本文選題：核磁共振 + SOFAST-HMQC��； 參考：《中國科學院大學(中國科學院武漢物理與數(shù)學研究所)》2017年碩士論文

【摘要】：膽堿及其衍生物是一類具有重要功生物能的分子。膽堿是磷脂酰膽堿和神經(jīng)遞質(zhì)乙酰膽堿的合成前體,被認為有助于人類腦部發(fā)育和記憶。磷脂酰膽堿和鞘磷脂是細胞膜的重要組分,對于細胞形態(tài)維持、物質(zhì)運輸和信號傳遞等具有重要作用。有研究表明膽堿衍生物含量變化與癌變過程有關(guān),已經(jīng)在癌癥診斷中作為癌變標志物。膽堿是人體的必需營養(yǎng)物質(zhì),部分可以在人體合成,但是更重要的來源卻是來自于飲食,膽堿類物質(zhì)常存在于牛奶、動物肝臟等食物中,也是嬰兒食品添加劑的成分之一。因此,發(fā)展快速準確的膽堿衍生物的檢測技術(shù)在食品分析、醫(yī)學診斷等領(lǐng)域具有重要的意義。膽堿衍生物的檢測方法眾多,有質(zhì)譜、酶比色法、雷氏鹽分光光度法等,這些方法通常需要復(fù)雜的樣品處理過程。PET和熒光是膽堿類物質(zhì)的原位檢測方法,但需要引入有放射性或細胞毒性的探針。核磁共振波譜作為一種非侵入性檢測方法,是針對復(fù)雜生物體系的理想的膽堿衍生物分析技術(shù)。一維1H和31PNMR是目前檢測復(fù)雜體系中膽堿衍生物的常用技術(shù),但譜線寬、信號重疊、導(dǎo)致解析困難,限制了其應(yīng)用。研究表明,1H-14NHSQC實驗可以實現(xiàn)牛奶、肝組織中的膽堿衍生物的高分辨、高靈敏檢測。然而,由于這些分子的縱向弛豫時間較長(T11s),通過常規(guī)采樣手段進行1H-14N二維HSQC采樣仍然需要較長的實驗時間,不利于在活體生物樣品中的應(yīng)用。在本論文中,我們將SOFAST-HMQC這—快速譜采集技術(shù)應(yīng)用于膽堿衍生物的檢測,通過快速1H-14N二維相關(guān)實驗提高相關(guān)物質(zhì)的檢測靈敏度。我們對1H-14N二維相關(guān)實驗的條件進行了優(yōu)化對比分析。以人工配制的3種不同膽堿衍生物的混合溶液作為標準樣品,通過一系列優(yōu)化實驗和模擬分析,獲得了適合三種膽堿衍生物同時檢測的1H-14N二維相關(guān)實驗的掃描間隔時間、極化轉(zhuǎn)移時間,以及SOFAST-HMQC實驗形狀脈沖的形狀和脈寬。在相同實驗條件下,將1H-14N SOFAST-HMQC實驗的膽堿衍生物檢測效果與文獻中選擇性同核去耦的1H-14NHSQC實驗的檢測效果進行了對比。結(jié)果表明,針對不同組分,1H-14N SOFAST-HMQC實驗的靈敏度是1H-14N HSQC實驗的1.47～2.05倍。通過實驗數(shù)據(jù)對比與理論分析,我們發(fā)現(xiàn)在1H-14N SOFAST-HMQC實驗中,縱向磁化的恢復(fù)效率更高,可以有效減少實驗的掃描間隔時間。最后,分別采用1H-14N SOFAST-HMQC技術(shù)和1H-14N HSQC技術(shù)對6個不同乳制品中的膽堿衍生物進行了分析。結(jié)果表明:與常規(guī)1H譜檢測相比,1H-14N二維相關(guān)技術(shù)對膽堿衍生物的檢測具有高選擇性,可消除復(fù)雜生物體系中其它背景信號的干擾;與1H-14NHSQC檢測技術(shù)相比,1H-14NSOFAST-HMQC技術(shù)具有更高的靈敏度,在單位時間內(nèi)信號增強可達1.32～3.00倍。此外,我們發(fā)現(xiàn),與牛奶樣品相比,奶粉溶液樣品中磷酸膽堿的相對含量明顯較低,而膽堿的含量卻相對較高,這可能是由奶粉中的膽堿添加劑所引起。
[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.
【學位授予單位】：中國科學院大學(中國科學院武漢物理與數(shù)學研究所)
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q6-33

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