基于電化學傳感與液質(zhì)聯(lián)用技術(shù)的生物活性物質(zhì)體內(nèi)分析研究
發(fā)布時間：2018-08-05 19:29
【摘要】：自然界中的食用或藥用植物為人類提供了大量的維持人類生命活動和治療人類疾病所必需的生物活性物質(zhì),這些物質(zhì)的檢測在現(xiàn)代分析科學中己顯示出越來越關(guān)鍵的作用,并廣泛地應(yīng)用于臨床診斷、治療監(jiān)測、食品安全、藥物安全性評價和質(zhì)量控制等諸多領(lǐng)域。在這些領(lǐng)域中,我們通常需要對復雜體系中的一種或多種生物活性組分進行體內(nèi)、體外分析,如食品分析中對重要功能性成分的檢測、中藥單方或復方制劑中重要活性成分的體外含量測定與體內(nèi)吸收分布考察等。而且,一些生物活性物質(zhì)在人體內(nèi)含量發(fā)生異常時會提示相應(yīng)的組織或器官病變,因而,在臨床診斷中往往還需要對作為疾病標志物的體內(nèi)生物活性物質(zhì)進行檢測。這就使得靈敏、高效、高選擇性且實用可靠的生物活性物質(zhì)體內(nèi)分析方法顯得尤為重要。本研究重點就建立生物活性物質(zhì)的體內(nèi)分析方法開展了以下五個方面的工作:1、通過直接碳化三聚氰胺泡沫(MF)制備了一種氮摻雜的三維(3D)彈性碳泡沫(NECF)材料,并以該NECF材料為基質(zhì),修飾以葡萄糖氧化酶(GOD)來構(gòu)建新型葡萄糖傳感器。在GOD/NECF電極中,其電極材料的前驅(qū)體MF來自于廚房保潔或建筑保溫材料,價格低廉,并且碳化工藝簡單。由于NECF具有三維(3D)互聯(lián)網(wǎng)絡(luò)結(jié)構(gòu)且結(jié)構(gòu)中含有大量的氮,從而大大增加了電極的有效表面積、機械穩(wěn)定性、活性位點與生物相容性。該體系所特有的結(jié)構(gòu)和所含有的大量的氮都有助于O2的催化還原反應(yīng),這對于通過監(jiān)測還原反應(yīng)中O2的消耗量來定量檢測葡萄糖是十分有利的。此外,NECF可以通過其良好的吸附性來結(jié)合GOD對葡萄糖進行檢測。GOD/NECF電極克服了傳統(tǒng)GOD修飾電極的機械穩(wěn)定性低和支撐材料的負效應(yīng)等缺點,且在對葡萄糖的電化學傳感分析中顯示出較寬的線性范圍(0.51μM-19.00 m M)、較低的檢出限(0.17μM)、良好的穩(wěn)定性和較高的靈敏度(98.46μA cm~(-2) mM~(-1))。最后,我們利用該電極實現(xiàn)了對人血清樣本中葡萄糖含量的檢測。2、基于球形MOF-5/三維多孔碳(3D-KSC)復合材料構(gòu)建了一種新型抗壞血酸無酶傳感器。3D-KSC是由天然植物洋麻桿高溫碳化制得的碳材料,具有三維多孔結(jié)構(gòu)及良好的生物兼容性等優(yōu)點,我們以3D-KSC為支撐材料通過一步水熱合成法制備了MOF-5/3D-KSC復合材料。在該復合材料中,具有球形多孔結(jié)構(gòu)的MOF-5微型材料均勻、牢固地生長在多孔的3D-KSC表面,且基于MOF-5/3D-KSC制備的一體電極與其他電極相比,由于兩種多孔材料的疊加效應(yīng),使其具有更大的電化學活性比表面積。并且MOF-5/3D-KSC一體電極對抗壞血酸脫氫氧化反應(yīng)顯示出巨大的催化能力。使其對抗壞血酸的檢測顯示出較寬的線性范圍(0.7μM-11.5 m M),且具有靈敏度高(68.4μA cm~(-2) m M-1)、檢測限低(0.24μM)和室溫下極其良好的穩(wěn)定性等優(yōu)點。同時,在本章中,該MOF-5/3D-KSC一體電極被成功的應(yīng)用于未經(jīng)稀釋的腸外營養(yǎng)液中抗壞血酸含量的檢測。3、我們基于GOD/KSC微電極與Ag/Ag Cl微參比電極設(shè)計了一種開路電位生物傳感器(OCPS),它僅包含一根工作電極和一根參比電極,并利用開路電位(Voc)的變化來監(jiān)測葡萄糖的含量。該傳感器的工作電極材料為環(huán)境友好的氮摻雜多孔碳(KSC)粉末,它是由天然植物洋麻桿(KS)高溫碳化并經(jīng)仔細研磨而來,具有良好的生物兼容性且每一微小碎片均具多孔結(jié)構(gòu)。與電流型傳感器相比,OCPS沒有外界電壓施加于電極之上,因而,大大降低了生物樣本及體內(nèi)環(huán)境中復雜成分的干擾。而且與傳統(tǒng)雙工作電極的自供能傳感器相比,OCPS僅有一根工作電極,簡化了修飾過程,極大地增加了傳感器的重現(xiàn)性。由于該檢測體系所需能量來自于生物樣品本身,不需要額外的能量供應(yīng),因此,OCPS更加易于小型化并且適用于活體分析。利用該傳感器,我們成功地檢測了小鼠淺表腫瘤組織和正常組織勻漿液當中的葡萄糖濃度。并且,在活體分析試驗中,我們還對如何利用OCPS長時間監(jiān)測大鼠皮下組織液的葡萄糖濃度進行了一次有益的嘗試。4、采用超高效液相色譜-四極桿-飛行時間質(zhì)譜(UPLC-Q-TOF-MS),根據(jù)時間依賴型掃描模式(MS~E)采集的超高精度樣品數(shù)據(jù),建立中藥復方制劑克咳膠囊活性物質(zhì)的高通量分析方法,在25 min內(nèi),對克咳膠囊中8類,41種復雜成分實現(xiàn)快速識別鑒定。并進一步追蹤活性物質(zhì)經(jīng)胃腸道吸收入血以及血-腦屏障跨越后的主要成分,形成體外物質(zhì)成分→入血活性物質(zhì)→入腦活性物質(zhì)的快速高效、整體評價方法。獲得克咳膠囊能夠吸收入血并移行入腦的物質(zhì)成分譜,為其臨床合理、安全用藥提供依據(jù)。5、采用超高速分離液相-三重四級桿串聯(lián)質(zhì)譜聯(lián)用(LC-MS-MS)技術(shù),建立同時測定生物樣品中嗎啡與差向異構(gòu)體麻黃堿、偽麻黃堿的分析方法。使用Agilent ZORBAX SB-C8柱,2 mmol L~(-1)醋酸銨的水溶液(含0.04%醋酸)與乙腈梯度洗脫,流速為0.2 mL min~(-1)。待測物在各自測定范圍內(nèi)線性關(guān)系良好(r~20.99),3種生物堿類成分和內(nèi)標的提取回收率均大于85.0%,方法的專屬性、準確度、精密度、基質(zhì)效應(yīng)和穩(wěn)定性均符合生物樣品的分析要求。所建立的方法快速測定了大鼠灌胃給予中藥克咳膠囊內(nèi)容物后3種生物堿類活性物質(zhì)的血漿濃度與不同腦組織濃度(包括運動前區(qū)皮質(zhì)、下丘腦、紋狀體、海馬、小腦和延髓),考察了這3種物質(zhì)在大鼠體內(nèi)的藥動學行為差異與腦部的組織分布特征。
[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.
【學位授予單位】：江西師范大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TP212.3
本文編號：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.
【學位授予單位】：江西師范大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TP212.3
【相似文獻】
相關(guān)博士學位論文 前1條
1 宋永貴;基于電化學傳感與液質(zhì)聯(lián)用技術(shù)的生物活性物質(zhì)體內(nèi)分析研究[D];江西師范大學;2017年
，本文編號：2166768
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