Fluorescence Detection of MMP-9. II. Ratiometric FRET-Based Sensing With Dually Labeled Specific Peptide

Title:Fluorescence Detection of MMP-9. II. Ratiometric FRET-Based Sensing With Dually Labeled Specific Peptide

Volume: 14 Issue: 13

Author(s): Rafal Fudala, Ryan Rich, Anindita Mukerjee, Amalendu P. Ranjan, Jamboor K. Vishwanatha, Anna K. Kurdowska, Zygmunt Gryczynski, Julian Borejdo and Ignacy Gryczynski

Affiliation:

Keywords: Fluorescence, FRET, labeled peptide, MMP-9, ratio-metric sensing.

Abstract: In our previous paper we showed that the MMP-9 enzyme recognizes a specific peptide sequence, Lys-Gly- Pro-Arg-Ser-Leu-Ser-Gly-Lys, and cleaves the peptide into two parts [1]. In this study, the peptide is labeled with two dyes, carboxyfluorescein (5-FAM) and Cy5. A highly efficient energy transfer of over 80% results in a dominant emission of Cy5 at ~670 nm with an excitation of 470 nm. Severance of the peptide by the MMP-9 enzyme eliminates Förster Resonance Energy Transfer (FRET) and strongly increases the fluorescence of the 5-FAM dye. In this manuscript we describe the strategy for a FRET-based method for MMP-9 enzyme detection. The basic aim is to apply a ratio-metric sensing technique in which a ratio of green/red fluorescence intensity is measured as a function of enzyme concentration. The ratio-metric method eliminates many experimental variables and enables accurate MMP-9 detection.

Cite this article as:

Fudala Rafal, Rich Ryan, Mukerjee Anindita, Ranjan P. Amalendu, Vishwanatha K. Jamboor, Kurdowska K. Anna, Gryczynski Zygmunt, Borejdo Julian and Gryczynski Ignacy, Fluorescence Detection of MMP-9. II. Ratiometric FRET-Based Sensing With Dually Labeled Specific Peptide, Current Pharmaceutical Biotechnology 2013; 14 (13) . https://dx.doi.org/10.2174/138920101413140605111109