desorption of surface ligands results in aggregation and precipitation of the QDs
over a period of several weeks. Also, a reduction in quantum efficiency occurs
when using these surface molecules, the extent of which is dependent on the
linker used and the chemical environment. In spite of these problems, this simple
procedure has allowed the production of fresh batches on an ‘‘as-needed’’ basis.
Again, well established protocols can be used for surface bioconjugation.
A variation of this direct adsorption method is to incubate the water soluble QDs
with thiol-containing biomolecules, which slowly replace the surface mercapto ligands via mass action. This has been demonstrated for DNA [18] and proteins [42]
in aqueous solutions as well as for other small biomolecules which can replace
the TOPO molecules in polar organic solvents [43]. In fact, in using this thiolexchange method, Van Orden and coworkers noted that the reduced quantum
yield, due to the direct adsorption of mercaptoacetic acid, was completely recovered
when thiolated bovine serum albumin (BSA) replaced it on the QD surface. Since
this protein-coated surface provides multiple functional groups, it has inherent
advantages over the bifunctional linkers in that it is amenable to a wide array of
techniques for covalent conjugation to biological molecules and biomaterials.
Several other strategies have been devised in recent years to obtain robust and
biocompatible QD capable of long term stability in aqueous solution (Fig. 12.3).
Among these, bioconjugation via electrostatic interactions has been used to attach
QDs to engineered recombinant proteins. This procedure involves the direct adsorption of dihydrolipoic acid (DHLA) onto the QD surface (leading to negative
charges), and then electrostatic self assembly to positively charged proteins [17,
44–46]. A strategy employing hydrophilic organic dendron ligands has been reported by Peng and coworkers [47], and a micellular encapsulation procedure has
been described by Dubertret et al. using phospholipids [48]. Importantly, amphiphilic polymer-coated QDs conjugated to streptavidin have become commercially
available [49]. These polymer-coated QD bioconjugates are extremely stable against
flocculation and highly luminescent. It should be noted that the QD surface modification and bioconjugation in all of these cases results in several macromolecules
being attached to one nanocrystal. As a result, the overall size of the QD bioconjugates can be increased 2–3 fold above the original (core)shell nanocrystal.
12.5
Applications
Quantum dot bioconjugates have been used for immunoassay [44, 45], FRET-based
binding assays [42] and DNA hybridization detection [18, 19, 38]. Nanocrystals are
also emerging as a new class of fluorescent labels for targeting cell surface receptors [43] and for in vivo targeting and imaging [48, 50]. An important aspect of
QD labels is their extremely high photostability, which allows real-time monitoring
or tracking of intracellular processes over long periods of time (hours to days) [46].
For example, QD bioconjugates have been injected into Xenopus embryos to monitor development to the tadpole stages [48] (Fig. 12.4). The potential for long term
12.5 Applications 413
over a period of several weeks. Also, a reduction in quantum efficiency occurs
when using these surface molecules, the extent of which is dependent on the
linker used and the chemical environment. In spite of these problems, this simple
procedure has allowed the production of fresh batches on an ‘‘as-needed’’ basis.
Again, well established protocols can be used for surface bioconjugation.
A variation of this direct adsorption method is to incubate the water soluble QDs
with thiol-containing biomolecules, which slowly replace the surface mercapto ligands via mass action. This has been demonstrated for DNA [18] and proteins [42]
in aqueous solutions as well as for other small biomolecules which can replace
the TOPO molecules in polar organic solvents [43]. In fact, in using this thiolexchange method, Van Orden and coworkers noted that the reduced quantum
yield, due to the direct adsorption of mercaptoacetic acid, was completely recovered
when thiolated bovine serum albumin (BSA) replaced it on the QD surface. Since
this protein-coated surface provides multiple functional groups, it has inherent
advantages over the bifunctional linkers in that it is amenable to a wide array of
techniques for covalent conjugation to biological molecules and biomaterials.
Several other strategies have been devised in recent years to obtain robust and
biocompatible QD capable of long term stability in aqueous solution (Fig. 12.3).
Among these, bioconjugation via electrostatic interactions has been used to attach
QDs to engineered recombinant proteins. This procedure involves the direct adsorption of dihydrolipoic acid (DHLA) onto the QD surface (leading to negative
charges), and then electrostatic self assembly to positively charged proteins [17,
44–46]. A strategy employing hydrophilic organic dendron ligands has been reported by Peng and coworkers [47], and a micellular encapsulation procedure has
been described by Dubertret et al. using phospholipids [48]. Importantly, amphiphilic polymer-coated QDs conjugated to streptavidin have become commercially
available [49]. These polymer-coated QD bioconjugates are extremely stable against
flocculation and highly luminescent. It should be noted that the QD surface modification and bioconjugation in all of these cases results in several macromolecules
being attached to one nanocrystal. As a result, the overall size of the QD bioconjugates can be increased 2–3 fold above the original (core)shell nanocrystal.
12.5
Applications
Quantum dot bioconjugates have been used for immunoassay [44, 45], FRET-based
binding assays [42] and DNA hybridization detection [18, 19, 38]. Nanocrystals are
also emerging as a new class of fluorescent labels for targeting cell surface receptors [43] and for in vivo targeting and imaging [48, 50]. An important aspect of
QD labels is their extremely high photostability, which allows real-time monitoring
or tracking of intracellular processes over long periods of time (hours to days) [46].
For example, QD bioconjugates have been injected into Xenopus embryos to monitor development to the tadpole stages [48] (Fig. 12.4). The potential for long term
12.5 Applications 413