Biotin-4-Fluorescein Based Fluorescence Quenching Assay for Determination of Biotin Binding Capacity of Streptavidin Conjugated Quantum Dots

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
Bioconjugate Chemistry (Impact Factor: 4.51). 02/2011; 22(3):362-8. DOI: 10.1021/bc100321c
Source: PubMed


The valency of quantum dot nanoparticles conjugated with biomolecules is closely related to their performance in cell tagging, tracking, and imaging experiments. Commercially available streptavidin conjugates (SAv QDs) are the most commonly used tool for preparing QD-biomolecule conjugates. The fluorescence quenching of biotin-4-fluorscein (B4F) provides a straightforward assay to quantify the number of biotin binding sites per SAv QD. The utility of this method was demonstrated by quantitatively characterizing the biotin binding capacity of commercially available amphiphilic poly(acrylic acid) Qdot ITK SAv conjugates and poly(ethylene glycol) modified Qdot PEG SAv conjugates with emission wavelengths of 525, 545, 565, 585, 605, 625, 655, 705, and 800 nm. Results showed that 5- to 30-fold more biotin binding sites are available on ITK SAv QDs compared to PEG SAv QDs of the same color with no systematic variation of biotin binding capacity with size.

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Available from: Marcel P Bruchez
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    • "Because the binding of HABA to mSA does not change its A 500 , the occupancy of mSA cannot be calculated in this way. Similarly, biotin-4-fluorescein (B4F) was used to estimate the available binding sites on streptavidin/avidin because binding quenches its fluorescence (Mittal and Bruchez 2011). In contrast , binding to mSA does not change the B4F fluorescence. "
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