生,得以重复利用。此外,该体系也可以有效地对环境加标样品中的汞离子进行检测。因此,该表面展示体系对环境中汞污染的修复与检测有着巨大的应用前景。
5.      基于近红外比率荧光探针Cy-NB来特异性地检测线粒体内半胱氨酸的浓度水平,并用于评估环境生物毒素胁迫下细胞及活体水平上的氧化应激状态,进而反映环境生物毒素。半胱氨酸作为细胞应对氧化应激的重要防御工具之一,它的浓度水平和细胞内的氧化应激状态息息相关。在半胱氨酸的存在下,探针Cy-NB的共轭电子体系会发生重排,导致了吸收光谱和荧光光谱的明显变化,进而实现了对半胱氨酸的检测。作为一个近红外比率型荧光探针,Cy-NB对线粒体内半胱氨酸的检测不受生物体自发荧光的干扰,因此有着较高的灵敏度。同时探针Cy-NB可以在五分钟内实现对半胱氨酸的检测。因此,探针Cy-NB可以敏感地检测到氧化胁迫状态下线粒体内半胱氨酸浓度水平的波动。此外探针Cy-NB还成功检测到了环境生物毒素刺激下,小鼠腹腔内半胱氨酸浓度水平的变化。因此,探针Cy-NB对于检测环境生物毒素导致的氧化胁迫,从而反映环境生物毒素的存在以及进一步探究半胱氨酸生理功能等方面具有广泛的应用前景。
6.      基于近红外荧光探针Cy-DES检测己烯雌酚。该探针可以有效地避免海水中可溶性有机物自发荧光的干扰。由于探针Cy-DES和己烯雌酚分子间的静电作用而发生分子间的集聚,进而探针Cy-DES的吸收光谱和荧光光谱性质发生了明显的改变。在1-8 μM己烯雌酚的浓度范围内,探针Cy-DES光谱性质的改变和己烯雌酚的浓度之间有着良好的线性关系(R = 0.999),检出限为0.2 μM。该检测方法成功地用于检测海水和生物样品中的己烯雌酚,并和传统的高效液相色谱法获得的数据相吻合。此外,肉眼可以直接通过观察到己烯雌酚存在下探针Cy-DES颜色的变化,因此该分析方法在己烯雌酚的现场检测方面有着巨大的应用前景。
Other AbstractOptical biosensors have been widely developed for the convenient detection of environmental pollutions and biological analysis, attributing to their outstanding advantages of high specificity and excellent biocompatibility. In this paper, we focused on the development of new biomaterials, new bio-reaction and new biological sensing strategies to establish novel biosensors to environmental analysis. These biosensors owned satisfied optical response performance, which could be utilized to transform the recognition behavior of specific targets to optical signals and achieve the detection of target objects. The detail contents of this dissertation are listed as follows:
1.      A sensitive fluorescent biosensor for the detection of copper ion inspired by biological recognition element pyoverdine
In this work, we have developed a fluorescent biosensor based on biological recognition element pyoverdine to selectively detect copper ion. The fluorescence of pyoverdine is quenched obviously after binding with copper ion. A good linearity within the range of 0.2–10 μM (R=0.997) is attained and the detection limit is 50 nM. The biosensor has been successfully utilized for the detection of copper ion in drinking water, seawater and bio-samples and the results agree well with those obtained by the inductively coupled plasma mass spectrometry. Therefore, the established biosensor is a creditable method to detect copper ion with high sensitivity and selectivity, which can be utilized as a powerful tool to monitor copper pollution in the environment.
2.      Pyoverdine secreted by Pseudomonas aeruginosa as a biological recognition element for the fluorescent detection of furazolidone
In this work, a method based on the rapid fluorescence quenching effect of furazolidone to pyoverdine for the detection of furazolidone was developed. Pyoverdine secreted by a Pseudomonas aeruginosa strain PA1 was purified through affinity chromatography and its fluorescent property was characterized. The fluorescence of pyoverdine could be specially quenched by furazolidone, and based on this phenomenon a fluorescent method to detect furazolidone was established. Fluorescence of pyoverdine was quenched by furazolidone due to the electrons transfer from pyoverdine to furazolidone. The optimal pH for the detection was 7.2 in 50 mM 3–(N–Morpholino) propanesulfonic acid solution, and the whole detection process could be completed within seconds. The linear range of the detection was 2–160 µM and the limit of detection (LOD) was 0.5 µM. This study was the first time to develop a fluorescent biosensor for furazolidone detection, and the rapid and specific fluorescent method can be potentially applied for furazolidone detection in the environmental aquatic samples.
3.      Ultrasensitive colorimetric detection of Cu2+ ion based on catalytic oxidation of L-cysteine
In this work, a simple, facile colorimetric sensor for the ultrasensitive determination of Cu2+ ion was developed based on the following principle: L-cysteine and 1-chloro-2,4-dinitrobenzene (CDNB) could be conjugated to form the yellow product 2,4-dinitrophenylcysteine (DNPC), which was measurable at absorbance of 355 nm; however, upon addition of Cu2+ ion, the absorbance of DNPC would be decreased owing to the Cu2+ ion catalytic oxidation of L-cysteine to L-cystine in the presence of O2. Thus, the colorimetric detection of Cu2+ ion could be achieved. The optimal pH, buffer, temperature and incubation time for the colorimetric sensor were obtained of pH 6.8 in 0.1 M HEPES solution, 90 oC and 50 min, respectively. A good linearity within the range of 0.8–10 nM (R=0.996) was attained, with a high detectability up to 0.5 nM. Analyses of Cu2+ ion in drinking water, lake water, seawater and biological samples were carried out and the method performances were found to agree well with that obtained by ICP-MS. The developed simple colorimetric sensor proved applicable for Cu2+ ion determination in real samples with high sensitivity and selectivity.
4.      Simultaneous bioremediation and biodetection of mercury ion through surface display of carboxylesterase E2 from Pseudomonas aeruginosa PA1
In this work, carboxylesterase E2 from mercury-resistant strain Pseudomonas aeruginosa PA1 has been successfully displayed on the outer membrane of E. coli Top10 bacteria to simultaneously adsorb and detect mercury ion. The transmission electron microscopy analysis shows that mercury ion can be absorbed by carboxylesterase E2 and accumulated on the outer membrane of surface-displayed E. coli bacteria. The adsorption of mercury ion is followed by a physicochemical, equilibrated and saturatable mechanism, which well fits the traditional Langmuir adsorption model. And the surface-displayed system can be regenerated by regulating pH values. In addition, the activity of carboxylesterase E2 can be inhibited by mercury ion, which has been successfully utilized to detect mercury ion. Therefore, the developed surface display system is of great potential in the simultaneous bioremediation and biodetection of environmental mercury pollution.
5.      A near-infrared ratiometric fluorescent probe for cysteine detection over glutathione indicating mitochondrial oxidative stress in vivo
In this work, we establish a near-infrared (NIR) ratiometric fluorescent probe Cy-NB for the selective detection of cysteine (Cys) over glutathione (GSH) and homocysteine (Hcy) in mitochondria to indicate oxidative stress. Heptamethine cyanine dye is chosen as the fluorophore of Cy-NB whose emission locates in NIR region. And p-nitrobenzoyl is employed as the fluorescent modulator due to its capability of selective-Cys response. Once triggered by Cys, the uncaged p-nitrobenzoyl rearranges the polymethine π-electron system of the fluorophore, which leads to a remarkable spectrum shifts in absorption and emission profiles. Taking advantage of these spectroscopic properties, we construct a ratiometric fluorescent signal for the detection of Cys with a detection limit of 0.2 µM within 5 min. Our probe Cy-NB can sensitively detect the mitochondrial Cys pool changes under different oxidative stress status in HepG2 cells. We also successfully employ Cy-NB to imaging Cys level changes in living mice. It suggests that mitochondrial Cys can be used as an oxidative stress biomarker with simple potential clinical applications. And our probe Cy-NB is of great potential for further utilizing in exploring the physiological function of Cys in biological systems.
6.      Cyanine-based colorimetric and fluorescent probe for the selective detection of diethylstilbestrol in seawater, shrimp and fish samples
In this work, we have developed a new colorimetric and fluorescent probe Cy-DES for the detection of DES with high sensitivity and selectivity. As a near-infrared probe, Cy-DES is able to avoid autofluorescence of dissolved organic compounds and maximize signal-to-background contrast. Taking advantage of the strong electrostatic interaction between the probe Cy-DES and DES, the spectroscopic properties of probe Cy-DES can be obviously changed in presence of DES. Under testing conditions, there is an excellent linearity within the range of 1 - 8 μM (r = 0.9997) and the detection limit is 0.2 μM. The probe Cy-DES is successfully applied for the detection of DES in spiked seawater, shrimp and fish samples. Additionally, the detection of DES can be directly achieved by naked eyes with the utilizing of probe Cy-DES. The developed method is of great potential for application in the on-site detection of DES.
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/13833
Collection中科院烟台海岸带研究所知识产出_学位论文
Affiliation中国科学院烟台海岸带研究所
First Author AffilicationYantai Institute of Coastal Zone Research, Chinese Academy of Sciences
Recommended Citation
GB/T 7714
殷堃. 新型光学生物传感方法的构建及其在环境检测中的应用[D]. 北京. 中国科学院大学,2016.
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