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In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy
Published on: January 19, 2011
Nonspecific particle-based method with two-photon excitation detection for sensitive protein quantification and cell
Sari Pihlasalo1, Anke Engbert, Eija Martikkala
1Laboratory of Biophysics and Medicity Research Laboratory, University of Turku, Tykistökatu 6A, 20520 Turku, Finland. sari.pihlasalo@utu.fi
Analytical Chemistry
|February 7, 2013
Summary
A new two-photon excitation (TPX) method enables sensitive protein quantification and cell counting in solution. This technique uses competitive adsorption onto particles, offering a 500-fold improvement in protein assay sensitivity.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Biophysics
Background:
- Accurate quantification of proteins and counting of cells are crucial in biological research and diagnostics.
- Existing methods often face limitations in sensitivity, dynamic range, or tolerance to sample complexity.
- There is a need for simple, sensitive, and robust assays for biomolecule and cell analysis.
Purpose of the Study:
- To develop and validate a novel, homogeneous assay using two-photon excitation (TPX) for protein quantification and eukaryotic cell counting.
- To demonstrate the high sensitivity and broad dynamic range of the developed TPX-based method.
- To assess the method's tolerance to common interfering substances found in biological samples.
Main Methods:
- A homogeneous assay based on competitive adsorption of fluorescently labeled proteins onto carboxylate-modified polystyrene microparticles.
- Detection of fluorescence changes resulting from the interaction of analyte proteins or cells with the particle surface.
- Optimization of assay conditions for both protein quantification and cell counting, validated by fluorescence microscopy.
Main Results:
- The protein quantification assay achieved picogram-level sensitivity (1.2 μg/L), over 500-fold improvement compared to sensitive commercial methods.
- The cell counting assay demonstrated sensitivity for as few as three cells with ~10% variation.
- The assay exhibited a broad dynamic range of approximately four orders of magnitude and tolerated high concentrations of neutral detergents.
Conclusions:
- The developed two-photon excitation (TPX) method provides a highly sensitive, simple, and versatile platform for protein quantification and cell counting.
- This technique offers significant advantages in sensitivity and dynamic range over existing methods.
- The TPX technology holds potential for broader applications in biological and diagnostic analyses.
