氣味結(jié)合蛋白生物傳感器研究
發(fā)布時(shí)間：2019-05-28 15:49
【摘要】：生物的嗅覺系統(tǒng)能夠靈敏、特異地檢測(cè)和識(shí)別復(fù)雜環(huán)境中痕量的氣味分子,是一種性能優(yōu)越的化學(xué)感受和檢測(cè)系統(tǒng)。在生物嗅覺的啟發(fā)下,研究者們一直致力于在體外模仿生物體的嗅覺感知能力,以期實(shí)現(xiàn)構(gòu)建高靈敏、高特異性的嗅覺生物傳感器,拓展其在環(huán)境監(jiān)測(cè)、食品檢測(cè),及疾病診斷等領(lǐng)域的應(yīng)用及發(fā)展。但是,目前常用的生物敏感材料(嗅覺組織、嗅覺細(xì)胞及嗅覺受體)存在著獲取困難、活性較難保持等問(wèn)題,限制了嗅覺傳感器的進(jìn)一步發(fā)展。氣味結(jié)合蛋白(odorant-bindingproteins,OBPs)作為一種胞外嗅覺蛋白,具有良好的氣味分子結(jié)合能力,同時(shí)其穩(wěn)定性較好,且易于實(shí)現(xiàn)體外表達(dá)和純化。因此,氣味結(jié)合蛋白傳感器對(duì)于嗅覺生物傳感的研究具有重要的現(xiàn)實(shí)意義和應(yīng)用前景。本論文以中華蜜蜂氣味結(jié)合蛋白(ASP2)、桔小實(shí)蠅氣味結(jié)合蛋白(BdorOBP2),及人氣味結(jié)合蛋白(OBP2a和OBP2b)為研究實(shí)例,結(jié)合不同的電化學(xué)檢測(cè)技術(shù)及生物敏感元件固定方法,完成了多種氣味結(jié)合蛋白傳感器的設(shè)計(jì)和構(gòu)建,實(shí)現(xiàn)了對(duì)化學(xué)信息素等氣味分子的檢測(cè)。結(jié)合電化學(xué)阻抗模型和分子對(duì)接技術(shù),探討了氣味結(jié)合蛋白與氣味分子之間的相互作用,分析了氣味結(jié)合蛋白傳感器的應(yīng)用及發(fā)展。論文的主要內(nèi)容及創(chuàng)新點(diǎn)如下:1.設(shè)計(jì)并構(gòu)建了昆蟲氣味結(jié)合蛋白傳感器,實(shí)現(xiàn)了對(duì)化學(xué)信息素的檢測(cè)。昆蟲具有非常靈敏的嗅覺系統(tǒng),這與其氣味結(jié)合蛋白結(jié)合氣味分子的能力密切相關(guān)。本論文以中華蜜蜂氣味結(jié)合蛋白(ASP2)和桔小實(shí)蠅氣味結(jié)合蛋白(BdorOBP2)為研究實(shí)例,設(shè)計(jì)并構(gòu)建了昆蟲氣味結(jié)合蛋白傳感器。首先成功表達(dá)和純化了這兩種氣味結(jié)合蛋白,然后利用物理吸附和層層自組裝的方法將其固定于叉指電極表面。通過(guò)電化學(xué)阻抗技術(shù),該傳感器實(shí)現(xiàn)了對(duì)不同化學(xué)信息素的檢測(cè),而且相較于其他類型氣味結(jié)合蛋白傳感器,其具有較寬的檢測(cè)范圍、較好的檢測(cè)靈敏度和檢測(cè)下限。2.基于氣味結(jié)合蛋白三維結(jié)構(gòu),設(shè)計(jì)了電化學(xué)阻抗模型,結(jié)合分子對(duì)接技術(shù)分析了氣味結(jié)合蛋白與化學(xué)信息素的相互作用。基于氣味結(jié)合蛋白結(jié)合氣味分子的主要場(chǎng)所是其結(jié)構(gòu)中心的疏水腔體,并且結(jié)合和釋放氣味分子的過(guò)程是需要蛋白構(gòu)象的改變來(lái)實(shí)現(xiàn)的這一機(jī)理,本論文構(gòu)建了昆蟲氣味結(jié)合蛋白檢測(cè)化學(xué)信息素的電化學(xué)阻抗模型,包括蛋白骨架和中心疏水腔兩部分,進(jìn)而對(duì)氣味結(jié)合蛋白構(gòu)象改變與傳感器阻抗變化之間的關(guān)系進(jìn)行了分析。同時(shí)結(jié)合分子對(duì)接的結(jié)果,分析了氣味結(jié)合蛋白的三級(jí)結(jié)構(gòu)中關(guān)鍵氨基酸殘基在結(jié)合化學(xué)信息素過(guò)程中所發(fā)揮的作用,這為研究氣味結(jié)合蛋白的特異性傳感結(jié)合能力奠定了基礎(chǔ)。3.設(shè)計(jì)并構(gòu)建了人氣味結(jié)合蛋白傳感器,實(shí)現(xiàn)了對(duì)不同鏈長(zhǎng)脂肪酸分子的檢測(cè)。脂肪酸是生物體的重要組成和儲(chǔ)能物質(zhì),也是一種重要的親脂性物質(zhì)。本論文利用人氣味結(jié)合蛋白對(duì)脂肪酸具有較好親和力的這一特性,設(shè)計(jì)了一種用于脂肪酸分子檢測(cè)的人氣味結(jié)合蛋白OBP2a傳感器。利用電化學(xué)還原的方法將氧化石墨烯還原為還原型氧化石墨烯并將其固定于印刷電極的碳工作電極表面,在增加電極導(dǎo)電性的同時(shí),利用殘留的含氧官能團(tuán)將氣味結(jié)合蛋白固定于傳感器表面。利用循環(huán)伏安檢測(cè)技術(shù),該傳感器實(shí)現(xiàn)了對(duì)二十二碳六烯酸、亞油酸,及月桂酸的測(cè)量。檢測(cè)結(jié)果表明人氣味結(jié)合蛋白對(duì)中、長(zhǎng)鏈脂肪酸具有較好的結(jié)合能力,可用于分析不同碳鏈長(zhǎng)的脂肪酸物質(zhì)。4.利用陽(yáng)極氧化鋁納米孔陣列,設(shè)計(jì)并構(gòu)建了氣味結(jié)合蛋白傳感器,實(shí)現(xiàn)了對(duì)苯甲醛、脂肪酸等分子的檢測(cè)。陽(yáng)極氧化鋁納米孔陣列是一種具有納米尺寸孔道的濾膜材料,其具有比表面積大的特點(diǎn),已經(jīng)廣泛應(yīng)用于溶液中目標(biāo)物質(zhì)的高靈敏檢測(cè)。本論文基于人氣味結(jié)合蛋白OBP2b對(duì)苯甲醛等氣味分子的結(jié)合能力,利用三電極體系,構(gòu)建了基于陽(yáng)極氧化鋁納米孔陣列的生物傳感器。將修飾有氣味結(jié)合蛋白的陽(yáng)極氧化鋁納米孔陣列置于工作電極與參比/對(duì)電極之間,使得納米孔陣列成為電化學(xué)檢測(cè)體系中電流及溶液流通的唯一通路。在苯甲醛及脂肪酸的作用下,固定于納米孔陣列上的氣味結(jié)合蛋白構(gòu)象發(fā)生改變,進(jìn)而引起納米孔中電流的變化。通過(guò)電化學(xué)阻抗檢測(cè),實(shí)現(xiàn)了對(duì)不同濃度苯甲醛及脂肪酸的檢測(cè)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TP212.3
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