State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, Jilin 130022, P. R. China.
University of Chinese Academy of Sciences , Beijing 100049, P. R. China.
ACS Appl Mater Interfaces. 2017 Dec 20;9(50):44241-44250. doi: 10.1021/acsami.7b15445. Epub 2017 Dec 11.
Matrix metalloproteinases (MMPs) are important biomarkers and potential therapeutic targets of tumor. In this report, a peptide microarray-based fluorescence assay is developed for MMPs inhibitors evaluation through immobilization of biotin-modified peptides on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush-modified glass slides. After biotin is recognized with cyanine 3 (Cy3)-modified avidin (Cy3-avidin), the microarrays can produce strong fluorescence signal. The biotin moieties detach from microarray, when the biotin-modified peptide substrates are specially cleaved by a MMP, resulting in decreased fluorescence intensity of the microarray. The decreasing level of fluorescence intensity is correlated with the MMP inhibition. Nine known MMP inhibitors against MMP-2 and MMP-9 are evaluated by the assay, and the quantitative determination of inhibitory potencies (half maximal inhibitory concentration) are obtained, which are comparable with the literatures. Two biocompatible fluorogenic peptides containing MMP-specific recognition sequences and FAM/Dabcyl fluorophore-quencher pair are designed as activatable reporter probes for sensing MMP-2 and MMP-9 activities in cell and in vivo. The peptide microarray-based results are well verified by the cell inhibition assay and in vitro fluorescence imaging, and further confirmed by the in vivo imaging of HT-1080 tumor-bearing mice.
基质金属蛋白酶（MMPs）是肿瘤的重要生物标志物和潜在治疗靶点。在本报告中，通过将生物素修饰的肽固定在聚（甲基丙烯酸缩水甘油酯-共-2-羟乙基甲基丙烯酸酯）（P（GMA-HEMA））刷修饰的玻片上，开发了一种基于肽微阵列的荧光分析方法，用于 MMPs 抑制剂的评估。生物素与 Cy3 修饰的亲和素（Cy3-avidin）结合后，微阵列会产生强烈的荧光信号。当 MMP 特异性切割生物素修饰的肽底物时，生物素部分会从微阵列上脱离，从而导致微阵列的荧光强度降低。荧光强度的降低水平与 MMP 抑制作用相关。该方法评估了 9 种针对 MMP-2 和 MMP-9 的已知 MMP 抑制剂，得到了抑制效力（半数最大抑制浓度）的定量测定结果，与文献值相当。设计了两种含有 MMP 特异性识别序列和 FAM/Dabcyl 荧光团-猝灭剂对的生物相容性荧光肽作为可激活的报告探针，用于感测细胞和体内 MMP-2 和 MMP-9 的活性。基于肽微阵列的结果通过细胞抑制实验和体外荧光成像得到了很好的验证，并通过 HT-1080 荷瘤小鼠的体内成像进一步得到了证实。
