Development of microchip liquid chromatography for ultra-sensitive analysis of trace amounts of bio-molecules.

本研究的目的是发展用于超灵敏分析痕量生物分子的微芯片液相色谱仪。首先，研制了具有苯并呋喃结构的水溶性荧光衍生化试剂。这些试剂有望防止分析物吸附到分析仪器上，并提高灵敏度。其次，合成了荧光双环核酸--脱氧胞苷衍生物。该结构可作为荧光衍生化试剂。利用合成的试剂和高效液相色谱法，我们建立了灵敏的生物分子和药物检测方法。这些方法适用于微芯片高效液相色谱的微量分析。质谱学是一种很有前途的微芯片液相色谱检测方法。因此，开发了MS衍生化试剂，并将其应用于生物样品中脂肪酸等生物分子的分析。这些方法也适用于微芯片液相色谱分析。对于小规模的分离，我们在毛细管柱和芯片上合成了整体柱。最后，我们尝试开发了激光激发的荧光检测系统。物镜附近的化合物是敏感可检测的。因此，在芯片流动区制备了小于5μm的狭窄空间，用于激光激发荧光检测。该方法能够对荧光化合物进行灵敏的检测。今后，本研究开发的衍生化试剂、微分离整体柱和荧光微检测系统等方法将在微芯片液相色谱微分析中得到总结。

The purpose of this study is development of microchip liquid chromatography for ultra-sensitive analysis of trace amounts of bio-molecules. At first, water-soluble fluorescent derivatization regents having benzofurazan structure were developed. These reagents are expected to prevent the adsorption of the analytes to the analytical instruments and improve the sensitivity. Next, fluorescent bi-cyclic nucleic acid, deoxycitidine derivative was synthesized. This structure might be useful as the fluorescent derivatization reagents. Using the synthesized reagents and high-performance liquid chromatography, we have developed the sensitive determination methods of bio-molecules and drugs. These methods are applicable for the microanalysis using microchip liquid chromatography. Mass spectrometry(MS) is promising for the detection method of microchip liquid chromatography. Thus, the derivatization reagents for MS were developed and applied to the analysis of bio-molecules such as fatty acids in biological samples. These methods are also applicable for the microchip liquid chromatography. For the small scale separation, we have synthesized monolithic column in capillary column and on microchip. Finally, we have tried to develop the fluorescence detection system by laser excitation. The compounds near the object lens are sensitively detectable. Therefore, the narrow space less than 5 μm was prepared in the flow area of the microchip and used for the laser excitation fluorescence detection. This method enabled the sensitive detection of the fluorescent compounds. In the future, the methods developed in this study, the derivatization reagents, the micro separation monolithic columns and the fluorescent micro detection system, will be summarized in the microanalysis using microchip liquid chromatography.