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.
Anal Chem. 2017 Jun 20;89(12):6749-6757. doi: 10.1021/acs.analchem.7b01037. Epub 2017 May 26.
Matrix metalloproteinases (MMPs) are closely associated with cancer cell invasion and metastasis. Herein, a fluorescence resonance energy transfer (FRET)-peptide microarray-based metal enhanced fluorescence (MEF) assay is proposed for multiple and sensitive profiling of MMPs activities on a novel Au/Ag@SiO substrate. The Au/Ag@SiO substrate is prepared by electroless deposition of silver on gold nanoparticle (GNP) seeds, followed by SiO shell coating and surface functionalization. The specific FRET peptides are spotted on the Au/Ag@SiO substrate to sensitively detect MMPs (MMP-2, -3, -7, -9, -14) via fluorescence recovery by the MMP cleavage of quenched peptide motifs and further enhanced by MEF. Under the optimal conditions, the limits of detection are 12.2 fg mL for MMP-2, 60 pg mL for MMP-3, 0.22 pg mL for MMP-7, 102 fg mL for MMP-9, and 0.68 ng mL for MMP-14, respectively. The practicability of the FRET-peptide microarray-based MEF assay is demonstrated by profiling of multiplexed MMPs activities in various cell lines and clinical thyroid tissue samples of papillary thyroid carcinoma (PTC) patients and thyroid nodules (TN) patients, and satisfactory results are obtained.
基质金属蛋白酶（MMPs）与癌细胞的侵袭和转移密切相关。在此，提出了一种基于荧光共振能量转移（FRET）肽微阵列的金属增强荧光（MEF）分析方法，用于在新型 Au/Ag@SiO 基底上对 MMPs 活性进行多种灵敏的分析。Au/Ag@SiO 基底通过在金纳米颗粒（GNP）种子上进行无电沉积银，然后进行 SiO 壳层涂覆和表面功能化来制备。特定的 FRET 肽被点在 Au/Ag@SiO 基底上，通过 MMP 切割猝灭肽基序来灵敏地检测 MMPs（MMP-2、-3、-7、-9、-14），并通过 MEF 进一步增强。在最佳条件下，MMP-2 的检测限为 12.2 fg mL，MMP-3 的检测限为 60 pg mL，MMP-7 的检测限为 0.22 pg mL，MMP-9 的检测限为 102 fg mL，MMP-14 的检测限为 0.68 ng mL。通过对各种细胞系和甲状腺乳头状癌（PTC）患者和甲状腺结节（TN）患者的临床甲状腺组织样本中多种 MMPs 活性的分析，验证了 FRET 肽微阵列 MEF 分析的实用性，得到了令人满意的结果。
