【作者】 张军波；

【导师】 叶建农；

【作者基本信息】 华东师范大学 ， 分析化学， 2011， 博士

【摘要】 毛细管电泳(Capillary Electrophoresis, CE),是二十几年来发展最快的分离分析技术之一,以毛细管为分离通道,以高压直流电场为驱动力,根据样品中各组分之间迁移速度的差异而实现分离的一种分离分析技术。它将分离柱效提高到上百万理论塔板数,进样量从微升水平进入纳升水平,且应用范围十分广泛,包括生物医药、分析化学、药物化学、环境化学、食品化学等诸多学科领域。本文探讨了毛细管电泳—安培/激光诱导荧光检测技术(CE-AD, CE-LIF)在疾病标志物检测和食品分析等方面的应用研究,主要内容如下：第一章绪论简单地概述毛细管电泳的特点、基本原理、分离模式、检测方式、发展趋势和应用现状,并扼要介绍本论文的目的和意义。第二章毛细管电泳-安培检测法测定尿样和水样中MGo和Go的含量本文首次利用2-硫代巴比妥酸(TBA)与甲基乙二醛(MGo)和乙二醛(Go)进行衍生,使原本无电化学活性的这两种生物标志物具有电化学活性,从而采用毛细管电泳-安培检测法(CE-AD)分离并检测了尿样及环境水样中的MGo与Go。对衍生方法与分析条件进行了优化,这两种物质在三个数量级上具有良好的线性关系(r2>0.9991)。MGo的最小检测限(LOD)与最低定量限(LOQ)分别为0.2μg/L与1.0μg/L,Go的LOD与LOQ分别为0.5μg/L和2.0μg/L,加标回收率和相对标准偏差(RSD)范围分别在90.9-101.3%与0.7-2.2%之间。CE-AD法提供了一种检测实际样品中MGo与Go的可靠、灵敏且成本较低的方法。第三章毛细管电泳-安培检测法快速测定尿样中儿茶酚胺及其代谢物含量本文利用毛细管电泳—安培检测法(CE-AD)检测了人体尿样中的混合代谢标志物,包括去甲肾上腺素(NE)、肾上腺素(E)、多巴胺(DA)、变肾上腺素(M)、香草扁桃酸(VMA)及高香草酸(HVA)。在80mmol/L硼砂—硼酸缓冲液(pH9.48)及16kV的分离电压条件下,这六种混合标志物与主要的干扰物(尿酸)能在24min内实现基线分离,而且在3个浓度数量级范围内有良好的线性相关性,最低检出限范围为1.0-5.0μg/L(S/N=3)。该CE-AD法能够对尿样中的六种标志物进行同步检测,能够同时得到多种疾病的相关诊断信息,避免了重复测量,降低了分析成本。因此,该方法在临床研究上有望成为一种快速简捷的检测新方法。第四章毛细管电泳-安培检测法测定尿样中8-OHdG和8-NO2Gua含量8-羟基脱氧鸟苷(8-hydroxy-2’-deoxyguanosine,8-OHdG)和8-硝基鸟嘌呤(8-nitroguanine,8-NO2Gua)在动物及人体研究中被广泛认同为DNA损伤生物标志物,但由于8-OHdG在实际样品中含量极低,且易被其他物质干扰,尿样中的8-OHdG与8-NO2Gua的检测工作极富挑战性。本文提出了用固相萃取技术富集、毛细管电泳(Capillary Electrophoresis, CE)分离-安培检测法(Amperometric Detection, AD)同步测定这两种标志物的方法；该方法灵敏度高,检测成本低。经过预处理的尿样中,8-OHdG与8-NO2Gua被成功地与尿样中的其他组分分离,在0.1-50μg/mL的浓度范围内呈良好的线性,最低检测限(LOD)分别为0.08μg/mL及0.10μg/mLo峰面积的相对标准偏差(RSD)分别为0.14%与1.58%,而迁移时间的RSD值分别为0.14%及0.20%。在优化条件下,该方法被应用于测定健康人与癌症病人尿样中的8-OHdG与8-NO2Gua。卖验结果表明,癌症病人与健康人尿样中的8-OHdG与8-NO2Gua的含量有明显不同。第五章毛细管电泳—激光诱导荧光检测法测定新生儿尿样中氨基酸含量本文利用异硫氰酸荧光素(FITC)与氨基酸衍生反应,采用毛细管电泳-激光诱导荧光检测法(CE-LIF)同时对与新生儿代谢异常相关的六种氨基酸类疾病标志物进行检测。对衍生方法与分析条件进行了优化,在0.01-5.0μmol/L的浓度范围内有良好的线性(r2>0.9970),最低检测限(LOD)在0.005-0.010μmol/L。加标回收率和相对标准偏差(RSD)范围分别在90.9-101.3%与0.7-2.2%之间。该方法灵敏度高,检测成本低,已成功应用于测定健康婴儿与苯丙氨酸代谢异常婴儿尿样中的氨基酸类标志物。第六章毛细管电泳—安培检测法测定食品中脂肪醛的含量本文首次采用毛细管电泳-安培检测法(CE-AD),发展了一种可同时检测食品中九种脂肪醛,即甲醛(C1)、乙醛(C2)、丙醛(C3)、戊醛(C4)、丁醛(C5)、戊醛(C6)、戊二醛(Gla)、2,3-丁二酮(Bud)和丙酮醛(MGo)的新方法。通过与电化学活性物质2-硫代巴比妥酸的衍生反应,九种无电活性的脂肪醛均能获得电化学活性,进而实现CE-AD法检测。本文优化了衍生条件以及CE-AD法的分离检测条件,实验方法符合人用药物注册技术要求国际协调会(ICH)的相关要求,回收率范围在82.8-123.8%之间。各脂肪醛在0.083-15.0mg/L范围内线性关系良好(r2≥0.9901),最低检出限(LOD)在0.008-0.074mg/L之间。该法所用仪器简单,分析成本低,为实际样品中非电活性低分子量单醛与二醛的检测提供了一种可靠、灵敏而简捷易行的方法。更多还原

【Abstract】 Capillary electrophoresis (CE) is one of the most important separation techniques in last twenty years. CE is the technique of performing electrophoresis in buffer-filled, narrow-bore capillaries, with a high-voltage power supply providing electric field; separation by electrophoresis relying on the differences in the speed of migration (or migration velocity) of charged species. In CE analysis, the volume of sample injection reaches nano liter level. CE has been widely applied in many fields, including analytical chemistry, medicinal chemistry, enviromental chemistry, food chemistry, biological chemistry, and so on. The present dissertation described the studies of CE, coupling amperimertric detection (AD) or laser induced fluoscence detection (LIF), application in the analysis of food contaminants and disease markers in real-world. The major contents are described as follows:1. PrefaceThe characteristics, the basic theories, the separation models, the detectors, the studies and applications of CE are introduced briefly. The goal and significance of this dissertation are also introduced.2. A novel capillary electrophoretic method for determining methylglyoxal and glyoxal in urine and water samplesTwo non-electroactive biomarkers methylglyoxal (MGo) and glyoxal (Go) in urine and environmental water samples were determined for the first time by capillary electrophoresis with amperometric detection (CE-AD) after derivatising with an electroactive compound2-thiobarbituric acid. Experimental conditions of derivatization and CE-AD detection were optimized. Highly linear response was obtained for these two biomarkers over three orders of magnitude with good correlation (r2>0.9991). The limits of detection (LODs) and limits of quantitation (LOQs) of MGo and Go were0.2μg/L and1.0μg/L,0.5μg/L and2.0μg/L, respectively. The average recovery and relative standard deviation (RSD) were within the range of90.9-101.3%and0.7-2.2%, respectively. The proposed CE-AD method provides a reliable and sensitive quantitative evaluation of MGo and Go in real sample matrices by employing relatively simple and inexpensive instrument.3. Fast analysis of catecholamines and metabolites in human urine by capillary chromatography with amperometric detectionThe urinary metabolic marker compounds, namely norepinephrine (NE), epinephrine (E), dopamine (DA), metanephrine (M), vanillylmandelic acid (VMA) and homovanillic acid (HVA) of cathecholamines were detected by capillary chromatography with amperometric detection (CE-AD). Electrophoretic runs were performed in an80mmol/L H3BO3-Na2B4O7running buffer (pH9.48) at a separation voltage of16kV, and the six marker compounds together with the major coexisting compound uric acid (UA) could be well separated within24min. Highly linear response was obtained for the six markers over three orders of magnitude with detection limits ranging from1.0to5.0μg/L (S/N=3). The proposed CE-AD method has been used to detect the six markers simultaneously in urine samples with the advantages of obtaining more diagnostic information of several diseases and avoiding redundant measurements and high assay cost. Thus, it could provide useful information for clinical researchers as a fast and simple analytical method.4. Determination of DNA damage markers:8-hydroxy-2’-deoxyguanosine and8-nitroguanine by capillary electrophoresis in urine8-hydroxy-2’-deoxyguanosine (8-OHdG) and8-nitroguanine (8-NO2Gua) have been widely used as markers of DNA damage in both animal models and human studies. Owing to the low level of8-OHdG and the complex matrix, measurement of8-OHdG and8-NO2Gua in urinary samples was a challenging work. In this paper, we have developed a sensitive and low-cost method for the determination of the two types of markers by capillary electrophoresis with amperometric detection (CE-AD) after solid phase extraction (SPE). In the spiked urine samples,8-OHdG and8-NO2Gua were well separated from other urine components, exhibiting a linear calibration over the concentration range of0.1～50μg/mL with the detection limits varied from0.08μg/mL to0.10μg/mL. The relative standard deviation (RSD) was in the range of0.14%and1.58%for peak area,0.14%and0.20%for migration time, respectively. Under optimized conditions, the proposed CE-AD method has been applied to quantitative evaluation of8-OHdG and8-NO2Gua in urinary samples from healthy volunteers and cancer patients. It was found that the levels of8-OHdG and8-NO2Gua in cancer patients were significantly higher than those in healthy ones.5. Measurements of amino acids by capillary electrophoresis with laser induced fluorescence--potential application in inborn errors of metabolismA method of capillary electrophoresis coupled with laser induced fluorescence detection has been developed for the determination of markers of six kind diseases of new-born babies. Factors influencing the separation and detection were examined and optimized. Highly linear response was obtained for markers with over three orders of magnitude with good correlation (r2>0.9970). The average revovery and RSD were within the range of90.9-103.4%and0.2-1.9%, respectively. The LODs were ranged from0.01-5.0nmol/L. The proposed CE-LIF method provides an inexpensive and sensitive quantitative evaluation of markers for the purpose of fast diagnose of some metabolic diseases of new-born.6. A novel capillary electrophoretic method for determining aliphatic aldehydes in food samples using2-thiobarbituric acid derivatizationA novel electrophoretic method for sensitive determination of nine aldehydes, including formaldehyde (C1), acetaldehyde (C2), propanal (C3), butanal (C4), pentanal (C5), hexanal (C6), glutaradehyde (Gla),2,3-butanedione (Bud) and methylgloxal (MGo) in food samples has been developed based on CE with amperometric detection (CE-AD). After derivatized with an electroactive compound2-thiobarbituric acid (TBA), these nine non-electroactive aldehydes were converted to electroactive adducts, therefore detectable by CE-AD approach. Experimental conditions of derivatization and CE-AD detection were optimized. The proposed method was validated according to International Conference on Harmonization (ICH) requirements with recovery results ranging from82.8to123.8%. Calibration plots of aliphatic aldehydes were linear (r2≥0.9901) in the concentration range from0.083 to15.0mg/L. The LODs were between0.008and0.074mg/L. The proposed CE-AD method provides a reliable and sensitive quantitative evaluation for non-electroactive low-molecular-mass monoaldehydes and dialdehydes in real sample matrices by employing relatively simple and inexpensive instrument.更多还原