On-chip assay of matrix metalloproteinase-3 activity using fluorescence-conjugated gelatin arrays

We developed a new high-throughput approach to measure matrix metalloproteinase (MMP)-3 activity using Cy3-conjugated gelatin arrays. We fabricated Cy3-conjugated gelatin arrays by immobilizing Cy3-conjugated gelatin onto well-type amine arrays. The ratio of Cy3-NHS to gelatin was optimized to improve the efficiency of the arrays. MMP-3 activity was determined by the decrease of fluorescence intensity due to MMP-3-mediated degradation of Cy3-conjugated gelatin. The gelatinolytic activity of MMP-3 increased in the presence of Brij-35, a non-ionic detergent, in a dose-dependent manner. Cy3-conjugated gelatin arrays were successfully applied for the analyses of MMP-3 activity, which showed a dose-dependent degradation of the Cy3-conjugated substrate. This chip-based activity assay using a Cy3-conjugated gelatin array is simple, rapid, cost-effective, and has a potential for activity-based proteomic researches and screening of MMP-3 inhibitors.