pH响应性聚合物涂层的制备及在蛋白质富集中的应用

In order to solve the low sensitivity during the separation of the low abundance protein by CE, the pH responsive polymer coating with on-line concentration function for low abundance protein will be prepared in this project. The coating will be able to adsorb the protein under determined pH value and ionic strength, and then the protein adsorbed by the coating will be desorbed completely while changing the pH value and ionic strength, and the quantities of the protein will be increased while passing through the detector at the same time during the separation process. Firstly, the coating was prepared by immobilizing poly(2-oxazoline)(POX), weak polyacid, and weak polybase mixed polymer brushes on the surface of the materials through polydopamine (PDA) anchored coating. In which POX has a strong high protein-resistant property; weak polyacid and weak polybase possess responsive properties in much larger range of pH value; and PDA can attach to all types of material surface under aqueous environment. The relations between the protein adsorption quantity and the coating composition, pH value and ionic strength of external environment will be investigated via quartz crystal microbalance with dissipation, and so on. Based on above studies, POX, weak polyacid, and weak polybase will be immobilized onto the capillary inner surface through PDA anchored coating to form polymer coating. The on-line protein concentration mechanism of the coating will be elucidated through the study of the relationships of the protein recovery, protein resolution, pH value, and ionic strength. From this work, we will find a new way for the enhancement of the sensitivity for the separation of low abundance protein.

针对毛细管电泳分离低丰度蛋白质灵敏度低的问题，本项目拟制备具有在线富集蛋白质功能的pH响应性聚合物涂层，即某一pH和离子强度下，涂层吸附蛋白质，当改变pH和离子强度时，能将所吸附的蛋白质全部释放，在分离过程中，同时通过检测器的蛋白质量增加。首先，通过聚多巴胺（PDA）将2-噁唑啉类聚合物(POX)和聚弱酸、聚弱碱接枝在材料表面形成由混合聚合物刷组成的涂层，其中POX赋予涂层优异的抗蛋白质吸附性能，聚弱酸和聚弱碱赋予涂层广泛的pH响应性，而PDA在潮湿的环境下对材料仍具有极强的黏附性。通过石英晶体微天平等研究涂层的蛋白质吸附量与涂层的组成、环境的pH值和离子强度之间的关系，然后以此为基础将POX、聚弱酸和聚弱碱通过PDA接在毛细管内壁形成涂层，通过蛋白质回收率和分离度等与pH及离子强度之间关系的研究，揭示涂层在线富集蛋白质的机理，为低丰度蛋白质检测灵敏度的提高提供一种新的途径。

提高低丰度蛋白质的分析灵敏度是毛细管电泳（CE）分离分析蛋白质的重要课题之一。本项目制备出一系列对蛋白质具有吸附和释放功能的二元聚合物混合刷涂层。研究了涂层的组成、构象等和蛋白质吸附/释放之间的关系。然后将这种涂层用于熔融硅毛细管内壁的修饰，通过蛋白质分离条件的改变，实现了CE对蛋白质的在线富集，提高了蛋白质的分析灵敏度，具体工作如下：.1. 采用阳离子开环聚合（CROP）及可逆加成断裂链转移（RAFT）聚合分别合成了氨基封端的聚2-甲基-2-噁唑啉（PMOXA-NH2）和巯基封端的聚丙烯酸（PAA-SH），然后通过聚多巴胺，将PMOXA-NH2和PAA-SH依次接枝在材料表面，形成PMOXA/PAA二元混合刷。结果表明，涂层可以通过改变环境的pH及离子强度（I）从亲水性向疏水性转变，当PAA的理论链长较PMOXA长时，在改变环境pH及I后，PMOXA/PAA涂层上吸附的牛血清蛋白和溶菌酶可以在短时间内快速释放。.2. 以上述方法为基础，制备出一系列PMOXA/PAA混合刷修饰的熔融硅毛细管。结果表明，PMOXA/PAA涂层毛细管可分别对牛血清蛋白和溶菌酶进行在线富集，相比于未修饰毛细管在普通CE分离模式下所得到的结果，PMOXA/PAA涂层毛细管可将检测到的溶菌酶信号放大26倍，最低检测限（LOD）达到4.5×10-9 mg/mL，检测灵敏度提高了1×105 倍；牛血清蛋白的富集倍数也超过了5000。.3. 通过CROP及RAFT聚合，分别合成了聚2-甲基-2-噁唑啉-r-甲基丙烯酸缩水甘油酯（PMOXA-r-GMA）和聚2-甲基-2-噁唑啉-b-聚甲基丙烯酸缩水甘油酯（PAA-b-PGMA）、聚4-乙烯基吡啶-b-聚甲基丙烯酸缩水甘油酯（P4VP-b-PGMA）。然后通过旋涂及热交联的方法在玻璃或硅的表面分别制备了PMOXA/PAA及PMOXA/P4VP混合刷涂层。结果表明，涂层的组成可以通过调节旋涂液比例进行调控，通过混合涂层组成及环境pH值、I的调节，可以调节涂层的亲水性，从而达到调节牛血清蛋白吸附量和释放量的目的。随后制备了热交联PMOXA/PAA涂层毛细管，将涂层毛细管用于毛细管电泳在线富集溶菌酶。结果表明，当PAA链长是PMOXA链长的2.2倍时，溶菌酶的灵敏度增强因子为17.69，LOD为8.7×10-5 g/L。.