Quantum Dots Support—Getting Started

Qdot™ nanocrystals offer many advantages over traditional fluorescent dyes:

• Qdot™ nanocrystals have a broad excitation range, and they can be excited by any wavelength below their emission peak. The lower the excitation wavelength, the higher the extinction coefficient and Qdot™ nanocrystal brightness. 
• Multicolor detection using Qdot™ nanocrystals can be done using a single excitation wavelength. 
• Qdot™ nanocrystals exhibit a large Stokes shift.
• Qdot™ nanocrystals have a narrow emission band. 
• Qdot™ nanocrystals have excellent photostability compared to traditional fluorescent dyes.

Qdot™ nanocrystals and bioconjugates are ideal for experiments requiring long-term photostability or single-excitation, multicolor analysis. Some example applications include:

• Flow cytometry
• Cell and tissue staining
• Cell tracking
• WesternDot™ western blotting
• In vivo imaging

We offer amino (PEG), carboxyl, and streptavidin-functionalized Qdot™ Innovator’s Tool Kit ITK™ Nanocrystals for the preparation of custom conjugates of proteins or other biomolecules. Amino (PEG)-derivatizedforms can be coupled to isothiocyanates and succinimidyl esters or with native carboxylic acids using water-soluble carbodiimides. Carboxyl-derivatized forms can be coupled to amine groups of proteins and modified oligonucleotides. Streptavidin-derivatized forms can be bound with biotinylated conjugates to form stable labeled complexes.

You can use Qdot™ nanocrystals with FRET applications in two scenarios:

• Qdot™ nanocrystals as donors with fluorescent dyes as acceptors
• Lanthanide (terbium, europium, etc.) as donors with Qdot™ nanocrystals as acceptors

Note: You cannot perform FRET experiments using Qdot™ nanocrystals as both donor and acceptor.

Qdot™ nanocrystals are most stable at pH 6–9, and marginal stability of Qdot™ nanocrystals is shown down to a pH 5. Qdot™ nanocrystals should not be used at pH > 9 due to the possibility of self-aggregation and clumping, and Qdot™ nanocrystals should not be used pH < 4 as the polymer and exposed core/shell will begin to dissociate. 

When stored at 4 degrees C, Qdot nanocrystals are stable for approximately 6 months. Qdot nanocrystals should never be frozen due to the possibility of aggregation. The temperature stability of Qdot nanocrystals is summarized below. Please note that fluorescence is not temperature dependent.

<0 degrees C: NEVER freeze Qdot nanocrystals - polymer induces aggregation at freezing temperatures.
>4 degrees C: Core/Shell/Polymer stable at 4 degrees C for ~ 6 months. May be filter sterilized using uncharged filters.
<60 degrees C: Core/Shell/Polymer stable at 60 degrees C (as in in situ hybridization).
<65 degrees C: Core/Shell/Polymer stable at 65 degrees C for only ~1 hour, beyond 1 hour, emission drops off.
<100 degrees C: Core/Shell/Polymer stable up to 100 degrees C brief exposure. OK for 5 minutes at 100 degrees C.
<360 degrees C: Only Core/Shell stable up to 360 degrees C.

Hydrophilic Qdot™ nanocrystals are stored and shipped in borate buffer pH 8.3–9.0, and organic Qdot™ nanocrystals are stored and shipped in decane. We have examined the stability of Qdot™ nanocrystals in a variety of other solvents; please contact customer support for more information.

Qdot™ nanocrystals do not require the use of antifades as they do not photobleach or fade in the same manner as a chemical dye. In our studies, Qdot™ nanocrystals work best with the following mountants:

• HistoMount™ medium (Cat No. 00-8030); best for long-term archiving
• Cytoseal™ 60 Mountant
• Clarion™ Mountant
• Most polyvinyl alcohol-based mountants (limited storage time, <weeks)
• Water-based mountants (limited storage time, <week)
• Up to 50% glycerol (limited storage time, <week)

Note: We do not recommend using ProLong™ mounting media with Qdot™ nanocrystals. 

Yes, you can visualize Qdot™ nanocrystals using a standard filter; they will excite at any wavelength below their emission. Keep in mind that the lower the excitation value the brighter the Qdot™ nanocrystal fluorescence output.

There are approximately 80–100 functional groups of each Qdot™ ITK™ nanocrystal. We use a type of immunosorbent assay to determine the EC₅₀ of each conjugate.

ITK™ Qdot™ nanocrystals use the original formulation of outer polymer provided in the first generation of the Qdot™ products; except for the Amine-PEG products, the outer polymer does not include PEG. The outer polymer of the standard Qdot™ nanocrystals includes PEG.

The number of molecules conjugated to one Qdot™ nanocrystal is based on the ratio of quantum dot:molecule used in the conjugation, the number of available binding sites on the Qdot™ nanocrystal, and the size of both the Qdot™ nanocrystal and the molecule of interest. In general, there are 2–3 antibodies, 4–5 biotin molecules, and 6–8 streptavidin molecules per Qdot™ nanocrystal.

The Qdot™ 605 Streptavidin conjugate has stable emission in a number of distinct buffers, across a range of pH conditions. At working concentrations, the quantum yield and colloidal dispersion of these materials have been found to be remarkably stable across pH 6–9 (not investigated outside this range) in Tris, HEPES, phosphate, and borate buffers. The Qdot™ 605 Streptavidin conjugate is stable and non-aggregated in buffered NaCl up to 200 mM at working concentrations. Higher salt concentrations may result in microscopic precipitation, but do not appear to cause bulk precipitation of the materials at working dilutions. In addition, a number of surfactants and additives such as Tween™ 20, Triton™ X-100, Pluronic™ F-68, NDSB-201, and EDTA, among others have been shown to maintain the fluorescence in 0.05% concentrations. In contrast, gelatin and dextran sulfate were both found to promote aggregation of the Qdot™ 605 Streptavidin conjugate at 0.05% concentrations, and should be avoided in labeling applications. In general, we recommend storage of the Qdot™ nanoparticle conjugate at the concentration at which it was shipped, rather than at a high dilution. Storing materials at working dilution over longer periods of time may result in substantial performance degradation. While we have not characterized the stability of the other Qdot™ conjugates in this variety of buffers, we anticipate similar levels of stability. 

Qdot™ conjugates have higher nonspecific binding in buffers that are not optimized for use with the materials. We have had successful staining results in a variety of buffer conditions, including TBS, PBS, RPMI media, and others, but have found that the performance in the Incubation Buffer is generally predictable and stable. 

We have not investigated the toxicity of the Qdot™ nanocrystals. The materials are provided in a solution which is ~2 mM total Cd concentration. We have demonstrated the utility of these materials in a variety of live-cell in vitro labeling experiments, but do not have systematic data investigating the toxicity of the materials to humans, to animals, or to cells in culture. 

The Qdot™ products contain cadmium and selenium (and tellurium, in the larger particles) in an inorganic crystalline form. We can only advise that you dispose of the material in compliance with all applicable local, state, and federal regulations for disposal of these classes of material. For more information on the composition of these materials, consult the Material Safety Data Sheet. 

We have not systematically investigated the energy transfer properties of the quantum dots, though the quantum dots may have useful properties as both energy transfer donors and acceptors. We have investigated the fluorescence of Qdot™ 605 Streptavidin conjugates that are coupled to each other through a bis-biotin linker, and found that the emission intensity of the materials was unperturbed at any concentration of biotin cross-linker. These results suggest that the interparticle quenching of these Qdot™ conjugates is negligible.