from Theron et al. in Nanotechnology in Water Treatment Applications

Quantum dots
Quantum dots (QDs) are colloidal, luminescent inorganic nanocrystals with unique photochemical properties, which include high quantum yields, large extinction coefficients, pronounced photostability, as well as broad absorption spectra coupled to narrow size-tunable photoluminescent emission spectra. A typical QD has a diameter of 2-8 nm and is usually composed of a core consisting of a semiconductor material, such as CdSe, enclosed in a shell of another semiconductor material with a larger spectral band-gap, such as ZnS. The shell prevents the surface quenching of excitons in the emissive core and thus increases the photostability and quantum yield for emission. Since QDs are usually synthesized from organometallic precursors and salts, they have no intrinsic aqueous solubility. Consequently, the native coordinating organic ligands on the surface of the QDs must either be exchanged or functionalized with a ligand that can impart both solubility and bioconjugation sites, if desired. Strategies for attaching biorecognition molecules to QDs comprise of direct chemical coupling to a functional group displayed on the QD surface or by non-covalent self-assembly in which proteins are engineered to express positively charged domains that interact through electrostatic attractions with the negative surface of modified QDs.

Although functionalized QDs have been used to detect complementary target nucleic acid sequences in chip-based assays and in fluorescent in situ hybridization (FISH) assays, are QDs increasingly being used in immunoassay detection. QD-based immunological assays have been applied to the detection of different bacterial and protozoan pathogens. For these immunoassays, the QDs are conjugated to organism-specific antibodies, or, alternatively, biotinylated organism-specific antibodies are used that are subsequently detected using a QD-streptavidin bioconjugate. These approaches have been used successfully for the detection of Cryptosporidium parvum (43 oocysts in spiked reservoir water), Giardia lamblia (1-5 x 10³ cysts) and Escherichia coli O157:H7 (2 x 10⁷ cells). Moreover, QDs appear to be especially suited for multiplex immunoassays. As a demonstration of the potential of QDs in multiplexed immunoassay formats, the simultaneous detection of E. coli O157:H7 and Salmonella enterica serovar Typhimurium bacteria (10⁴ cells in 2 h), and of C. parvum and G. lamblia (5 x 10³ cysts) in environmental water samples, using different coloured QDs as immunoassay labels, has been reported.

QDs have also been applied in flow cytometry because of their broad absorption spectra and narrow size-tunable photoluminescent emission spectra. The broad absorption band enables semiconductor QDs to simplify and improve the detection of multiple bacterial targets in flow cytometry samples. Each organic dye needs a separate excitation source resulting in multiple excitation sources and complicated experimental setups when monitoring more than one organism. In comparison is only a single excitation source in the UV range needed to excite all the visible colours of CdSe QDs. QDs have been used to simultaneously enumerate pathogenic E. coli O157:H7 and harmless E. coli DH5alpha in water. Cross spectral talk of QDs are also drastically lower than the organic dyes traditionally used in flow cytometry due to their narrow emission spectra. Flow cytometric measurements of QDs have been compared with the widely used fluorochrome, fluorescein isothiocyanate (FITC). The minimum fluorophore concentration for detection was a 100-fold lower when paramagnetic beads were labeled with QDs.

Despite their capability for single molecule and multiplexed detection, it is, however, unlikely that QDs will completely replace traditional organic fluorophores as biological labels. Some of the challenges that have yet to be overcome include financial costs, since QDs are expensive in comparison to organic dyes and there is an initial financial investment required regarding instruments optimized for use with QDs. Non-specific binding and aggregation are two factors that can lower QD fluorescence as in the case of antibody stained Cryptosporidium oocysts. Also, QDs are an order of magnitude larger than organic dyes and thus the extent to which their presence perturbs the recognition event being detected, must be determined. This is particularly important when multiplex assays are desired, since labelling several biomolecules with QDs of different sizes could result in varying degrees of perturbation due to the large differences in the QD sizes. In contrast, most organic dyes are of similar size in spite of their large differences in absorption/emission characteristics.

Tags: Microbial Detection | Pathogen Detection | Biodiagnostics | Biodetection Assays | Biomolecular Detection