Capillary isoelectric focusing of proteins with liquid core waveguide laser-induced fluorescence whole column imaging detection
Capillary isoelectric focusing of proteins with liquid core waveguide laser-induced fluorescence whole column imaging detection
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DOI：
10.1021/ac034587m
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发表时间：
2003-09-15
影响因子：
7.4
通讯作者：
Pawliszyn, J
中科院分区：
文献类型：
--
作者：
Liu, Z;Pawliszyn, J
研制了一种毛细管等电聚焦液芯波导（LCW）激光诱导荧光全柱成像检测系统。采用聚四氟乙烯af2400毛细管作为分离通道和轴向照明LCW。激发光从毛细管的一端引入，在毛细管内向前传播。由于Teflon AF 2400毛细管的折射率（n = 1.29-1.31）低于水的折射率（n = 1.33），因此内反射非常明显。采用Teflon AF 2400毛细管避免了使用高折射率添加剂，如甘油，使系统具有更广泛的应用。由于其惰性化学性质，毛细管对蛋白质的吸附和电渗透性有限；因此，可以避免用聚合物溶液进行毛细管预处理和将聚合物添加剂添加到样品混合物中。用这种方法检测了三种类型的蛋白质：天然荧光蛋白、共价标记蛋白和非共价标记蛋白。此外，还对变性条件下的CIEF进行了研究，并发现了其优于原生模式的几个优点。与市售紫外检测仪相比，分离效率和峰值容量相近，检测灵敏度提高了3-5个数量级。
A capillary isoelectric focusing (CIEF) system with liquid core waveguide (LCW) laser-induced fluorescence whole column imaging detection was developed in this study. A Teflon AF 2400 capillary was used as both the separation channel and the axially illuminated LCW. The excitation light was introduced at one end of the capillary, and propagated forward within the capillary. As the Teflon AF 2400 capillary has a refractive index (n = 1.29-1.31) lower than that of water (n = 1.33), total internal reflection was very apparent. The employment of the Teflon AF 2400 capillary avoided the use of high refractive index additives such as glycerol, accommodating the system to wider applications. Due to its inert chemical properties, the capillary exhibited limited protein adsorption and electroosmotic flow; thus, the need for capillary preconditioning with polymeric solution and the addition of polymeric additives into the sample mixture can be avoided. Three types of proteins, naturally fluorescent proteins, covalently labeled proteins, and noncovalently labeled proteins, were examined using this method. CIEF under denaturing conditions was also explored, and several advantages over the native mode were found. When compared to a commercially available instrument with UV detection, the separation efficiency and peak capacity were similar while the detection sensitivity was enhanced by 3-5 orders of magnitude.