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The
use of luminescent quantum dot bioconjugates as
a
reporter in microarray assays
Advances in nonisotopic detection
methods have significantly influenced many areas of
research ranging from fundamental molecular biology
to clinical diagnostics. Most optical methods require
fluorescent probes to detect and monitor microscopic
interactions. Examples of these labels include many
well-known organic fluorophores and naturally occurring
chromophores such as green or red fluorescent protein
and phycoerytherin. Despite their obvious success in
applied research, traditional fluorophores exhibit many
undesirable characteristics including relatively weak
spectral emissions, short fluorescence lifetimes, narrow
excitation and broad emission spectra. Luminescent semiconductor
nanocrystals (quantum dots) provide a promising alternative
to traditional fluorescent labels. Compared to organic
dyes, quantum dots can be orders of magnitude brighter
and completely resistant to fading. Dependent only on
their physical size, quantum dots also exhibit narrow
and symmetrical emission spectra which are excitable
over a broad range of wavelengths. These novel optical
properties render quantum dots ideal for use in a wide
array of biotechnological applications.
One technology particularly dependent
on organic fluorophores as a reporter system is microarrays
or "DNA-chips". Microarrays are powerful tools commonly
used for high-density genotyping, characterization of
gene expression profiles, and more recently, genomic
resequencing. However, the sensitivity of conventional
microarrays is reduced by poor signal to noise ratios
caused, in part, by poor quantum yields and rapid fading
of the commonly used fluorophores CY3 and CY5. In terms
of the reporter assay itself, biotin end-labeling of
nucleic acid, fluorophore-streptavidin bioconjugation,
and steric hindrances between DNA and protein molecules
further contribute substantial variability to the assay.
Each of these phenomena dramatically influences the
accuracy and precision within and between microarray
experiments. Therefore, a more desirable system would
not only include an enhanced reporter but also a simpler
assay.
My primary aim as a GSTP postdoctoral
fellow is to incorporate quantum dots into microarray
technologies. Specifically, I will develop 1) a means
of rapid size-selection of newly synthesized quantum
dots, 2) water-soluble surface chemistries compatible
with relevant bioconjugation chemistries, and 3) a one-step
procedure to deliver bioreactive quantum dots to DNA
probes immobilized on a solid substrate. Once completed,
I predict that the accuracy and reliability of microarray-based
experiments will increase substantially. Advantageously,
the use of quantum dots as a reporter assay in microarray
experiments will also serve as a model to enhance the
performance of other technologies.
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