Quantum dots are fluorophore nanocrystals whose excitation and emission is fundamentally different than traditional organic fluorophores. Instead of electronic transitions from one valence orbital to another, quantum-dot fluorescence involves exciting an electron from the bulk valence band of the semiconductor material across an energy gap, making it a conduction electron and leaving behind a hole. The electron–hole pair (also known as an exciton) is quantum-confined by the small size of the nanocrystal (smaller than the exciton Bohr radius). When the electron–hole pair eventually recombines, a characteristic photon is emitted. Minute changes to the size of the confining crystal alter the energy bandgap, thus determining the color of the fluorescence photon. In general, the smaller the quantum dot, the larger the bandgap energy for a given material, and thus, the shorter the wavelength of the emitted fluorescence. Of the many types of quantum dots that can be made from various semiconductor materials, CdSe/ZnS quantum dots are presently the most common commercially available as secondary antibody conjugates. They are composed of a core of cadmium selenide ranging from about 10 to 50 atoms in diameter and about 100 to 100,000 atoms in total, and as mentioned, the size of the core determines the fluorescence emission spectra. They have a thin zinc sulfide passivating layer that improves the fluorescence quantum efficiency and stability of the quantum dots and an organic polymer coating to make them water soluble and enabling bioconjugation to targeting molecules such as anti-IgG (immunoglobulin G) secondary antibodies, Fab fragments, peptides, or streptavidin ().
FIGURE 1 (a) Diagram representing the composition of a CdSe/ZnS quantum dot showing the core, shell, coating, and targeting molecules. The overall size is about 15 to 20 nm. (b) Micromolar aqueous solutions of 525, 565, 585, 605, and 655 quantum dots (left to (more ...)
They exhibit high fluorescence quantum yields, and as would be expected from a solid-state material, they are extremely resistant to reactive oxygen-mediated photobleaching. They have large absorption cross-sections and broad absorption spectra with narrow band fluorescence emission that can be tuned over a broad range from blue to near-infrared. Under ambient light, micromolar solutions are nearly colorless, but under UV excitation, they exhibit brilliant and distinct fluorescence (). Very different from traditional fluorophores, they have symmetrical Gaussian-shaped emission spectra, and more importantly, all have exceptionally large Stoke’s shifts and can be excited equally well at a single UV wavelength, making them excellent for multiple labeling experiments (Chan et al., 2002
; Klostranec and Chan, 2006
). Typically, commercially available quantum dots are named for their peak emission wavelength (i.e., 525, 565, 585, 605, and 655 nm). Additionally, quantum dot cores have sufficient electron density to be directly visible by electron microscopy in the 60-keV to 400-keV accelerating voltage range without any special contrasting treatments () and can be discriminated by their different sizes and shapes (525-, 565-, and 585-nm Cd/Zn quantum dots are spherical, and 605- and 655-nm quantum dots are oblong).