由多个移动反应界面所形成的新型等速电泳高效富集原理与方法研究

As an important model for separation and stacking, isotachophoresis (ITP) not only can be applied in many separation techniques, but also be employed with the other analytical technologies. With ITP technique, the detection efficient can be improved significantly. The previous research results which was obtained by the applicant indicated that the mechanism of the stacking metal ions for the ITP formed by multiple moving reaction boundaries(mMRB) was not the difference of the mobilily, which is the stacking mechanism of the traditional ITP, but the difference in the reaction equilibrium constant. The project focuses on the investigation of the stacking theory and methodology on the novel ITP model formed by mMRB system, mainly including: (1) the theory investigations and relevant experiments on the novel ITP mode and the mathmatical model; (2) the theoretical and experimental study on the controlling mechanism of the novel ITP model; (3) the theoretical and experimental study on the quantitative analysis of the targets in the novel ITP model. The aims of the project are to extablish the mathmatical mode, theory and methodology of the novel ITP, manifest the intrinsic principle of ITP phenomenon, promot the research of ITP mechanism, and provid the theoretical basis for the applicant of the new ITP mode. The significances of the project are that developing ITP theory, expanding the space for the development of ITP, discovering the new area of MRB, and providing a new method for the high sensitivity detection.

等速电泳(ITP)作为重要的富集、分离模式，广泛应用于多种分离技术，或与其他技术联用，实现样品高效富集，提高检测灵敏度。申请人前期研究表明由多个移动反应界面(mMRB)所形成的ITP高效富集的机理不能用经典ITP理论（即淌度差异）解释，而是与络合反应平衡常数差异有关。本课题开展由mMRB所形成的ITP高效富集原理与方法研究，内容包括：1、mMRB数学模型及由该mMRB所形成的新型ITP模式理论和实验研究；2、ITP新模式的控制理论与实验研究；3、在ITP新模式中定量分析方法的理论与实验研究。研究目标是建立基于mMRB的ITP数学模型、理论和方法，阐明新型ITP现象背后的本质原理，为这一ITP新模式高效、简便富集痕量目标物的实际应用提供理论基础。研究意义在于：创新ITP理论，拓展ITP发展空间，同时发掘MRB研究新领域，为痕量目标化合物分析检测提供潜在新方法。

由多个移动反应界面(mMRB)所形成的ITP高效富集的机理完全不同于经典ITP理论，本课题开展由mMRB所形成的ITP高效富集原理与方法研究，以带有颜色、便于肉眼观察的金属钴离子和铜离子与金属螯合剂EDTA作为模型离子，通过调控多个实验参数对mMRB所形成的ITP的移动速率及金属离子富集效率进行测定，由此建立该mMRB所形成的新型ITP模式理论和富集效率控制方法。在ITP新模式中，利用PDMS材料设计制作多通道微流控直读芯片，基于EDTA 光催化原理及凝血酶与适配体之间的特异性结合，分别设计了ET-NB传感器和磁性纳米颗粒-凝血酶-辣根过氧化物酶复合结构。结合新型ITP定量分析及富集效率控制的理论，将所开发的芯片和实验方法应用于实际样品检测。ET-NB传感器用于乳制品中三聚氰胺含量测定，其测定婴幼儿奶粉和牛奶三聚氰胺的检测限（LOD）值分别为0.1μM和0.2μM，比FDA规定的婴儿配方奶粉和牛奶的安全限值（8μM，20μM）低80和100倍；牛奶的定量加标回收率为87％-108％，婴幼儿配方奶为90％-107％。构建的凝血酶检测方法用于尿样以及血清样本中外标凝血酶进行检测，平均回收率分别为99.62%和100.23%，检测方法能够有效地对抗复杂生物样品中各种组分对于检测的干扰。所有实验结果表明课题所建立的基于mMRB的ITP数学模型、理论和方法，为这一ITP新模式高效、简便富集痕量目标物的实际应用提供理论基础，同时发掘MRB研究新领域，为痕量目标化合物分析检测提供潜在新方法。