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.

Download full-text


Available from: Marcel P Bruchez,
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We recently reported the engineering of monomeric streptavidin (mSA) for use in monomeric detection of biotinylated ligands. Although mSA can be expressed functionally on the surface of mammalian cells and yeast, the molecule does not fold correctly when expressed in Escherichia coli. Refolding from inclusion bodies is cumbersome and yields a limited amount of purified protein. Improving the final yield should facilitate its use in biotechnology. We tested the expression and purification of mSA fused to GST, MBP, thioredoxin, and sumo tags to simplify its purification and improve the yield. The fusion proteins can be expressed solubly in E. coli and increase the yield by more than 20-fold. Unmodified mSA can be obtained by proteolytically removing the fusion tag. Purified mSA can be immobilized on a solid matrix to purify biotinylated ligands. Together, expressing mSA as a fusion with a solubilization tag vastly simplifies its preparation and increases its usability in biotechnology.
    Applied Microbiology and Biotechnology 04/2014; 98(14). DOI:10.1007/s00253-014-5682-y · 3.34 Impact Factor
  • Source
    • "All of these properties are taken advantage of for mcSPT using our HSM. However, QDs do have practical limiting properties [40] such as fluorescence intermittency, the potential for non-fluorescent QDs [50] and a difficultly in producing monovalent probes [51]. For example, the presence of non-fluorescent QDs leads to the possibility of underestimating the oligomer size of complexes in mcSPT experiments. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins. However, since the spatial scale of these interactions is below the diffraction limit of the light microscope, the dynamics of these interactions have been difficult to study on living cells. We have developed a novel high-speed hyperspectral microscope (HSM) to perform single particle tracking of up to 8 spectrally distinct species of quantum dots (QDs) at 27 frames per second. The distinct emission spectra of the QDs allows localization with ∼10 nm precision even when the probes are clustered at spatial scales below the diffraction limit. The capabilities of the HSM are demonstrated here by application of multi-color single particle tracking to observe membrane protein behavior, including: 1) dynamic formation and dissociation of Epidermal Growth Factor Receptor dimers; 2) resolving antigen induced aggregation of the high affinity IgE receptor, FcεR1; 3) four color QD tracking while simultaneously visualizing GFP-actin; and 4) high-density tracking for fast diffusion mapping.
    PLoS ONE 05/2013; 8(5):e64320. DOI:10.1371/journal.pone.0064320 · 3.23 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Colloidal semiconductor nanocrystals (SC-NC) present unique and well-controlled physical and chemical properties that make them most suitable, as fluorescent agents, for high resolution imaging and direct follow-up in biological milieus. SC-NC present intense fluorescence that is tunable by size, composition, and shape. In addition, they have large molar extinction coefficients and thermal and photochemical stability, thus avoiding the relatively fast photobleaching characteristic of traditional fluorescent dyes. Nevertheless, constructing SC-NC–labeled, biologically active macromolecules and especially enzymes is challenging. Ensuring the enzyme’s stability, sustaining its biological functions, and achieving the desired application all require specific design of the nanoparticles (NPs) properties, their surface coating, and the labeling process. Here, we report an in-depth investigation of the experimental steps involved in the development of a flexible SC-NC labeling toolkit for recombinant human cholinesterases (ChEs), including acetylcholinesterase-R (AChE-R), and the homologous enzyme butyrylcholinesterase (BChE). AChE and BChE, the major acetylcholine (ACh) hydrolyzing enzymes, regulate ACh-mediated neurotransmission, signal transduction, and anti-inflammatory reactions. Impaired ChE functioning is causally involved in many pathologies, including Alzheimer’s and Parkinson’s diseases, cardiovascular disorders, myasthenia gravis, and Sjogren’s syndrome. In addition, exposure to anti-ChE drugs, insecticides, and poisonous nerve agents present prevalent health and security issues. Therefore, recombinant ChEs have been developed both as therapeutic agents and for biomedical research purposes. The labeling toolkit presented here enables optimization of highly emitting SC-NC- ChE conjugates with hydrolytic activity and capacity to bind anti-ChEs, suitable for use for biomedical applications and allows sensitive long-term follow-up of the locations and interactions of these important enzymes in biological systems.
    BioNanoScience 03/2013; 3(1). DOI:10.1007/s12668-012-0072-3
Show more