基于活性光接枝化学的聚合物基三维蛋白质芯片设计制备及其性能研究

Protein biochips have great potential applications in the fields of disease detection and diagnosis, drug discovery, personalized medicine and proteomics. Surface properties of substrates are crucial parameters determining sensitivity of protein chips. In our study, polymers were chose as substrate instead of widely used glass. Functional polymer brushes with excellent resistant to non-specific protein absorption property were firstly introduced onto substrate via living photograft polymerization. Then three dimensional (3D) cross-linked microarrays were further fabricated on the first polymer brush layer by secondary graft polymerization to covalently immobilize proteins. This elaborate design aims to achieve followed results: antifouling background surface could significantly reduce the background fluorescence intensity, and the 3D protein microarrays exhibited higher signal fluorescence intensity due to higher protein immobilization density. We will explore the characteristics of photograft polymerization on polymer substrates associated with our design. On this basis we will further tune the physical and chemical properties of 3D microarrays and methods for protein immobilization, finally developing a new strategy of surface modification to realize high protein binding capacity, high biological activity preservation and low background signal for protein biochip. This research will open up new avenues to develop high sensitive polymer-based protein biochips.

蛋白质芯片在临床诊断、新药筛选、个体化医疗及蛋白质组学等领域有广阔的应用前景。基材表面物理化学特性是影响蛋白质芯片检测灵敏度的重要因素，本课题拟以聚合物材料代替传统玻璃基材，通过活性光接枝技术，在其表面先接枝具有抗蛋白质非特异性吸附的功能聚合物，再二次接枝具有交联结构的三维微阵列以固定蛋白，由此制备能够高密度固定蛋白且背景信号低的高灵敏度蛋白质芯片。研究活性光接枝法改性聚合物基材的特点和规律，在此基础上调整三维微阵列的物理特性、化学结构及蛋白质固定方式，探索能够实现高蛋白固定量、高蛋白活性保持率和低背景信号的表面改性新策略，为研发高灵敏度聚合物基蛋白质芯片提供新思路。

蛋白质芯片技术在临床诊断、药物开发、蛋白质组学等领域有重要的应用前景，基材表面物理化学特性是影响蛋白质芯片检测灵敏度的重要因素。按照原定计划，本项目利用可见光活性接枝技术，通过表面聚合反应设计发展了多种在聚合物表面构筑三维结构的方法，以此为基材成功制备了系列高灵敏度蛋白质芯片。主要结果如下：（1）基于紫外/可见光活性接枝聚合，开发了一种在抗蛋白污染层改性聚合物基材表面制备三维凝胶微阵列的技术，通过控制凝胶微阵列结构和高度，实现了对探针分子的高密度固定。（2）利用可见光交联接枝聚合反应发展了一种制备多层次复杂结构三维蛋白微阵列的技术，可用于两种及以上目标蛋白的检测。在此基础上开发了在聚合物载体表面分隔固定化酶方法，实现了相互抑制酶的高活性分隔固定。（3）基于可见光引发接枝聚合方法，通过调控聚合物表面润湿性和可交联微液滴组成，开发了可见光引发原位交联接枝聚合制备生物芯片新策略。（4）开发了一种通过光接枝聚合制备三维凝胶层高效固定生物分子探针的方法。通过调节环氧基团在三维交联层中的含量及交联层厚度，可以方便控制探针分子的固定效率和密度。与传统的二维生物芯片相比，三维芯片的探针固定效率显著提高，可达80%以上。.本项目在《Chemical Communication》、《Macromolecular Rapid Communications》、《Journal of Materials Chemistry B》、《Langmuir》等期刊发表SCI收录论文11篇，获得2项授权发明专利, 申请发明专利3项。培养博士研究生4人，硕士研究生1人。