title: "One-step" detection of matrix metalloproteinase activity using a fluorogenic peptide probe-immobilized diagnostic kit.
abstract: Matrix metalloproteinases (MMPs) have been shown to be abundant in pathological conditions such as cancer, osteoarthritis (OA), and rheumatoid arthritis (RA). The extent of MMPs detected in biological samples provides important clinical information for diagnosis, prognosis, and therapeutic monitoring of various diseases relating with MMPs. Herein, we developed a new high-throughput MMP diagnostic kit (MMP-D-KIT) based on a 96-well plate by immobilizing MMP-13 specific fluorogenic peptide probes (MMP peptide probe), which is a pair consisting of a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3), onto the biocompatible glycol chitosan (GC) polymer anchored 96-well plate. When MMP enzymes were simply added and incubated in a MMP-D-KIT, the fluorescence of each well was recovered and the fluorescence intensity showed distinct difference within minutes through NIR fluorescence imaging system. The fluorescence was recovered not only by MMP-13 activity, but also by other MMPs activity. Furthermore, recovery of NIR fluorescent signals in MMP-D-KIT was proportional to concentrations of immobilized MMP peptide probe-GC conjugates and, importantly, MMP concentration. The MMP-D-KIT is most specific for target MMP, compared with other enzymes including caspase-3 and 20s proteasome. Additionally, the MMP-D-KIT was used to detect MMP activity in biological samples such as synovial fluid from 12 OA patients (grades 1-4 based on the Kellgren-Lawrence grading scale). It was found that the fluorescence intensity measured using MMP-D-KIT decidedly correlates with the progression of OA. The MMP-D-KIT could be applicable in detecting MMP activities in various biological samples and evaluating the effects of MMP inhibitors in a rapid and easy fashion.
authors.affiliation: Biomedical Research Center, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul, South Korea.
authors.forename: Ju Hee
authors.initials: JH
authors.lastname: Ryu
authors.forename: Aeju
authors.initials: A
authors.lastname: Lee
authors.forename: Seulki
authors.initials: S
authors.lastname: Lee
authors.forename: Cheol-Hee
authors.initials: CH
authors.lastname: Ahn
authors.forename: Jong Woong
authors.initials: JW
authors.lastname: Park
authors.forename: James F
authors.initials: JF
authors.lastname: Leary
authors.forename: Sangjin
authors.initials: S
authors.lastname: Park
authors.forename: Kwangmeyung
authors.initials: K
authors.lastname: Kim
authors.forename: Ick Chan
authors.initials: IC
authors.lastname: Kwon
authors.forename: In-Chan
authors.initials: IC
authors.lastname: Youn
authors.forename: Kuiwon
authors.initials: K
authors.lastname: Choi
mesh_terms: D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D020782:Matrix Metalloproteinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D015335:Molecular Probes / Q000032:analysis / Q000737:chemistry*; D020370:Osteoarthritis, Knee / Q000201:enzymology; D010455:Peptides / Q000737:chemistry*; D011933:Reagent Kits, Diagnostic; D019265:Spectroscopy, Near-Infrared
chemical_list: D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D015335:Molecular Probes; D010455:Peptides; D011933:Reagent Kits, Diagnostic; D020782:Matrix Metalloproteinases