School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, 430205, PR China; State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, PR China.
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, PR China.
Anal Chim Acta. 2019 Oct 31;1078:112-118. doi: 10.1016/j.aca.2019.05.071. Epub 2019 Jun 1.
Herein, an array-based in situ fluorescence assay is proposed for high-throughput analysis and localization of multiplex matrix metalloproteinases (MMPs) activities in cell monolayers and tissue sections. Five specific MMPs (MMP-2, -3, -7, -9, and -14) peptide substrates containing FAM/Dabcyl fluorescent resonance energy transfer (FRET) pair are directly spotted on the surface of cell monolayers or tissue sections, and hydrolyzed by localized MMPs, resulting in fluorescence recovery of FAM. MMPs activities are determined by the fluorescence intensity of stained cells/tissues due to the cellular internalization of peptide fragments with FAM moiety. We demonstrate that the array-based in situ fluorescence assay is suitable for identifying the MMPs expression patterns of cells, as well as determining the secreted MMPs activities in cell monolayer with high sensitivity (as low as hundreds of cells per square centimeter). The feasibility of the assay is further confirmed by evaluating inhibition potencies of six compounds toward five MMPs. Profiling of five MMPs activities in the localized parts of 32 thyroid tissues is performed without separation or extraction procedures, demonstrating the good practicality of the method.
本文提出了一种基于阵列的原位荧光分析方法，用于高通量分析和定位细胞单层和组织切片中多种基质金属蛋白酶（MMPs）的活性。将含有 FAM/Dabcyl 荧光共振能量转移（FRET）对的五种特定 MMPs（MMP-2、-3、-7、-9 和 -14）肽底物直接点样在细胞单层或组织切片表面，通过局部 MMPs 水解，导致 FAM 的荧光恢复。由于具有 FAM 部分的肽片段被细胞内化，因此可以通过染色细胞/组织的荧光强度来确定 MMPs 活性。我们证明，基于阵列的原位荧光分析适用于鉴定细胞中 MMPs 的表达模式，以及以高灵敏度（低至每平方厘米数百个细胞）确定细胞单层中分泌的 MMPs 活性。通过评估六种化合物对五种 MMPs 的抑制能力进一步证实了该测定的可行性。在无需分离或提取步骤的情况下，对 32 个甲状腺组织的局部部位的五种 MMPs 活性进行了分析，证明了该方法的良好实用性。
