基于电化学传感与液质联用技术的生物活性物质体内分析研究
发布时间：2018-08-05 19:29
【摘要】：自然界中的食用或药用植物为人类提供了大量的维持人类生命活动和治疗人类疾病所必需的生物活性物质,这些物质的检测在现代分析科学中己显示出越来越关键的作用,并广泛地应用于临床诊断、治疗监测、食品安全、药物安全性评价和质量控制等诸多领域。在这些领域中,我们通常需要对复杂体系中的一种或多种生物活性组分进行体内、体外分析,如食品分析中对重要功能性成分的检测、中药单方或复方制剂中重要活性成分的体外含量测定与体内吸收分布考察等。而且,一些生物活性物质在人体内含量发生异常时会提示相应的组织或器官病变,因而,在临床诊断中往往还需要对作为疾病标志物的体内生物活性物质进行检测。这就使得灵敏、高效、高选择性且实用可靠的生物活性物质体内分析方法显得尤为重要。本研究重点就建立生物活性物质的体内分析方法开展了以下五个方面的工作:1、通过直接碳化三聚氰胺泡沫(MF)制备了一种氮掺杂的三维(3D)弹性碳泡沫(NECF)材料,并以该NECF材料为基质,修饰以葡萄糖氧化酶(GOD)来构建新型葡萄糖传感器。在GOD/NECF电极中,其电极材料的前驱体MF来自于厨房保洁或建筑保温材料,价格低廉,并且碳化工艺简单。由于NECF具有三维(3D)互联网络结构且结构中含有大量的氮,从而大大增加了电极的有效表面积、机械稳定性、活性位点与生物相容性。该体系所特有的结构和所含有的大量的氮都有助于O2的催化还原反应,这对于通过监测还原反应中O2的消耗量来定量检测葡萄糖是十分有利的。此外,NECF可以通过其良好的吸附性来结合GOD对葡萄糖进行检测。GOD/NECF电极克服了传统GOD修饰电极的机械稳定性低和支撑材料的负效应等缺点,且在对葡萄糖的电化学传感分析中显示出较宽的线性范围(0.51μM-19.00 m M)、较低的检出限(0.17μM)、良好的稳定性和较高的灵敏度(98.46μA cm~(-2) mM~(-1))。最后,我们利用该电极实现了对人血清样本中葡萄糖含量的检测。2、基于球形MOF-5/三维多孔碳(3D-KSC)复合材料构建了一种新型抗坏血酸无酶传感器。3D-KSC是由天然植物洋麻杆高温碳化制得的碳材料,具有三维多孔结构及良好的生物兼容性等优点,我们以3D-KSC为支撑材料通过一步水热合成法制备了MOF-5/3D-KSC复合材料。在该复合材料中,具有球形多孔结构的MOF-5微型材料均匀、牢固地生长在多孔的3D-KSC表面,且基于MOF-5/3D-KSC制备的一体电极与其他电极相比,由于两种多孔材料的叠加效应,使其具有更大的电化学活性比表面积。并且MOF-5/3D-KSC一体电极对抗坏血酸脱氢氧化反应显示出巨大的催化能力。使其对抗坏血酸的检测显示出较宽的线性范围(0.7μM-11.5 m M),且具有灵敏度高(68.4μA cm~(-2) m M-1)、检测限低(0.24μM)和室温下极其良好的稳定性等优点。同时,在本章中,该MOF-5/3D-KSC一体电极被成功的应用于未经稀释的肠外营养液中抗坏血酸含量的检测。3、我们基于GOD/KSC微电极与Ag/Ag Cl微参比电极设计了一种开路电位生物传感器(OCPS),它仅包含一根工作电极和一根参比电极,并利用开路电位(Voc)的变化来监测葡萄糖的含量。该传感器的工作电极材料为环境友好的氮掺杂多孔碳(KSC)粉末,它是由天然植物洋麻杆(KS)高温碳化并经仔细研磨而来,具有良好的生物兼容性且每一微小碎片均具多孔结构。与电流型传感器相比,OCPS没有外界电压施加于电极之上,因而,大大降低了生物样本及体内环境中复杂成分的干扰。而且与传统双工作电极的自供能传感器相比,OCPS仅有一根工作电极,简化了修饰过程,极大地增加了传感器的重现性。由于该检测体系所需能量来自于生物样品本身,不需要额外的能量供应,因此,OCPS更加易于小型化并且适用于活体分析。利用该传感器,我们成功地检测了小鼠浅表肿瘤组织和正常组织匀浆液当中的葡萄糖浓度。并且,在活体分析试验中,我们还对如何利用OCPS长时间监测大鼠皮下组织液的葡萄糖浓度进行了一次有益的尝试。4、采用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS),根据时间依赖型扫描模式(MS~E)采集的超高精度样品数据,建立中药复方制剂克咳胶囊活性物质的高通量分析方法,在25 min内,对克咳胶囊中8类,41种复杂成分实现快速识别鉴定。并进一步追踪活性物质经胃肠道吸收入血以及血-脑屏障跨越后的主要成分,形成体外物质成分→入血活性物质→入脑活性物质的快速高效、整体评价方法。获得克咳胶囊能够吸收入血并移行入脑的物质成分谱,为其临床合理、安全用药提供依据。5、采用超高速分离液相-三重四级杆串联质谱联用(LC-MS-MS)技术,建立同时测定生物样品中吗啡与差向异构体麻黄碱、伪麻黄碱的分析方法。使用Agilent ZORBAX SB-C8柱,2 mmol L~(-1)醋酸铵的水溶液(含0.04%醋酸)与乙腈梯度洗脱,流速为0.2 mL min~(-1)。待测物在各自测定范围内线性关系良好(r~20.99),3种生物碱类成分和内标的提取回收率均大于85.0%,方法的专属性、准确度、精密度、基质效应和稳定性均符合生物样品的分析要求。所建立的方法快速测定了大鼠灌胃给予中药克咳胶囊内容物后3种生物碱类活性物质的血浆浓度与不同脑组织浓度(包括运动前区皮质、下丘脑、纹状体、海马、小脑和延髓),考察了这3种物质在大鼠体内的药动学行为差异与脑部的组织分布特征。
[Abstract]:The edible or medicinal plants in nature provide a large number of bioactive substances for human life and the treatment of human diseases. The detection of these substances has shown more and more key roles in modern analytical science and is widely used in clinical diagnosis, treatment monitoring, food safety, and drug safety assessment. In many fields, such as price and quality control, we usually need to carry out in vivo, in vitro analysis of one or more bioactive components in complex systems, such as detection of important functional components in food analysis, determination of the content of important active components in Chinese medicine single or compound preparation in vitro and in vivo absorption distribution And, in addition, some bioactive substances may indicate the corresponding tissue or organ lesions when the content of the human body is abnormal in the human body. Therefore, in the clinical diagnosis, it is often necessary to detect the bioactive substances in the body as the marker of the disease. This makes the sensitive, efficient, highly selective and practical bioactive substance analysis in vivo. This method is particularly important. This study focuses on the following five aspects: 1, a nitrogen doped three-dimensional (3D) elastic carbon foam (NECF) material was prepared by direct carbonization of melamine foam (MF), and the NECF material was used as the matrix to modify the glucose oxidase (GOD). A new type of glucose sensor is built. In the GOD/NECF electrode, the precursor of the electrode material MF comes from the kitchen cleaning or the building insulation material, the price is low, and the carbonization process is simple. Because NECF has a three-dimensional (3D) network structure and contains a lot of nitrogen in the structure, the effective surface area, mechanical stability and life of the electrode are greatly increased. Sexual loci and biocompatibility. The structure specific to the system and the amount of nitrogen contained in the system will contribute to the catalytic reduction of O2, which is very beneficial for the quantitative detection of glucose by monitoring the consumption of O2 in the reduction reaction. In addition, NECF can be used to detect.GOD/NEC with GOD through its good desorption. F electrode overcomes the disadvantages of low mechanical stability of traditional GOD modified electrode and negative effect of supporting material, and shows a wide linear range (0.51 mu M-19.00 m M) in the electrochemical sensing analysis of glucose, a lower detection limit (0.17 mu M), good stability and high sensitivity (98.46 mu A cm~ (-2) mM~ (-1)). Finally, we Using this electrode, the glucose content in human serum samples was detected by.2. A new ascorbic acid free enzyme sensor.3D-KSC based on spherical MOF-5/ three-dimensional porous carbon (3D-KSC) composite was constructed, which was made by carbonization of natural plant hemp rod at high temperature. It has three dimensional porous structure and good biocompatibility. We have prepared a MOF-5/3D-KSC composite by one step hydrothermal synthesis using 3D-KSC as the supporting material. In this composite, the MOF-5 micro material with a spherical porous structure is uniformly and firmly on the porous 3D-KSC surface, and the integrated electrode prepared on the basis of MOF-5/3D-KSC is compared with the other electrodes because of the superposition of two porous materials. Adding effect, it has a larger electrochemical activity specific surface area. And the MOF-5/3D-KSC one electrode shows a great catalytic ability against the dehydrogenation oxidation of the acid, which shows a wide linear range (0.7 mu M-11.5 m M), with a high sensitivity (68.4 A cm~ (-2) m M-1), and a low detection limit (0.24 mu M). In this chapter, the MOF-5/3D-KSC one electrode has been successfully applied to the detection of ascorbic acid content in the undiluted parenteral nutrient solution.3. We designed an open circuit potential biosensor (OCPS) based on GOD/KSC microelectrode and Ag/Ag Cl microelectrode, which contains only one. Working electrode and a reference electrode are used to monitor the content of glucose by the change of open circuit potential (Voc). The working electrode material of this sensor is environmentally friendly nitrogen doped porous carbon (KSC) powder. It is carbonized by high temperature of natural plant hemp rod (KS) and is carefully grinded, with good biocompatibility and small fragmentation. The film has a porous structure. Compared with the current sensor, OCPS has no external voltage on the electrode, thus greatly reducing the interference of the complex components in the biological samples and in the internal environment. And compared with the traditional dual working electrode, the OCPS only has one working electrode, simplifies the modification process and greatly increases the transmission. As the energy required for the detection system comes from the biological sample itself and does not require additional energy supply, the OCPS is more easily miniaturized and is suitable for living body analysis. In vivo analysis, we also made a useful attempt on how to use OCPS for long time monitoring of glucose concentration in subcutaneous tissue fluid of rats,.4, using ultra high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS), ultra high precision sample data collected by time dependent scanning mode (MS~E), to establish the compound of Chinese medicine. The high throughput analysis method of the active substance of KKT capsule, in 25 min, was used to identify the 8 types and 41 complex components of kKE capsule, and to further trace the main components of the active substances after the absorption of blood into the gastrointestinal tract and the crossing of the blood brain barrier, forming the substances in vitro, the active substance into the blood and the brain active substance. Rapid and high quality, overall evaluation method. The material composition spectrum of Keke capsule can be absorbed into the blood and moved into the brain. It provides a basis for its clinical rational and safe use of.5, and uses ultra high speed separation liquid phase three weight four stage tandem mass spectrometry (LC-MS-MS) to establish the morphine and differential isomer ephedrine in the same time measurement biological samples. The method of analysis of pseudoephedrine. Using Agilent ZORBAX SB-C8 column, 2 mmol L~ (-1) ammonium acetate aqueous solution (containing 0.04% acetic acid) and acetonitrile gradient elution, the flow rate is 0.2 mL min~ (-1). The linear relationship between the measured objects is good (r~20.99), the extraction recovery of the 3 alkaloids and internal standards is greater than 85%, the specificity of the method, Accuracy, precision, matrix effect and stability were all consistent with the requirements of biological samples. The methods established to quickly determine the plasma concentration of 3 alkaloid active substances and different brain tissue concentrations (including the premotor cortex, hypothalamus, striatum, hippocampus, cerebellum and medulla) in rats after gavage of Chinese medicine KKT capsule content. The pharmacokinetic behavior of these 3 substances in rats and their brain tissue distribution characteristics were investigated.
【学位授予单位】：江西师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TP212.3
本文编号：2166768
[Abstract]:The edible or medicinal plants in nature provide a large number of bioactive substances for human life and the treatment of human diseases. The detection of these substances has shown more and more key roles in modern analytical science and is widely used in clinical diagnosis, treatment monitoring, food safety, and drug safety assessment. In many fields, such as price and quality control, we usually need to carry out in vivo, in vitro analysis of one or more bioactive components in complex systems, such as detection of important functional components in food analysis, determination of the content of important active components in Chinese medicine single or compound preparation in vitro and in vivo absorption distribution And, in addition, some bioactive substances may indicate the corresponding tissue or organ lesions when the content of the human body is abnormal in the human body. Therefore, in the clinical diagnosis, it is often necessary to detect the bioactive substances in the body as the marker of the disease. This makes the sensitive, efficient, highly selective and practical bioactive substance analysis in vivo. This method is particularly important. This study focuses on the following five aspects: 1, a nitrogen doped three-dimensional (3D) elastic carbon foam (NECF) material was prepared by direct carbonization of melamine foam (MF), and the NECF material was used as the matrix to modify the glucose oxidase (GOD). A new type of glucose sensor is built. In the GOD/NECF electrode, the precursor of the electrode material MF comes from the kitchen cleaning or the building insulation material, the price is low, and the carbonization process is simple. Because NECF has a three-dimensional (3D) network structure and contains a lot of nitrogen in the structure, the effective surface area, mechanical stability and life of the electrode are greatly increased. Sexual loci and biocompatibility. The structure specific to the system and the amount of nitrogen contained in the system will contribute to the catalytic reduction of O2, which is very beneficial for the quantitative detection of glucose by monitoring the consumption of O2 in the reduction reaction. In addition, NECF can be used to detect.GOD/NEC with GOD through its good desorption. F electrode overcomes the disadvantages of low mechanical stability of traditional GOD modified electrode and negative effect of supporting material, and shows a wide linear range (0.51 mu M-19.00 m M) in the electrochemical sensing analysis of glucose, a lower detection limit (0.17 mu M), good stability and high sensitivity (98.46 mu A cm~ (-2) mM~ (-1)). Finally, we Using this electrode, the glucose content in human serum samples was detected by.2. A new ascorbic acid free enzyme sensor.3D-KSC based on spherical MOF-5/ three-dimensional porous carbon (3D-KSC) composite was constructed, which was made by carbonization of natural plant hemp rod at high temperature. It has three dimensional porous structure and good biocompatibility. We have prepared a MOF-5/3D-KSC composite by one step hydrothermal synthesis using 3D-KSC as the supporting material. In this composite, the MOF-5 micro material with a spherical porous structure is uniformly and firmly on the porous 3D-KSC surface, and the integrated electrode prepared on the basis of MOF-5/3D-KSC is compared with the other electrodes because of the superposition of two porous materials. Adding effect, it has a larger electrochemical activity specific surface area. And the MOF-5/3D-KSC one electrode shows a great catalytic ability against the dehydrogenation oxidation of the acid, which shows a wide linear range (0.7 mu M-11.5 m M), with a high sensitivity (68.4 A cm~ (-2) m M-1), and a low detection limit (0.24 mu M). In this chapter, the MOF-5/3D-KSC one electrode has been successfully applied to the detection of ascorbic acid content in the undiluted parenteral nutrient solution.3. We designed an open circuit potential biosensor (OCPS) based on GOD/KSC microelectrode and Ag/Ag Cl microelectrode, which contains only one. Working electrode and a reference electrode are used to monitor the content of glucose by the change of open circuit potential (Voc). The working electrode material of this sensor is environmentally friendly nitrogen doped porous carbon (KSC) powder. It is carbonized by high temperature of natural plant hemp rod (KS) and is carefully grinded, with good biocompatibility and small fragmentation. The film has a porous structure. Compared with the current sensor, OCPS has no external voltage on the electrode, thus greatly reducing the interference of the complex components in the biological samples and in the internal environment. And compared with the traditional dual working electrode, the OCPS only has one working electrode, simplifies the modification process and greatly increases the transmission. As the energy required for the detection system comes from the biological sample itself and does not require additional energy supply, the OCPS is more easily miniaturized and is suitable for living body analysis. In vivo analysis, we also made a useful attempt on how to use OCPS for long time monitoring of glucose concentration in subcutaneous tissue fluid of rats,.4, using ultra high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS), ultra high precision sample data collected by time dependent scanning mode (MS~E), to establish the compound of Chinese medicine. The high throughput analysis method of the active substance of KKT capsule, in 25 min, was used to identify the 8 types and 41 complex components of kKE capsule, and to further trace the main components of the active substances after the absorption of blood into the gastrointestinal tract and the crossing of the blood brain barrier, forming the substances in vitro, the active substance into the blood and the brain active substance. Rapid and high quality, overall evaluation method. The material composition spectrum of Keke capsule can be absorbed into the blood and moved into the brain. It provides a basis for its clinical rational and safe use of.5, and uses ultra high speed separation liquid phase three weight four stage tandem mass spectrometry (LC-MS-MS) to establish the morphine and differential isomer ephedrine in the same time measurement biological samples. The method of analysis of pseudoephedrine. Using Agilent ZORBAX SB-C8 column, 2 mmol L~ (-1) ammonium acetate aqueous solution (containing 0.04% acetic acid) and acetonitrile gradient elution, the flow rate is 0.2 mL min~ (-1). The linear relationship between the measured objects is good (r~20.99), the extraction recovery of the 3 alkaloids and internal standards is greater than 85%, the specificity of the method, Accuracy, precision, matrix effect and stability were all consistent with the requirements of biological samples. The methods established to quickly determine the plasma concentration of 3 alkaloid active substances and different brain tissue concentrations (including the premotor cortex, hypothalamus, striatum, hippocampus, cerebellum and medulla) in rats after gavage of Chinese medicine KKT capsule content. The pharmacokinetic behavior of these 3 substances in rats and their brain tissue distribution characteristics were investigated.
【学位授予单位】：江西师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TP212.3
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1 宋永贵;基于电化学传感与液质联用技术的生物活性物质体内分析研究[D];江西师范大学;2017年
，本文编号：2166768
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