Peptide substrate-based inkjet printing high-throughput MMP-9 anticancer assay using fluorescence resonance energy transfer (FRET)

A peptide probe-based MMP-9 inhibitory assay was developed to assess anticancer activity of compounds on parchment paper using FRET-based inkjet printing. The fluorescent peptide containing 5-FAM (donor) and QXL520 (acceptor) was used as a substrate with the sequence of Pro-Leu-Gly-Cys-His-Ala-Arg-Lys specific to MMP-9. The suitability of the peptide as a substrate of MMP-9 was confirmed by HPLC. When both MMP-9 and the peptide were printed on the identical reaction spot of parchment paper, the peptide was cleaved and fragmented by MMP-9. The fluorescence was then recovered by 5-FAM and detected at Ex/Em = 490 +/- 20 nm/520 +/- 20 nm. Based on the successful performance of FRET on the parchment paper without any surface treatment, the assay platform was applied to test compounds, and a successful anticancer assay could be accomplished. This methodology holds notable strengths in versatile applications to the MMP enzyme family, as well as control of ejection volume, reduction of consumed enzyme amount, and rapid reaction time. Hence, we suggest the FRET peptide-based assay using inkjet printing as a viable replacement for sensitive and high-throughput evaluation of anticancer compounds. (C) 2017 Elsevier B.V. All rights reserved.