Nanocrystals Ppt

[Cortney Ruic]

Multicolormultiplex assays are a particularly important application of Qdot nanocrystal conjugates. The emission from Qdot nanocrystals is narrow and symmetrical; therefore, overlap with other emission colors is minimal, producing less bleed through into adjacent detection channels and allowing many more colors to be used simultaneously. Because each type of Qdot nanocrystal is based on the same underlying material (they differ primarily in core size, as described below), the conjugation and assay methods for one color are easily extrapolated to other colors, simplifying protocol development. Furthermore, every Qdot nanocrystal can be excited using a single light source (e.g., 405 nm violet diode laser), reducing the need for multiple lasers or laborious alignments and compensations when performing three- or four-color detection assays.

Qdot nanocrystals are nanometer-scale atom clusters comprising a core, shell and surface coating (Figure 6.6.1). The core is made up of a few hundred to a few thousand atoms of a semiconductor material (cadmium selenide (CdSe) or cadmium telluride (CdTe)). A semiconductor shell (typically zinc sulfide (ZnS)) surrounds and stabilizes the core, improving both the optical and physical properties of the material.

In most of our Qdot nanocrystal conjugates, the affinity reagent is coupled to the amphiphilic polymer coating via a functionalized polyethylene glycol (PEG) linker. PEG linkers have been shown to reduce nonspecific binding in flow cytometry and imaging assays, thereby improving signal-to-noise ratios. Qdot primary and secondary antibody conjugates, Qdot streptavidin conjugates, Qtracker non-targeted quantum dots, and Qdot ITK amino (PEG) quantum dots, as well as the reactive nanocrystals provided in the Qdot Antibody Conjugation Kits, all utilize this PEG linker chemistry.

Although Qdot nanocrystals are composed of semiconductor materials, their small size results in spectroscopic properties that are radically different from those of bulk semiconductors. Absorption of a photon causes an electron to move from the semiconductor valence band to the conductance band, creating an exciton (electron-hole pair). Absorption occurs as long as the energy of the incident photons is higher than the semiconductor bandgap energy; thus, excitons can be created over a wide range of energies within the nanocrystal core. The higher-energy excitons relax to the lowest bandgap energy before they recombine and emit a photon. Therefore, the wavelength range of the absorption spectrum is broad, whereas that of the emission spectrum is narrow (Figure 6.6.3, Figure 6.6.4, Figure 6.6.5).

Due to the small size of the nanocrystal, the electron-hole separation is confined to be smaller than the Bohr radius of the semiconductor. As the nanocrystal core is made progressively smaller, more energy is required to confine the exciton, and the energy of the emitted photons increases. Therefore, smaller nanocrystals exhibit shorter-wavelength emission. By controlled variation of the core size, we have developed a range of nanocrystals with distinctively different emission spectral characteristics (Figure 6.6.5) but largely overlapping absorption spectra (Figure 6.6.4) and generally similar physical properties. Currently, we offer eight Qdot nanocrystal types identified by 3-digit numerals representing the peak emission wavelength in nanometers (e.g., Qdot 655 nanocrystal, emission peak = 655 nm).

Furthermore, Qdot nanocrystals exhibit fluorescence intermittency, commonly referred to as blinking, whereby continuously excited single fluorophores will spontaneously alternate between emitting and non-emitting states across timescales from milliseconds to many seconds. By its nature, blinking does not significantly impact ensemble measurements. Instead, it is most readily observable in single molecule detection trajectories where the binary nature of the "on" and "off" emission levels provides a useful confirmatory indication that the signals represent single fluorophores rather than clustered multiples.

Since the first reported applications of quantum dot nanocrystals for biomolecular detection in 1998, many reviews have been published that may be consulted for further information on their properties and applications, both actual and prospective.

For example, nucleic acid hybridization can be detected by complexation of terminally biotinylated oligonucleotides with Qdot streptavidin conjugates (Figure 6.6.6). Qdot streptavidin conjugates in combination with biotinylated antibodies provide increased multiplexing capacity for immunodetection of proteins on both imaging and flow cytometry platforms. Single-particle tracking of molecular motion is a particularly compelling application of Qdot streptavidin conjugates that takes advantage of their extraordinary fluorescence output capacity. The principal subjects for these measurements are cell-surface receptors and molecular motors such as myosin. Single-molecule detection uncovers details of molecular motion and assembly that are obscured by the averaging effects of bulk measurements. Qdot nanocrystals are quite essential in such applications as fluorescence photon output ultimately propagates into the spatial and temporal resolution of the measurements.

This formulation results in a smaller overall particle size and an increase in the number of streptavidins per nanocrystal relative to PEG-linked conjugates. These characteristics are advantageous in fluorescence resonance energy transfer (FRET) assays for detecting hybridization of target nucleic acid sequences to biotinylated oligonucleotides captured on the Qdot nanocrystal surface. Qdot ITK streptavidin conjugates are supplied in 250 L units of 2 M solution in kits that also provide Qdot incubation buffer (50 mM borate buffer, pH 8.3, containing 2% BSA and 0.05% sodium azide). Qdot incubation buffer is also available in separately packaged 30 mL units (Q20001MP).

With recent advances in Qdot probe technology and fluorescence imaging platforms, researchers are now able to obtain detection sensitivities from fluorescence-based detection comparable with those obtained from ECL (Figure 6.6.7). Furthermore, unlike ECL techniques, fluorescence-based detection methods provide the capability to detect multiple antigens on a single blot without stripping and reprobing. Importantly, probes with long-wavelength (red to near-IR) emission allow signal collection beyond the typical autofluorescence emitted by standard nitrocellulose and PVDF membranes, resulting in low background fluorescence and superior signal-to-noise ratios.

WesternDot secondary antibody conjugates provide a powerful alternative to traditional methods for western blot detection. The WesternDot secondary antibody conjugates allow detection of proteins that have been immobilized on membranes (nitrocellulose or PVDF) following western transfer. These conjugates utilize Qdot nanocrystals enhanced with VIVID technology, making them brighter than the original Qdot antibody conjugates. The unique fluorescent properties of WesternDot antibody conjugates allow simultaneous detection of multiple proteins on a single blot without stripping and reprobing or producing duplicate gels and blots. The stability of the WesternDot reagents makes it possible to take multiple images and store dried blots for months with minimal loss of fluorescent signal. Protein bands can be captured with fluorescent imagers equipped with UV, violet or deep-blue excitation wavelengths, including gel imagers with a UV transilluminator. Our current selection of WesternDot secondary antibody conjugates includes:

Figure 6.6.12 Distribution of Qdot nanocrystals in cytoplasmic vesicles after labeling cells with the Qtracker 655 Cell Labeling Kit (Q25021MP). HeLa cells were labeled with the Qtracker 655 Cell Labeling Kit and then observed using a Leica TCS SP2 confocal microscope (excitation at 488 nm). This representative image shows the Qdot nanocrystals distributed in vesicles throughout the cytoplasm.

Qtracker non-targeted quantum dots are designed for small animal in vivo imaging, and especially for studying vascular structure after microinjection (Figure 6.6.13). Our selection of Qtracker non-targeted quantum dots includes:

These nanocrystals exhibit intense red or near-infrared fluorescence emission, enabling maximum transmission through tissues while avoiding interference from background autofluorescence. Qtracker non-targeted quantum dots have polyethylene glycol (PEG) surface coatings to minimize nonspecific binding interactions and associated inflammatory responses. Because the PEG surface coating does not contain reactive functional groups, the Qtracker non-targeted quantum dots are retained in circulation longer and can be imaged for up to 3 months without additional injections. Qtracker non-targeted quantum dots are supplied as 2 M solutions in 50 mM borate buffer, pH 8.3 in units of 200 L.

Figure 6.6.13 Chick embryo injected through the major vitelline vein with Qtracker non-targeted quantum dots. Following a few minutes of circulation of the Qtracker 705 non-targeted quantum dots (Q21061MP), fluorescence images of the embryo were captured at increasing magnification using 460 nm excitation and a digital imaging system equipped with appropriate emission filters. These Qtracker reagents revealed highly detailed vascular structure at all levels of magnification. Images contributed by Greg Fisher, Carnegie Mellon University.

Qdot ITK amino (PEG) quantum dots have amine-derivatized PEG covalently attached to the amphiphilic inner coating and react efficiently with succinimidyl ester derivatives and other amine-reactive compounds. Our Qdot ITK amino (PEG) quantum dots are provided as 8 M solutions and include:

Qdot ITK organic quantum dots have an aliphatic hydrocarbon surface coating instead of an amphiphilic polymer coating. They are provided as a suspension in decane and are specifically designed for applications requiring organic solvents. Our Qdot ITK organic quantum dots are provided as 1 M solutions and include:

3a8082e126