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提高毛细管电泳灵敏度的新方法研究
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摘要
高效毛细管电泳(High performance capillary electrophoresis, HPCE)具有分离效率高、分析速度快、运行成本低、样品用量少等优点,在分离科学尤其是生物大分子和微环境样品微量组分的分离测定中得到了广泛关注和迅猛发展,已成为一种重要的现代分离分析技术。但是,CE的进样量仅为纳升级,检测光程一般为几十微米,导致其灵敏度低于液相色谱。因此,发展有效的方法提高CE的灵敏度已成为毛细管电泳的一个重要研究方向。鉴于此,本学位论文在前人工作基础上,围绕提高CE的灵敏度,开展了以下创新性研究工作:
     1.将大量样品电动堆积技术与甲醇辅助胶束坍塌诱导的MSS (micelle to solvent stacking)富集技术相结合,建立了一种新的富集技术。该技术可显著提高CE的灵敏度,并可用于富集分离尿液中的小檗碱和茶碱。
     2.以NBD-F为衍生试剂,建立了在柱衍生-MEKC-LIF (micellar electrokinetic chromatography-laser induced fluorescence)新方法,并首次将其用于灵敏、快速测定牛奶、奶粉、乳制品饮料和豆浆粉中的羟脯氨酸,据此可判断乳制品中是否含有皮革水解物。
     3.制备了可用于CE样品前处理的胍基修饰的磁性纳米颗粒。该纳米颗粒可选择性萃取和富集蛋白质混合溶液中的牛血清白蛋白,显著提高了CE检测牛血清白蛋白(bovine serum albumin, BSA)的灵敏度。
     4.合成了氧化石墨烯-四氧化三铁复合纳米材料,考察了将其作为CE样品前处理中固相萃取吸附剂对葡萄糖氧化酶(glucose oxidase, GOx)进行选择性富集的可行性,提高了CE检测葡萄糖氧化酶的灵敏度。
     本论文共分为六章:
     第一章:对提高毛细管电泳检测灵敏度的方法进行了综述,重点介绍了近年来CE在线富集技术和萃取技术的研究进展和发展趋势。
     第二章:将大量样品电动进样堆积技术与甲醇辅助胶束坍塌诱导的MSS技术有机结合,建立了一种新型在线联合富集方法。对方法的富集原理进行了探讨,考察了影响富集效率的各个因素。该方法对小檗碱和茶碱的富集倍数分别为6400和630,塔板数分别为2.9×106/m和6.5×105/m。该方法已成功用于尿液中小檗碱和茶碱的测定。
     第三章:以NBD-F为衍生试剂,建立了一种检测乳制品中皮革标记物羟脯氨酸的在柱衍生-MEKC-LIF新方法。该方法可以在7min内完成电泳分离过程,检测限为1.6±0.5ng/mL,已成功用于奶粉、牛奶、乳制品饮料和豆浆粉中羟脯氨酸的检测。
     第四章:首次合成了胍基修饰的磁性Fe304纳米颗粒,利用透射电镜、X-射线衍射仪、红外光谱仪、振动样品磁量计和ζ电势仪等手段对其形貌和性质进行了表征,探讨了影响吸附和解吸附效率的因素。将此多功能纳米材料作为固相萃取吸附剂进行样品前处理,可使CE检测BSA的灵敏度提高15倍。
     第五章:合成了氧化石墨烯-四氧化三铁(GO-Fe3O4)复合纳米材料,利用透射电镜、红外光谱仪和振动样品磁量计对其进行了表征,考察了其作为CE样品前处理中固相萃取吸附剂的可行性。结果表明,该材料可选择性富集GOx,富集倍数为26,藉此可提高CE检测GOx的灵敏度。此外,首次深入研究了GO-Fe3O4复合材料的类过氧化物酶活性,并将其用于H2O2和葡萄糖的比色法测定,拓展了其在生命分析化学领域内的应用。
     第六章:结论
Capillary electrophoresis (CE) is a liquid phase separation technique with the advantages of high resolution, high effieciency, short analysis time, simplicity, low sample and solvent consumption. The theory and application of CE have proved to be one of the most active research fileds in analytical chemistry. A capillary with smaller inside diameter is preferred for rapid and high-resolution separation by applying higher voltages. However, it is also responsible for the major limitation of the technique. The small detection volume and short optical pathlength typically result in low sensitivity. Development and establishment of novel methods or strategies to overcome the shortcoming are important. In order to improve the sensitivity in CE, the following major innovative researches were carried out in this thesis on the basis of the previous works.
     1. Combination of MSS with LASEKSI, a method was developed based on methanol assited micelle collapse for the first time, and it was applied to concentrate the berberine and theophylline in urine sample and higher focusing efficiency was obtained.
     2. NBD-F was selected as in-capillary derivatization reagent to label the hydroxyproline, which is marker of college. A simple, rapid and sensitive method was established based on in-capillary derivatization by MEKC-LIF for fully automated detection of hydroxyproline in dairy products for food safety purpose.
     3. Guanidine group functionalized Fe3O4nanoparticles were prepared and characterized. The novel nanomaterials were used as solid phase extration material for enrichment of BSA and the sensitivity of CE was improved.
     4. Graphene oxide-FesO4nanocomposites (GO-Fe3O4) were proved to be a novel solid phase extraction materials for GOx to improve the sensitivity of CE. In addition, the nanocomposites were exploited their peroxidase-like activity and applied for colorimetric detection of H2O2and glucose.
     This dissertation consists of six chapters.
     Chapter1:Approaches to improve the sensitivity of CE were briefly described. The recent developments of the methods help address this problem were reviewed.
     Chapter2:A new on-line method coupling MSS technique with large amount sample electrokinetic stacking injection (LASEKSI) for the analysis of cationic molecules was established. In this MSS-LASEKSI, an equilibrium state was formed and could be maintained for a long time, leading to the continuous stacking of the analytes on the basis of MSS. Extremely large amount of sample was permitted to be injected and then an improved enrichment fold could be achieved, comparing with the each case. Under the optimized conditions,6.4×103-and6.3×102-fold enrichment in peak heights upon normal CZE method and number of plates of2.9×106and6.5×105/m were attained for berberine and theophylline, respectively. The developed method may provide prospect for exploiting a new concentration technique to achieve higher enrichment factor.
     Chapter3:A rapid, sensitive and automated approach for the determination of hydroxyproline (Hyp) by MEKC-LIF was developed for the first time. The new method employed NBD-F as the fluorescent reagent for in-capillary derivatization and the main parameters were discussed. The CE process could be completed within7min and the detection limit for Hyp was1.6±0.5ng/mL. This approach was applied on the analysis of Hyp in milk powder, liquid milk, milk drink and soymilk powder samples for food safety purpose.
     Chapter4:Guanidine group functionalized magnetic nanoparticles were prepared and characterized by transmission electron microscopy, X-ray diffraction, fourier transform infrared spectra, vibration sample magnetometer and zeta potential analyzer. The novel multifunctional nanoparticles were served as a solid-phase extraction sorbent for easy isolation and preconcentration of acidic protein from aqueous solution. Fifteen-fold enrichement efficiency was achieved and detection limit was45ng/mL for BSA by CE.
     Chapter5:Graphene oxide-Fe3O4nanocomposites (GO-Fe3O4) were perepared and characterized. It was found that the GO-Fe3O4nanocomposite can be used as a solid phase sorbent for GOx in CE. The GOx can be enriched for26fold using the nanomaterials and the detection sensitivity of CE was improved. In addition, the nanocomposite was demonstrated to possess intrinsic peroxidase-like activity for the first time, and can be used for colorimetric detection of H2O2and glucose.
     Chapter6:Conclusion.
引文
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