Fluorescence detection of MMP-9. II. ratiometric FRET-based sensing with dually labeled specific peptide

Rafal Fudala, Ryan Rich, Anindita Mukerjee, Amalendu P. Ranjan, Jamboor K. Vishwanatha, Anna K. Kurdowska, Zygmunt Gryczynski, Julian Borejdo, Ignacy Gryczynski

Research output: Contribution to journalArticle

11 Scopus citations

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.

Original languageEnglish
Pages (from-to)1134-1138
Number of pages5
JournalCurrent Pharmaceutical Biotechnology
Volume14
Issue number13
DOIs
StatePublished - 1 Jan 2014

Keywords

  • FRET
  • Fluorescence
  • Labeled peptide
  • MMP-9
  • Ratio-metric sensing

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