光纤LED诱导荧光毛细管电泳检测技术的研究与应用
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摘要
高效毛细管电泳(HPCE)是广泛应用于生命科学领域(如氨基酸、多肽、蛋白质、核酸、单细胞分析)及其它领域(包括食品化学、药物化学、环境化学、毒物学、医学和法医学等)的一种新型分离分析技术。具有自动化程度高、分离速度快、样品用量少、分离效率高、检测模式多样等特点。HPCE中的荧光检测技术因其具有高的灵敏度而成为检测痕量样品的首选。本文以发展光纤发光二极管(光纤LED)诱导荧光HPCE检测技术为出发点,并将其运用于生物体液中游离氨基酸、烟叶中游离氨基酸的分离分析研究。实验结果证明此新的荧光检测技术不仅简单易行、耗费低,而且具有高的检测灵敏度和低的背景噪音,方法准确可靠。1.光纤LED诱导荧光HPCE检测仪测定生物体液样品中游离的兴奋性氨基酸。兴奋性氨基酸作为脑内主要的兴奋性神经递质对正常的神经生理活动有着十分重要的作用,它和某些神经系统疾病尤其是中枢神经系统疾病密切相关,因此研究体液中的兴奋性氨基酸有着重要的意义。本文实验中所选择的生物体液样品包括兔血清、人血清、人体脑脊液。以2,3-萘二甲醛(NDA)标记的游离氨基酸衍生产物作为检测对象,通过对分离电压、缓冲溶液浓度和pH值实验条件进行优化,取得了良好的分离效果。优化的实验条件为:有效长度为50cm的非涂层熔融硅毛细管,内径为75μm;电泳缓冲液为10mmol/L的四硼酸钠缓冲液(pH9.3);分离电压20kV;检测温度为室温;检测器为自组装的光纤LED诱导荧光检测器,采用最大发射波长为425nm的LED。在此条件下,谷氨酸和天冬氨酸的最低检测限分别达到2.1×10~(-8)和2.3×10~(-8)mol/L;相对标准偏差均小于5.0%;谷氨酸和
The high performance capillary electrophoresis (HPCE) is an new separating and analyzing technique, which has been widely applied to the determination of samples in the life sciences field ( including amino acids, polypeptide, protein, nucleic acid, single cell ) and other fields (such as food chemistry, medicinal chemistry, environmental chemistry, toxicology, iatrology, medical jurisprudence and so on ). HPCE has some advantages of high automation, short run time, minimum sample cost, high separation efficiency and diversification of detection mode etc. The high sensitivity makes fluorescence detector in HPCE an attractive technique for the determination of small quantities of matter. Developing a new type of capillary electrophoresis optical fiber light-emitting diode(CE-optical fiber LED) induced fluorescence detector is an important topic. It’s utility was successfully demonstrated by the separation and determination of free amino acids in biological fluids and tobacco. The experimental results indicated that this new detector offers some advantages, such as simplicity and flexibility, low back ground noise, low cost, high sensitivity and so on.
1. Determination of excitatory amino acids (EAAs) in biological fluids by CE-optical fiber LED induced fluorescence detection. The EAAs are the important
The high performance capillary electrophoresis (HPCE) is an new separating and analyzing technique, which has been widely applied to the determination of samples in the life sciences field ( including amino acids, polypeptide, protein, nucleic acid, single cell ) and other fields (such as food chemistry, medicinal chemistry, environmental chemistry, toxicology, iatrology, medical jurisprudence and so on ). HPCE has some advantages of high automation, short run time, minimum sample cost, high separation efficiency and diversification of detection mode etc. The high sensitivity makes fluorescence detector in HPCE an attractive technique for the determination of small quantities of matter. Developing a new type of capillary electrophoresis optical fiber light-emitting diode(CE-optical fiber LED) induced fluorescence detector is an important topic. It’s utility was successfully demonstrated by the separation and determination of free amino acids in biological fluids and tobacco. The experimental results indicated that this new detector offers some advantages, such as simplicity and flexibility, low back ground noise, low cost, high sensitivity and so on.
1. Determination of excitatory amino acids (EAAs) in biological fluids by CE-optical fiber LED induced fluorescence detection. The EAAs are the important
引文
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