复杂生物体系胆碱及其衍生物的快速核磁共振检测

发布时间：2018-04-15 02:32

本文选题：核磁共振 + SOFAST-HMQC　；参考：《中国科学院大学(中国科学院武汉物理与数学研究所)》2017年硕士论文

【摘要】：胆碱及其衍生物是一类具有重要功生物能的分子。胆碱是磷脂酰胆碱和神经递质乙酰胆碱的合成前体,被认为有助于人类脑部发育和记忆。磷脂酰胆碱和鞘磷脂是细胞膜的重要组分,对于细胞形态维持、物质运输和信号传递等具有重要作用。有研究表明胆碱衍生物含量变化与癌变过程有关,已经在癌症诊断中作为癌变标志物。胆碱是人体的必需营养物质,部分可以在人体合成,但是更重要的来源却是来自于饮食,胆碱类物质常存在于牛奶、动物肝脏等食物中,也是婴儿食品添加剂的成分之一。因此,发展快速准确的胆碱衍生物的检测技术在食品分析、医学诊断等领域具有重要的意义。胆碱衍生物的检测方法众多,有质谱、酶比色法、雷氏盐分光光度法等,这些方法通常需要复杂的样品处理过程。PET和荧光是胆碱类物质的原位检测方法,但需要引入有放射性或细胞毒性的探针。核磁共振波谱作为一种非侵入性检测方法,是针对复杂生物体系的理想的胆碱衍生物分析技术。一维1H和31PNMR是目前检测复杂体系中胆碱衍生物的常用技术,但谱线宽、信号重叠、导致解析困难,限制了其应用。研究表明,1H-14NHSQC实验可以实现牛奶、肝组织中的胆碱衍生物的高分辨、高灵敏检测。然而,由于这些分子的纵向弛豫时间较长(T11s),通过常规采样手段进行1H-14N二维HSQC采样仍然需要较长的实验时间,不利于在活体生物样品中的应用。在本论文中,我们将SOFAST-HMQC这—快速谱采集技术应用于胆碱衍生物的检测,通过快速1H-14N二维相关实验提高相关物质的检测灵敏度。我们对1H-14N二维相关实验的条件进行了优化对比分析。以人工配制的3种不同胆碱衍生物的混合溶液作为标准样品,通过一系列优化实验和模拟分析,获得了适合三种胆碱衍生物同时检测的1H-14N二维相关实验的扫描间隔时间、极化转移时间,以及SOFAST-HMQC实验形状脉冲的形状和脉宽。在相同实验条件下,将1H-14N SOFAST-HMQC实验的胆碱衍生物检测效果与文献中选择性同核去耦的1H-14NHSQC实验的检测效果进行了对比。结果表明,针对不同组分,1H-14N SOFAST-HMQC实验的灵敏度是1H-14N HSQC实验的1.47～2.05倍。通过实验数据对比与理论分析,我们发现在1H-14N SOFAST-HMQC实验中,纵向磁化的恢复效率更高,可以有效减少实验的扫描间隔时间。最后,分别采用1H-14N SOFAST-HMQC技术和1H-14N HSQC技术对6个不同乳制品中的胆碱衍生物进行了分析。结果表明:与常规1H谱检测相比,1H-14N二维相关技术对胆碱衍生物的检测具有高选择性,可消除复杂生物体系中其它背景信号的干扰;与1H-14NHSQC检测技术相比,1H-14NSOFAST-HMQC技术具有更高的灵敏度,在单位时间内信号增强可达1.32～3.00倍。此外,我们发现,与牛奶样品相比,奶粉溶液样品中磷酸胆碱的相对含量明显较低,而胆碱的含量却相对较高,这可能是由奶粉中的胆碱添加剂所引起。
[Abstract]:Choline and its derivatives are a class of molecules with important biological energy.Choline, a synthetic precursor of phosphatidylcholine and neurotransmitter acetylcholine, is thought to contribute to human brain development and memory.Phosphatidylcholine and sphingomyelin are important components of cell membrane, which play an important role in cell morphology maintenance, material transport and signal transduction.Some studies have shown that the changes of choline derivatives are related to the carcinogenesis process and have been used as cancer markers in cancer diagnosis.Choline is an essential nutrient in human body, which can be synthesized in human body, but the more important source is from diet. Choline is often found in milk, animal liver and other foods, and it is also one of the ingredients of infant food additives.Therefore, the development of rapid and accurate detection technology of choline derivatives is of great significance in food analysis, medical diagnosis and other fields.There are many methods for the detection of choline derivatives, such as mass spectrometry, enzyme colorimetry, Rayleigh salt spectrophotometry and so on. These methods usually require complex sample treatment process. PET and fluorescence are in situ detection of choline.But a radioactive or cytotoxic probe needs to be introduced.Nuclear magnetic resonance spectroscopy (NMR), as a noninvasive method, is an ideal analytical technique for choline derivatives in complex biological systems.One-dimensional 1H and 31PNMR are commonly used techniques for the detection of choline derivatives in complex systems, but their applications are limited because of their wide spectral lines and overlapping signals.The results showed that 1H-14NHSQC assay could be used to detect choline derivatives in milk and liver tissues with high resolution and sensitivity.However, due to the longer longitudinal relaxation time of these molecules, it still takes a long time to carry out 1H-14N two-dimensional HSQC sampling by conventional sampling method, which is not conducive to the application in living biological samples.In this thesis, SOFAST-HMQC, a fast spectrum acquisition technique, is applied to the detection of choline derivatives, and the sensitivity of the related substances is improved by a fast 1H-14N two-dimensional correlation experiment.We have optimized and analyzed the conditions of 1H-14N two-dimensional correlation experiment.Using the mixture solution of three different kinds of choline derivatives as standard sample, the scanning interval time of 1H-14N two-dimensional correlation experiment suitable for simultaneous detection of three kinds of choline derivatives was obtained by a series of optimization experiments and simulation analysis.The polarization transfer time and the shape and width of the pulse in the SOFAST-HMQC experiment.Under the same experimental conditions, the detection effect of choline derivatives from 1H-14N SOFAST-HMQC experiment was compared with that of 1H-14NHSQC experiment with selective homonuclear decoupling in literature.The results show that the sensitivity of 1H-14N SOFAST-HMQC test for different components is 1.47 ~ 2.05 times higher than that of 1H-14N HSQC experiment.Through the comparison of experimental data and theoretical analysis, we find that longitudinal magnetization has higher recovery efficiency in 1H-14N SOFAST-HMQC experiment, which can effectively reduce the scanning interval.Finally, the choline derivatives in 6 different dairy products were analyzed by 1H-14N SOFAST-HMQC and 1H-14N HSQC respectively.The results show that the 1H-14N two-dimensional correlation technique has a high selectivity for the detection of choline derivatives, which can eliminate the interference of other background signals in complex biological systems, and has a higher sensitivity than that of the 1H-14NHSQC technique, and the 1H-14NSOFAST-HMQC technique is more sensitive than the conventional 1H-14N correlation technique in the detection of choline derivatives, and can eliminate the interference of other background signals in complex biological systems.The signal enhancement can reach 1.32 ~ 3.00 times in unit time.In addition, we found that the relative content of choline phosphate in milk powder solution was significantly lower than that in milk sample, but the content of choline was relatively high, which may be caused by the choline additive in milk powder.
【学位授予单位】：中国科学院大学(中国科学院武汉物理与数学研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q6-33

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