气味结合蛋白生物传感器研究
发布时间：2019-05-28 15:49
【摘要】：生物的嗅觉系统能够灵敏、特异地检测和识别复杂环境中痕量的气味分子,是一种性能优越的化学感受和检测系统。在生物嗅觉的启发下,研究者们一直致力于在体外模仿生物体的嗅觉感知能力,以期实现构建高灵敏、高特异性的嗅觉生物传感器,拓展其在环境监测、食品检测,及疾病诊断等领域的应用及发展。但是,目前常用的生物敏感材料(嗅觉组织、嗅觉细胞及嗅觉受体)存在着获取困难、活性较难保持等问题,限制了嗅觉传感器的进一步发展。气味结合蛋白(odorant-bindingproteins,OBPs)作为一种胞外嗅觉蛋白,具有良好的气味分子结合能力,同时其稳定性较好,且易于实现体外表达和纯化。因此,气味结合蛋白传感器对于嗅觉生物传感的研究具有重要的现实意义和应用前景。本论文以中华蜜蜂气味结合蛋白(ASP2)、桔小实蝇气味结合蛋白(BdorOBP2),及人气味结合蛋白(OBP2a和OBP2b)为研究实例,结合不同的电化学检测技术及生物敏感元件固定方法,完成了多种气味结合蛋白传感器的设计和构建,实现了对化学信息素等气味分子的检测。结合电化学阻抗模型和分子对接技术,探讨了气味结合蛋白与气味分子之间的相互作用,分析了气味结合蛋白传感器的应用及发展。论文的主要内容及创新点如下:1.设计并构建了昆虫气味结合蛋白传感器,实现了对化学信息素的检测。昆虫具有非常灵敏的嗅觉系统,这与其气味结合蛋白结合气味分子的能力密切相关。本论文以中华蜜蜂气味结合蛋白(ASP2)和桔小实蝇气味结合蛋白(BdorOBP2)为研究实例,设计并构建了昆虫气味结合蛋白传感器。首先成功表达和纯化了这两种气味结合蛋白,然后利用物理吸附和层层自组装的方法将其固定于叉指电极表面。通过电化学阻抗技术,该传感器实现了对不同化学信息素的检测,而且相较于其他类型气味结合蛋白传感器,其具有较宽的检测范围、较好的检测灵敏度和检测下限。2.基于气味结合蛋白三维结构,设计了电化学阻抗模型,结合分子对接技术分析了气味结合蛋白与化学信息素的相互作用。基于气味结合蛋白结合气味分子的主要场所是其结构中心的疏水腔体,并且结合和释放气味分子的过程是需要蛋白构象的改变来实现的这一机理,本论文构建了昆虫气味结合蛋白检测化学信息素的电化学阻抗模型,包括蛋白骨架和中心疏水腔两部分,进而对气味结合蛋白构象改变与传感器阻抗变化之间的关系进行了分析。同时结合分子对接的结果,分析了气味结合蛋白的三级结构中关键氨基酸残基在结合化学信息素过程中所发挥的作用,这为研究气味结合蛋白的特异性传感结合能力奠定了基础。3.设计并构建了人气味结合蛋白传感器,实现了对不同链长脂肪酸分子的检测。脂肪酸是生物体的重要组成和储能物质,也是一种重要的亲脂性物质。本论文利用人气味结合蛋白对脂肪酸具有较好亲和力的这一特性,设计了一种用于脂肪酸分子检测的人气味结合蛋白OBP2a传感器。利用电化学还原的方法将氧化石墨烯还原为还原型氧化石墨烯并将其固定于印刷电极的碳工作电极表面,在增加电极导电性的同时,利用残留的含氧官能团将气味结合蛋白固定于传感器表面。利用循环伏安检测技术,该传感器实现了对二十二碳六烯酸、亚油酸,及月桂酸的测量。检测结果表明人气味结合蛋白对中、长链脂肪酸具有较好的结合能力,可用于分析不同碳链长的脂肪酸物质。4.利用阳极氧化铝纳米孔阵列,设计并构建了气味结合蛋白传感器,实现了对苯甲醛、脂肪酸等分子的检测。阳极氧化铝纳米孔阵列是一种具有纳米尺寸孔道的滤膜材料,其具有比表面积大的特点,已经广泛应用于溶液中目标物质的高灵敏检测。本论文基于人气味结合蛋白OBP2b对苯甲醛等气味分子的结合能力,利用三电极体系,构建了基于阳极氧化铝纳米孔阵列的生物传感器。将修饰有气味结合蛋白的阳极氧化铝纳米孔阵列置于工作电极与参比/对电极之间,使得纳米孔阵列成为电化学检测体系中电流及溶液流通的唯一通路。在苯甲醛及脂肪酸的作用下,固定于纳米孔阵列上的气味结合蛋白构象发生改变,进而引起纳米孔中电流的变化。通过电化学阻抗检测,实现了对不同浓度苯甲醛及脂肪酸的检测。
[Abstract]:The biological olfactory system can be used to detect and identify trace amount of odorous molecules in complex environment, which is an excellent chemical sensing and detection system. Inspired by the sense of biological smell, the researchers have been working on the ability to mimic the sense of smell of living organisms in vitro, with a view to achieving a high-sensitivity and high-specific sense of smell biosensor, and to expand its application and development in the fields of environmental monitoring, food detection and disease diagnosis. However, the most commonly used biosensitive materials (olfaction, olfactory and olfactory receptors) are difficult to obtain, and the activity is difficult to maintain, which limits the further development of the olfactory sensor. Odor-bindingprotein (OBPs), as an extracellular olfactory protein, has good odor-binding ability, and its stability is good, and it is easy to realize in vitro expression and purification. Therefore, the smell-binding protein sensor has important practical significance and application prospect for the study of the sense of smell and biological sensing. In this paper, the odor-binding protein (ASP2), the odor-binding protein (BdorOBP2) and the human odor-binding protein (OBP2a and OB2b) of the Chinese bee-odor-binding protein (ASP2) and the human odor-binding protein (OBP2a and OB2b) are used as the research examples, and the different electrochemical detection technology and the method for fixing the biological sensing element are combined. The design and construction of a plurality of odor-binding protein sensors are completed, and the detection of the odor molecules such as the chemical pheromone and the like is realized. Combined with the electrochemical impedance model and the molecular docking technology, the interaction between the odor-binding protein and the odor molecule is discussed, and the application and development of the smell-binding protein sensor are analyzed. The main content and innovation point of the thesis are as follows:1. The insect-odor-binding protein sensor is designed and constructed, and the detection of the chemical information is realized. The insect has a very sensitive olfactory system, which is closely related to the ability of the odor-binding protein to bind to the odor molecule. In this paper, an insect-odor-binding protein sensor was designed and constructed with the odor-binding protein (ASP2) of the Chinese bee and the odor-binding protein (BdorOBP2) of the fruit fly. The two kinds of odor-binding proteins are successfully expressed and purified, and then are fixed on the surface of the interdigital electrode by means of physical adsorption and layer-by-layer self-assembly. Through the electrochemical impedance technology, the sensor realizes the detection of different chemical pheromones, and has wider detection range and better detection sensitivity and lower detection limit compared with other types of odor-binding protein sensors. The electrochemical impedance model was designed based on the three-dimensional structure of the odor-binding protein, and the interaction between the odor-binding protein and the chemical information was analyzed by means of the molecular docking technique. The main site based on the odor-binding protein-binding odor molecule is the hydrophobic cavity of its structure center, and the process of binding and releasing the odor molecules is the mechanism that requires a change in the protein conformation, In this paper, the electrochemical impedance model of the insect-odor-binding protein was constructed, including the two parts of the protein framework and the central hydrophobic cavity, and then the relationship between the change of the conformation of the odor-binding protein and the change of the impedance of the sensor was analyzed. At the same time, the role of the key amino acid residues in the three-stage structure of the odor-binding protein in the process of binding to the chemical information is analyzed by the result of the molecular docking, which lays a foundation for the study of the specific sensing and binding capacity of the odor-binding protein. The human odor-binding protein sensor is designed and constructed, and the detection of the fatty acid molecules with different chain lengths is realized. The fatty acid is an important component of an organism and an energy-storing substance, and is also an important lipophilic substance. In this paper, a human odor-binding protein OBP2a sensor for fatty acid molecule detection was designed by using human odor-binding protein to have a good affinity for fatty acid. The oxidized graphene is reduced to the reduced oxidized graphene by the method of electrochemical reduction and is fixed on the surface of the carbon working electrode of the printing electrode, and the odor-binding protein is fixed on the surface of the sensor by the residual oxygen-containing functional group at the same time of increasing the conductivity of the electrode. The sensor realizes the measurement of docosahexaenoic acid, linoleic acid, and lauric acid by using cyclic voltammetry. The results show that the human odor-binding protein pair has better binding ability to long-chain fatty acid, and can be used for analyzing the fatty acid material with different carbon chain length. An odor-binding protein sensor was designed and constructed with an anode alumina nanopore array, and the detection of the molecules such as benzaldehyde, fatty acid and the like was realized. Anodic alumina nanopore array is a kind of filter membrane material with nano-size channel, which has the characteristics of large specific surface area, and has been widely used in high sensitive detection of target substances in solution. Based on the binding capacity of human odor-binding protein OBP2b to the odor molecules such as benzaldehyde and the like, a biosensor based on the anodic aluminum oxide nanopore array was constructed by means of the three-electrode system. The anode aluminum oxide nano-hole array modified with the odor-binding protein is arranged between the working electrode and the reference/ counter electrode, so that the nano-hole array is the only path of the current and the solution flow in the electrochemical detection system. Under the action of benzaldehyde and fatty acid, the conformation of the odor-binding protein fixed on the nano-hole array changes, thus causing the change of the current in the nano-hole. And the detection of the benzaldehyde and the fatty acid at different concentrations is realized through the electrochemical impedance detection.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TP212.3
[Abstract]:The biological olfactory system can be used to detect and identify trace amount of odorous molecules in complex environment, which is an excellent chemical sensing and detection system. Inspired by the sense of biological smell, the researchers have been working on the ability to mimic the sense of smell of living organisms in vitro, with a view to achieving a high-sensitivity and high-specific sense of smell biosensor, and to expand its application and development in the fields of environmental monitoring, food detection and disease diagnosis. However, the most commonly used biosensitive materials (olfaction, olfactory and olfactory receptors) are difficult to obtain, and the activity is difficult to maintain, which limits the further development of the olfactory sensor. Odor-bindingprotein (OBPs), as an extracellular olfactory protein, has good odor-binding ability, and its stability is good, and it is easy to realize in vitro expression and purification. Therefore, the smell-binding protein sensor has important practical significance and application prospect for the study of the sense of smell and biological sensing. In this paper, the odor-binding protein (ASP2), the odor-binding protein (BdorOBP2) and the human odor-binding protein (OBP2a and OB2b) of the Chinese bee-odor-binding protein (ASP2) and the human odor-binding protein (OBP2a and OB2b) are used as the research examples, and the different electrochemical detection technology and the method for fixing the biological sensing element are combined. The design and construction of a plurality of odor-binding protein sensors are completed, and the detection of the odor molecules such as the chemical pheromone and the like is realized. Combined with the electrochemical impedance model and the molecular docking technology, the interaction between the odor-binding protein and the odor molecule is discussed, and the application and development of the smell-binding protein sensor are analyzed. The main content and innovation point of the thesis are as follows:1. The insect-odor-binding protein sensor is designed and constructed, and the detection of the chemical information is realized. The insect has a very sensitive olfactory system, which is closely related to the ability of the odor-binding protein to bind to the odor molecule. In this paper, an insect-odor-binding protein sensor was designed and constructed with the odor-binding protein (ASP2) of the Chinese bee and the odor-binding protein (BdorOBP2) of the fruit fly. The two kinds of odor-binding proteins are successfully expressed and purified, and then are fixed on the surface of the interdigital electrode by means of physical adsorption and layer-by-layer self-assembly. Through the electrochemical impedance technology, the sensor realizes the detection of different chemical pheromones, and has wider detection range and better detection sensitivity and lower detection limit compared with other types of odor-binding protein sensors. The electrochemical impedance model was designed based on the three-dimensional structure of the odor-binding protein, and the interaction between the odor-binding protein and the chemical information was analyzed by means of the molecular docking technique. The main site based on the odor-binding protein-binding odor molecule is the hydrophobic cavity of its structure center, and the process of binding and releasing the odor molecules is the mechanism that requires a change in the protein conformation, In this paper, the electrochemical impedance model of the insect-odor-binding protein was constructed, including the two parts of the protein framework and the central hydrophobic cavity, and then the relationship between the change of the conformation of the odor-binding protein and the change of the impedance of the sensor was analyzed. At the same time, the role of the key amino acid residues in the three-stage structure of the odor-binding protein in the process of binding to the chemical information is analyzed by the result of the molecular docking, which lays a foundation for the study of the specific sensing and binding capacity of the odor-binding protein. The human odor-binding protein sensor is designed and constructed, and the detection of the fatty acid molecules with different chain lengths is realized. The fatty acid is an important component of an organism and an energy-storing substance, and is also an important lipophilic substance. In this paper, a human odor-binding protein OBP2a sensor for fatty acid molecule detection was designed by using human odor-binding protein to have a good affinity for fatty acid. The oxidized graphene is reduced to the reduced oxidized graphene by the method of electrochemical reduction and is fixed on the surface of the carbon working electrode of the printing electrode, and the odor-binding protein is fixed on the surface of the sensor by the residual oxygen-containing functional group at the same time of increasing the conductivity of the electrode. The sensor realizes the measurement of docosahexaenoic acid, linoleic acid, and lauric acid by using cyclic voltammetry. The results show that the human odor-binding protein pair has better binding ability to long-chain fatty acid, and can be used for analyzing the fatty acid material with different carbon chain length. An odor-binding protein sensor was designed and constructed with an anode alumina nanopore array, and the detection of the molecules such as benzaldehyde, fatty acid and the like was realized. Anodic alumina nanopore array is a kind of filter membrane material with nano-size channel, which has the characteristics of large specific surface area, and has been widely used in high sensitive detection of target substances in solution. Based on the binding capacity of human odor-binding protein OBP2b to the odor molecules such as benzaldehyde and the like, a biosensor based on the anodic aluminum oxide nanopore array was constructed by means of the three-electrode system. The anode aluminum oxide nano-hole array modified with the odor-binding protein is arranged between the working electrode and the reference/ counter electrode, so that the nano-hole array is the only path of the current and the solution flow in the electrochemical detection system. Under the action of benzaldehyde and fatty acid, the conformation of the odor-binding protein fixed on the nano-hole array changes, thus causing the change of the current in the nano-hole. And the detection of the benzaldehyde and the fatty acid at different concentrations is realized through the electrochemical impedance detection.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TP212.3
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