Troubleshooting and Things You Must Know About Quantum Dots

1. Troubleshooting and Things You Must Know About Quantum Dots CD 1. What are Quantum Dots? Quantum dots (QDs) are nanoparticles of semiconductors, which were theorized in the 1970s and initially created in the early 1980s. If semiconductor particles are made small enough, quantum effects come into play, which limits the energies at which electrons and holes can exist in the particles. Because energy is associated with wavelength or color, this means that the optical properties of the particle can be finely tuned depending on its size. As artificial nanostructures, by controlling their size, material, and shape, QDs can be made to emit or absorb specific wavelengths or colors of light or obtain varied properties. There are three main types of quantum dots: · · · III-V-semiconductors: made of elements of main group III of the periodic table of the elements (boron, aluminum, gallium, indium) and main group V (nitrogen, phosphorus, arsenic, antimony, bismuth) II-VI- semiconductors: made of elements of transition metal group II (zinc, cadmium) and main group VI (oxygen, sulfur, selenium, tellurium) Silicon (Si), the standard material of the semiconductor and chip industry With a reliable manufacturing technology, Creative Diagnostics is capable of churning out large quantities of nanocrystals where each batch is produced according to the exact same parameters. Our DiagNano™ products are now widely used in a wide-ranging number of applications including catalysis, electronics, photonics, composites, sensing, solar cells, fluorescent biological labels and optical probes for biological and medical imaging. Tel: 1-631-624-4882 Email: info@cd-bioparticles.com

2. 2. Basic Structure of DiagNano™ Nanocrystals Core: determines the color of the DiagNano™ nanocrystal Shell: improves brightness and stability of the DiagNano™ nanocrystal Coating: provides water solubility and/or functional groups for conjugation Biomolecules: covalently attached to polymer shell and can include immunoglobins, streptavidin, receptor ligands, or oligonucleotides 3. Advantages of DiagNano™ Nanocrystals over Traditional Fluorescent Dyes · QDs 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 nanocrystal brightness. When using QDs, multicolor detection can be completed by using a single excitation wavelength. DiagNano™ nanocrystals exhibit a large Stokes shift. DiagNano™ nanocrystals have a narrow emission band. DiagNano™ nanocrystals possess excellent photostability compared to traditional fluorescent dyes. · · · · 4. DiagNano™ Nanocrystal Applications QDs and bioconjugates are ideal for experiments which need long-term photostability or single-excitation, multicolor analysis, such as flow cytometry, cell and tissue staining, cell tracking, western blotting and in vivo imaging. In addition, we also offer amino, carboxyl, GSH, MPA, etc. functionalized quantum dots for custom covalent conjugation with proteins, antibodies, and other biomolecules. Quantum dots labeling and conjugation kits with highly efficient and site-specific labeling and conjugating abilities are available too. Tel: 1-631-624-4882 Email: info@cd-bioparticles.com

3. 5. pH Range of Stable DiagNano™ Nanocrystals pH Not recommended QDs start to self-aggregate or clump pH > 9 6 < pH < 9 Most optimal stability is in this pH range Marginal stability is shown in this range 5 < pH < 6 Not recommended The polymer will dissociate pH < 4 6. Temperature Range of Stable DiagNano™ Nanocrystals Temperature Never freeze DiagNano™ Nanocrystals <=0°C Core/Shell/Polymer stable at 4°C for about 6 months >=4°C <=60°C Core/Shell/Polymer stable at 60°C Core/Shell/Polymer stable at 65°C for only about 1 hour <=65°C Core/Shell/Polymer stable up to 100°C for brief exposure. OK for 5 minutes at 100°C. <=100°C <=360°C Only Core/Shell stable up to 360°C Tel: 1-631-624-4882 Email: info@cd-bioparticles.com

4. 7. Solvents Stability of DiagNano™ Nanocrystals Chemical Chemical Acetonitrile Alcohols Alkanes Cacodylate (arsenic-based buffer) Chloroform DMF DMSO 5% Tween 4% Saponin Triton X-100 NP-40 Formaldehyde/Paraformaldehyde Formalin Glutaraldehyde Glycerol Unstable OK up to <10% Stable Unknown Unstable Stable Stable Stable Stable Unknown Unknown Unknown Unknown Unknown OK up to 50% H2O2 Heavy Metals MgCl2 Marine salt (sea salt concentrations) NaCl NaI Paraffin-embedding PBS Buffer Phenol Picric Acid Organic solvents Sodium azide Thimerosal Xylene Unstable Variable Stable OK Stable up to 1 M Stable up to 1 M Stable Unstable Unknown Unknown Stable Stable Unknown Stable 8. Mounting Media for DiagNano™ Nanocrystals In addition to commercial mounting media, there are several media ideal for short-term storage, namely most polyvinyl alcohol-based mountants (<weeks), water-based mountants (<week) and glycerol (up to 50%, <week). Troubleshooting 1. Why is there a small amount of aggregation of my DiagNano™ product even though I stored it properly? You may see a small amount of aggregation of the DiagNano™ product during correct storage. Before official usage, we suggest centrifuging the vial at 2000 × g for 1 minute to remove the aggregates. Pipette only the supernatant and avoid the pellet. 2. If my DiagNano™ product is completely aggregated, what should I do? If that happens, we suggest you order a new product because QDs cannot be dispersed back into solution. Moreover, since freezing will cause the aggregation, do not freeze your QDs. Tel: 1-631-624-4882 Email: info@cd-bioparticles.com

5. 3. Why is there a loss of fluorescence after I mounted my samples even though it was brightly fluorescent before that? It's important to select an appropriate mounting media to retain the fluorescence of DiagNano™ nanocrystals. As mentioned before, choose commercial media or proper mountant according to your desired storage time. 4. Is it normal that I found my DiagNano™ product blinking? All single-luminescent molecules blink (including organic dyes), which is an inherent property of QDs. The brightness and photostability of DiagNano™ nanocrystals make the blinking more visibly apparent. Actually, the higher energy excitation, the faster they blink. Try β-mercaptoethanol to reduce blinking. 5. Suggestions for very high background DiagNano™ streptavidin QD conjugate • Check if the sample has a high level of endogenous biotin Some tissues, for example, spleen and kidney sections may contain endogenous biotin, resulting in the nonspecific signal. Thus, check your sample first and then block the sample using an avidin-biotin pre-blocking step (BSA or normal animal serum) to decrease nonspecific binding. • Find grainy staining or clumps of fluorescent material in the background This is caused by the slight aggregation of BSA. You may spin your samples by a benchtop centrifuge or a filter to remove microscopic precipitates. In addition, a high level of NaCl or other salts in the incubation buffer may contribute to this too, so try to reduce the overall salt concentration. • Optimize the concentration of biotinylated secondary antibodies Although high levels of biotinylated antibody are required to obtain specific labeling, overly high levels will lead to nonspecific binding of the antibody to the sample. It's necessary to optimize the level of biotinylated antibody to achieve specific signal in staining. • Optimize the concentration of your streptavidin QD conjugate The level of the final probe should be optimized for each conjugate. Generally, concentrations at or slightly below saturation should have the optimal signal-to-background ratio, while concentrations substantially higher than saturation may lead to high background levels. For more information, view our website: www.cd-bioparticles.com Tel: 1-631-624-4882 Email: info@cd-bioparticles.com Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Fax: 1-631-938-8221 5