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

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

Journal Name: Current Pharmaceutical Biotechnology

Volume 14 , Issue 13 , 2013

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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.

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

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Article Details

VOLUME: 14
ISSUE: 13
Year: 2013
Page: [1134 - 1138]
Pages: 5
DOI: 10.2174/138920101413140605111109
Price: $58

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