基于二硫化碳改性表面的无细胞表达系统构建蛋白质芯片感应表面的研究

The protein chip based on total internal reflection imaging ellipsometry has been a novel but promising technique for protein-protein interaction analysis. However, several drawbacks in its biosensing interface remain a significant barrier for its development and further application in the field of fundamental biology and clinical laboratory test. In order to overcome these problems, the studies on the expression method and immobilization approach of protein ligand, as well as surface modification strategy for protein chip interface are planned to carry out in this investigation. Focusing on the crucial function of biosensing interface, cell-free protein synthesis system and carbon disulfide surface modification method are combined to construct a practical biosensing interface which is expected to present good bio-activity of protein ligand and the capability to inhibit non-specific adsorption. Thereafter, according to the feature of the biosensing interface and the principle of ellipsometry signal response, the setup of the optical system is optimized to improve its detection sensitivity and dynamic range, prompting its biology and clinic application. The desired achievements of this project can not only be used for the protein chip based on total internal reflection imaging ellipsometry, but also fulfill the need for various kinds of protein analysis methods which employ gold slides as the substrates.

全内反射椭偏成像蛋白质芯片作为一种新型蛋白质相互作用检测分析方法，具有重大应用前景。但是，该蛋白质芯片的感应表面，仍存在着配基活性表现不佳和非特异性吸附的问题，阻碍了该蛋白质芯片进一步的应用。针对现有不足，本项目拟从配基分子表达、芯片表面改性和配基装配方法入手，着重考虑如何提高配基分子活性表现和阻隔非特异性吸附等科学问题，同时关注蛋白质芯片反应界面性质和光学信号传导等因素，耦合利用无细胞表达系统和二硫化碳表面改性技术，尝试在金表面上进行配基分子的表达和原位活性装配，探索构建配基分子数量可控、具有高生物活性表现和能够抑制非特异性吸附的实用化感应表面的方法。在此基础上，针对感应表面的性质，优化椭偏光学器件设置，以期提高该蛋白质芯片的检测灵敏度和动态范围等性能指标，推进其在生物学和临床医学领域的应用。本项目的预期研究结果具有广泛适用性，能够用于以金表面为基底的其它蛋白质相互作用分析手段。

全内反射椭偏成像蛋白质芯片作为一种新型蛋白质相互作用检测分析方法，具有重大应用前景。但是，该蛋白质芯片的感应表面，仍存在着配基活性表现不佳和非特异性吸附的问题，阻碍了该蛋白质芯片进一步的应用。针对现有不足，本项目针对如何提升配基分子活性表现和如何抑制非特异性吸附两个核心科学问题，从配基分子表达、芯片表面改性和配基装配方法入手，系统研究了二硫化碳与金表面的相互作用、配基分子装配的控制与感应表面的构建、感应表面抑制非特异性吸附的能力以及感应表面与光学信号传导的关系四方面的内容，利用无细胞表达系统在二硫化碳改性表面上构建了感应表面，提高了全内反射椭偏成像蛋白质芯片的检测性能，推进其在基础生物学和临床医学领域的应用，完成了项目研究计划的全部既定工作任务同时达到了预期目标。