检测生物样品中肿瘤标志物环氧合酶-2的管内磁富集-毛细管电泳方法研究

Cyclooxygenase-2 (COX-2) is an inducible enzyme, whose expression is low or undetectable in most cells and tissues, but increases dramatically upon stimulation, inflammatory and pain. Furthermore, an elevated COX-2 level is a prominent finding in most of human malignant tumors and is correlated with tumor inflammogenesis, progression, metastasis, and radiosensitive. Therefore, COX-2 is a kind of the ideal tumor marker. Up to now, there have been two categories of methods developed for the detection of COX-2: one is based on the antigen and antibody response, however, its operation is tedious; the other is based on the reaction between COX-2 and its inhibitor, but it is limited by the targeting property of inhibitors, especially the interference from COX-1 or carbonic anhydrase is hard to eliminate. In a word, it is very urgent to develop a sensitive, selective and high throughout method for the detection of COX-2 in biological sample with small volume.. Capillary electrophoresis (CE) is one of the principal separation and analysis techniques with versatile advantages of high efficiency, short analysis time, low sample and reagent consumption. The development of various kinds of CE separation modes and on-column preconcentration strategies makes it a powerful tool for a wide range of applications from small organic molecules to biomacromolecules. Based on this, CE will also be an attractive tool to separate and determine COX-2 in biological samples. .Among the existing preconcentration technologies for the low abundance proteins, the magnetic separation based on functionalized magnetic nanoparticles has gained popularity as a facile method for the simultaneous capture and enrichment of bound molecules. In this project, the multifunctional magnetic nanoparticles (M-F-I NPs) will be prepared by immobilizing both fluorescent molecules and COX-2 inhibitor onto the surface of magnetic nanoparticles after coating with SiO2 layer through a linker molecule with three branched chains. The prepared M-F-I NPs are expected to possess three functions, i.e., magnetic response, fluorescence labeling, and targeted marker for COX-2. By adding M-F-I NPs into a biological sample solution, COX-2 will bind selectively onto the M-F-I NPs surface via specific interaction with inhibitor and be captured from most interferences. .The capillary column will be used as the preconcentration channel, which is especially suited for the biological sample with small volume. When the incubated products of M-F-I NPs and COX-2 are injected by applying pressure as a long plug into the capillary under the magnetic field, the in-tube trapping and preconcentration of COX-2 bound M-F-I NPs is expected and further application of the pressure provides a foregoing discharge of other unbound components, which will result in the purification of the trapped target analytes. Thus, a high-throughput magnetic in-tube preconcentration strategy is developed, and it can simultaneously capture the magnetic nanoparticles from the different samples on the different capillary columns. Next, the COX-2 bound fluorescent molecules will be eluted from M-F-I NPs surface owing to the breakage of the disulfide bond (S-S) in the linker molecule, and separated by capillary electrophoresis (CE) and detected with laser-induced fluorescence (LIF). Furthermore, the developed CE system combined with magnetic in-tube preconcentration will be applied to the determination of COX-2 in different biological samples such as plasma, cell and animal tissue. The relation between the COX-2 content and the different physiological status such as normality, inflammation and cancer will be deeply discussed..The developed CE system combined with magnetic in-tube preconcentration is very suited to the biological sample with small volume. The successful implementation of the project will provide a simple, selective and high throughout method to find COX-2 for the early detection of cancer.

环氧合酶-2（COX-2）是一种诱导酶，在正常组织、细胞中不表达或低表达，但当组织受损、发炎时表达增强，且与众多癌症的发生、发展、转移及术后有关，是一种理想的肿瘤标志物。目前，检测COX-2的方法有两类，一类是基于抗原-抗体反应的方法，但操作繁琐；另一类是基于COX-2与其抑制剂反应的方法，但受限于抑制剂的靶向性。因此，建立高灵敏、高选择、高通量、简便地检测极小体积生物样品中COX-2的方法是十分必要的。本项目拟以抑制剂为靶向分子、荧光分子为信号输出单元、磁性纳米颗粒为分离载体，制备可从复杂样品中选择性捕获COX-2的多功能磁性纳米复合材料，建立高通量的管内磁富集-毛细管电泳体系，用于血液、细胞及动物组织中COX-2的测定，并深入探讨COX-2浓度与动物正常、炎症、癌症等生理状态的关系。研究成果对于癌症的早期发现和诊断具有重要意义。

本项目利用蛋白相变体可以自组装形成具有粘附性薄膜的特性，发展了可以简单、快速地制备蛋白涂层毛细管柱的新方法。经一步法原位制备了溶菌酶、牛血清白蛋白及-乳白蛋白相变超分子薄膜涂层毛细管柱，并以所制备的涂层毛细管为分离通道，建立了分离药物、神经递质及氨基酸等对映异构体的毛细管电色谱新方法。基于共价有机框架材料(COFs)孔隙率高、比表面积大、热稳定性良好等特性，通过改变COFs制备条件，发展了室温下制备腙键类COFs和3D COFs涂层毛细管柱的原位生长法，制得的COFs涂层均匀、致密，以其为分离通道，提升了开管毛细管电色谱方法对中性、弱极性及异构体物质的分离能力，如COF-LZU8涂层毛细管已用于几种常见的塑料制品中邻苯二甲酸酯(PAEs)的检测。设计并合成了几种可进行细胞及活体内目标物检测和成像研究的新型荧光探针，如DPAS-Cys可定位脂滴并检测其中的半胱氨酸，双通道荧光探针MB-NAP-N3可通过红色和绿色荧光通道分别和连续检测细胞及斑马鱼外源性和内源性产生的HClO和H2S。开发了COFs材料的光敏氧化酶催化活性，如在光照条件下，COF-300-AR可催化氧化无色底物TMB快速转变为蓝色产物TMBox，而交替进行开-关灯源的操作，会导致TMBox的吸光度产生类似台阶状的变化，进一步验证了该催化反应的光控性。基于COF-300-AR的光敏氧化酶催化特性，发展了测定细胞裂解液中谷胱甘肽和大鼠血清中五羟色胺含量的新方法。发展了以COF-300-AR为模板原位制备贵金属纳米颗粒的新方法，所制得PtNPs@COF-300-AR负载了粒径均一、分布均匀的超细Pt NPs，在无光照情况下，也具有较高的类氧化酶催化活性。进一步深入研究发现Cu2+可以有效调节TpBpy的光敏氧化酶活性，并且大大拓宽了其pH使用范围，即使在中性溶液中，TpBpy仍有一定的催化能力。合成了一些新型硅量子点并研究了其在生命分析化学中的应用。建立了几种基于等温核酸扩增检测糖苷酶的新方法。发展了几种高灵敏分析检测复杂样品中色氨酸及其代谢物、嘌呤类化合物、脱氧核苷及其氧化产物的液相色谱/毛细管电泳-高分辨质谱新方法。本项目的开展显著提高了毛细管电泳尤其毛细管电色谱的理论和应用研究，也拓宽了纳米材料和等温核酸扩增的应用范围。该项目的研究结果对生命分析化学和纳米分析化学的发展具有重要意义。