Wu Yeping, Wang Anzhi, Ding Xiaokang, Xu Fu-Jian
State Key Laboratory of Chemical Resource Engineering, §Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education, and ⊥Beijing Laboratory of Biomedical Materials, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology , Beijing 100029, China.
ACS Appl Mater Interfaces. 2017 Jan 11;9(1):127-135. doi: 10.1021/acsami.6b12033. Epub 2016 Dec 22.
The development of new materials for fast and sensitive protease assay is in demand for timely diagnosis of diseases, such as cardiovascular disease, cancers, and Alzheimer disease. Herein, poly(methacrylic acid) (PMAA) brushes were synthesized from the surfaces of silica nanoparticles via surface-initiated atom transfer radical polymerization (ATRP), and functionalized with series of proteolytically cleavable peptides for highly sensitive protease assay. Upon the proteolytic cleavage of the peptides, a short peptide fragment with fluorescent tag (GGK-FITC) is released to the solution, which can be easily detected with a benchtop fluorescence microscope. The grafting densities of PMAA brushes and peptides can be readily tuned by controlling the monomer concentrations of sodium methacrylate in the ATRP reaction. Because of the three-dimensional architecture of PMAA brushes, the loading amount of peptides can reach 21.4% of the total weight of functionalized silica particles (22.4 peptides/nm), which is much higher than direct immobilization on silica nanoparticles without polymer brushes. Because of the high loading density of peptides, the limit of detection (LOD) of trypsin can reach 1.4 pM in buffer solution or 2.6 nM in nondiluted serum. By rational design of peptide substrates, the peptide-functionalized PMAA brushes can be readily expanded to detect other proteases, such as matrix metalloproteinase-2 (MMP-2), a virtual biomarker for many cancers, with an LOD of 1.1 pM. The proteolytically cleavable peptide-functionalized PMAA brushes offer a starting point for fast and sensitive protease assay.
开发用于快速灵敏蛋白酶检测的新材料对于及时诊断疾病（如心血管疾病、癌症和阿尔茨海默病）至关重要。在此，通过表面引发的原子转移自由基聚合（ATRP）从二氧化硅纳米颗粒表面合成了聚（甲基丙烯酸）（PMAA）刷，并通过一系列可被蛋白酶裂解的肽进行功能化，以实现高灵敏蛋白酶检测。当肽被蛋白酶裂解后，带有荧光标签的短肽片段（GGK-FITC）被释放到溶液中，可通过台式荧光显微镜轻松检测到。通过控制ATRP反应中甲基丙烯酸钠的单体浓度，可以很容易地调节PMAA刷和肽的接枝密度。由于PMAA刷的三维结构，肽的负载量可达功能化二氧化硅颗粒总重量的21.4%（22.4个肽/nm），这远高于直接固定在没有聚合物刷的二氧化硅纳米颗粒上的情况。由于肽的高负载密度，胰蛋白酶在缓冲溶液中的检测限（LOD）可达1.4 pM，在未稀释血清中可达2.6 nM。通过合理设计肽底物，肽功能化的PMAA刷可以很容易地扩展用于检测其他蛋白酶，如基质金属蛋白酶-2（MMP-2），这是许多癌症的虚拟生物标志物，其LOD为1.1 pM。可被蛋白酶裂解的肽功能化PMAA刷为快速灵敏的蛋白酶检测提供了一个起点。
