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Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye
Published on: November 5, 2010
Metal-enhanced PicoGreen fluorescence: application for double-stranded DNA quantification.
A I Dragan1, E S Bishop, J R Casas-Finet
1Institute of Fluorescence, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA.
Analytical Biochemistry
|September 15, 2009
Summary
PicoGreen (PG) dye enhances double-stranded DNA (dsDNA) detection sensitivity when combined with silver nanoparticles. This metal-enhanced fluorescence significantly amplifies dsDNA quantification capabilities.
Area of Science:
- Biochemistry
- Nanotechnology
- Analytical Chemistry
Background:
- PicoGreen (PG) is a standard fluorescent probe for double-stranded DNA (dsDNA) detection.
- Existing methods have limitations in sensitivity for dsDNA quantification.
Purpose of the Study:
- To investigate the spectral properties of PicoGreen (PG) in proximity to silver nanoparticles.
- To enhance the sensitivity of PG-based dsDNA detection and quantification.
Main Methods:
- Studied spectral properties of free and DNA-bound PG with silver island films (SiFs).
- Analyzed PG fluorescence at various concentrations (20 pM–3.5 µM) to confirm monomeric state.
- Measured fluorescence enhancement due to metal-enhanced fluorescence (MEF) effect.
Main Results:
- PG fluorescence increases ~1100-fold upon binding to dsDNA.
- PG/dsDNA on SiFs shows a 7-fold increase due to MEF, totaling 7700-fold vs. free dye on glass.
- Free PG on SiFs exhibited a 5-fold brightness decrease.
- Total enhancement of ~38,000-fold achieved for PG on dsDNA on silver compared to free PG on SiFs.
Conclusions:
- Silver nanoparticles significantly enhance PicoGreen fluorescence for dsDNA detection.
- Metal-enhanced fluorescence offers a powerful method for amplified dsDNA quantification.
