Related Experiment Video
Updated: Jun 15, 2026
08:34
A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening
Published on: October 16, 2015
A peptide-based fluorescent ratiometric sensor for quantitative detection of proteins
Laurence Choulier1, Volodymyr V Shvadchak, Aletia Naidoo
1Biosensor Group, CNRS/Université de Strasbourg, IREBS, 67412 Illkirch Cedex, France. laurence.choulier@unistra.fr
Analytical Biochemistry
|March 2, 2010
Summary
A novel ratiometric fluorescent sensor using a peptide and 3-hydroxychromone (3HC) was developed for antibody fragment detection. This peptide-based sensor enables sensitive and quantitative detection of antibody fragments in complex biological samples.
Area of Science:
- Biochemistry
- Chemical Biology
- Analytical Chemistry
Background:
- Ratiometric fluorescent sensors offer advantages for quantitative analysis due to their internal referencing.
- Peptide-based constructs can be engineered to bind specific biomolecules.
- 3-hydroxychromone (3HC) derivatives are environmentally sensitive fluorophores with potential for sensing applications.
Purpose of the Study:
- To develop a ratiometric fluorescent sensor for detecting antibody fragments.
- To characterize the sensor's performance, including binding affinity and detection limits.
- To demonstrate the sensor's utility in complex biological media.
Main Methods:
- Solid-phase synthesis of a peptide labeled with a 3HC derivative.
- Characterization of the peptide-fluorophore construct and its interaction with an antibody fragment (scFv1F4(Q34S)) using surface plasmon resonance (SPR).
- Measurement of fluorescence emission ratio changes upon antibody binding and competition assays.
Main Results:
- The synthesized sensor exhibited a significant change (up to 47%) in its fluorescence emission ratio upon binding to the antibody fragment.
- The dye minimally impacted the binding kinetics and equilibrium of the antibody-peptide interaction.
- A detection limit of 15 nM for the antibody fragment was achieved, with detection in the 50-100 nM range in the presence of bovine serum albumin.
Conclusions:
- Fluorescent ratiometric sensing based on small synthetic peptides is a promising approach for quantitative target detection.
- The developed sensor demonstrates high sensitivity and specificity for antibody fragments.
- Further improvements in dye spectroscopic properties could enhance the sensor's capabilities.
Related Concept Videos
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
01:24Tagging and Fusion Proteins
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...