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The VNP sensors were then evaluated in a DNA microarray as outlined
in Fig. 4.20 (Soto
, 2006). The target DNA on the chip is probed with
biotinylated test DNA. If the sequences are complementary (i.e., the
nucleotide sequences anneal to each other by virtue of base pairing), the
test DNA binds to the target DNA via nucleic acid hydridization. Next,
the dual-functionalized VNPs are added; binding is accomplished via
interaction of the conjugated neutravidin and the biotinylated test DNA.
Detection is carried out via the fluorescent signal derived from the
fluorophores attached to the VNP. Signal sensitivity of CPMV sensors was
compared to the signal intensity given when using: (i) dye-labeled test
DNA molecules (this is one-step detection method; the dye-labeled DNA
is used instead of biotinylated DNA) and (ii) dye-labeled streptavidin. The
CPMV sensors outperformed the conventionally used detection devices
(Soto
et al.
, 2006).
In a second example, dye- and antibody-conjugated CPMV probes were
utilized in an immunoassay. Again high sensitivity and specificity were
confirmed (Sapsford
et al.
, 2006). These techniques, of course, have the
potential to be applied to other VNPs as well.
et al.
Figure 4.20
DNA microarray detection scheme. (A) DNA oligonucleotides 1 and
2 (probes) are immobilized in a microarray format on glass slides. (B) DNA probe
1 hybridizes with a previously amplified and biotinylated target DNA molecule.
This hybridization event is detected using (C) streptavidin-Cy5 (Cy5 = fluorescent
dye) or (D) NA-Cy5-CPMV (NA = neutravidin). (E) Quantification post-detection
indicates a true-positive signal for the NA-Cy5-CPMV detection method (blue
spot, hybridization with DNA probe 1) or a true-negative signal (gray spot,
non-hybridization with DNA probe 2). (F) A false-negative signal (gray spot) is
observed for streptavidin-Cy5 as the total number of fluorophores at the DNA
probe 1 spot results in the generation of a signal that is below the detection threshold
(or background). Reproduced with permission from Soto, C. M., Blum, A. S., Vora, G.
J., Lebedev, N., Meador, C. E., Won, A. P., Chatterji, A., Johnson, J. E., and Ratna, B. R.
(2006) Fluorescent signal amplification of carbocyanine dyes using engineered viral
nanoparticles,
J. Am. Chem. Soc.
,
128
(15), 5184-5189.
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