毛细管电泳激光诱导荧光分离检测脑汇编.ppt

毛细管电泳激光诱导荧光分离检测脑肠肽 研究意义 生物活性肽是一类具有重要生物学功能的多肽，生物活性肽的研究是生命科学，神经生理学、药理学和临床医学领域的前沿课题，开展生物活性肽的分离分析研究，寻求灵敏，高效，快速和简便的分析方法具有重要意义。生物活性多肽在内源性物质中占有非常重要的地位。作为激素、神经递质、免疫调节器、辅酶或酶阻剂、药物、毒素和生物抗体，它们参与控制和调节生物体中的许多重要的生理过程。当它们在体内分泌失常或代谢失调，就导致严重的病变。脑肠肽主要分布在中枢神经系统例如下丘脑及肠消化道内。脑肠肽能调节胃肠的蠕动，调节胃肠的分泌作用和吸收作用，在调节胃肠道系统的生理活性过程中起重要作用。神经紧张肽等具有降低血压，止痛，能控制肠道收缩，增加血管的通透性等重要生理作用。已有的测定方法 RIA CCK-4 Gel Chromatography-RIA CE-MS NT or NT8-13 CZE-UV TLC HPLC-UV ELISA CE-MS CE-LIF的优点 CE由于具有高的分离效率,样品用量少,快速,简便,环保等特点,因而是分离生物活性物质如蛋白质,多肽和核酸的强有力的工具.由于紫外检测受毛细管短光程的限制,CE-UV的检测灵敏度为10-510-6mol/L,CE-LIF检测是CE最灵敏的检测方法之一.Ar+激光器(激发波长488nm,发射波长520nm)是最常用的一种商品化的VIS区的激光源.利用自身的荧光进行检测,仅局限与那些具有芳香环氨基酸残基(色氨酸和酪氨酸)的多肽分子.对于大部分的肽来说,事先要经过衍生化处理使其转化为具有荧光基团的化合物.FITC（荧光素异硫氰酸盐）1,NT8-13:5.0 x10-9 M;2,NT:1.0 x10-8M,NKB:5.0 x10-9 M,CCK:5.0 x10-9 M PH 9.5 110mM borate,20%methanol 0.5psi-10s Peptide Linear Regression equation Correlation coefficient Linear Range Detection limit NT 8-13 Y=1160.1+2662.5 0.9953 120nM 0.5nM NT Y=688.24+375.81 0.9963 240nM 1.0nM NKB Y=455.6+464.14 0.9983 220nM 1.0nM CCK Y=754.87+1645.8 0.9970 120nM 0.8nM 提高毛细管检测灵敏度的方法 1.通过设计特殊类型的检测池,扩展在毛细管内的检测光程.2.发展高灵敏度的检测器,如激光诱导荧光检测,化学发光检测,电化学检测等等 3.采用样品浓缩富集技术,包括电堆积、场放大、等速电泳、pH梯度、不连续缓冲液富集、固相萃取、膜富集等。Transient pseudo-isotachophoresis for sample concentration in capillary electrophoresis Many water miscible organic solvents such as acetonitrile,acetone and small alcohols can function as a terminating ion in transient isotachophoresis,which leads to sample concentration on the capillary.It is suggested that this method could be termed transient“pseudo-sotachophoresis”(pseudo-ITP).Because of their low conductivity,these water miscible organic solvents provide the high field strength necessary for band sharpening similar to that provided by the terminating ion.Salts,when present in such samples act briefly as leading ions,migrating rapidly in the organic solvent until they are slowed at the interface of the separation buffer.1,NT8-13:5.0 x10-9 M;2,NT:1.0 x10-8M,NKB:5.0 x10-9 M,CCK:5.0 x10-9 M The concentration of NaCl inthe aqueous phase was held constant at 1%(w/v)Running buffer:PH 9.5 110mM borate,20%methanol 5psi-25s 1,NT8-13:5.0 x10-9 M;2,NT:1.0 x10-8M,NKB:5.0 x10-9 M,CCK:5.0 x10-9 M The percentage of methanol in the sample was kept constant at 70%(v/v)Running buffer:PH 9.5 110mM borate,20%methanol 5psi-25s 1,NT8-13:1.0 x10-9 M;2,NT:2.0 x10-9M,NKB:1.0 x10-9 M,CCK:1.0 x10-9 M The percentage of methanol in the sample was kept constant at 70%(v/v);the concentration of NaCl inthe aqueous phase was held constant at 1%(w/v)Running buffer:PH 9.5 110mmM borate,20%methanol 5psi-25s Peptide Linear Regression equation Correlation coefficient Linear Range Detection limit NT 8-13 Y=238855-48646 0.9945 0.2510nM 0.04nM NT Y=67587-13525 0.9972 0.2510nM 0.1nM NKB Y=63808-18370 0.9998 0.310nM 0.2nM CCK Y=85934-31931 0.9974 0.410nM 0.08nM 富集后的线性及检出限富集后的线性及检出限 Y:H X:nM