Biomedical Engineering Reference
In-Depth Information
that carries both a fluorochrome and a non fluorescent quencher chromophore at opposite
ends of a strand. The two ends are near each other in the absence of a complementary
strand. As a complementary nucleic acid binds to the loop, the molecular conformation is
changed, thereby removing the quencher from the fluorochrome's vicinity. An unquenched
fluorescence results. They further explain that DABCYL (a nonfluorescent chromophore;
(4-((1-4-dimethylamino)-phenyl)-azo-benzoic acid) is used typically to quench the fluorescence.
Sha et al. (2007) report that metal nanoparticles may also be used to quench fluorescence
rather than any organic moiety (
Dubertret and Kramer, 2001; Maxwell et al., 2002; Du
et al., 2003; Sha et al., 2005; Stoermer et al., 2006
).
Lakowicz (2001)
has indicated that fluo-
rescence from dyes located within a few nanometers from a surface may be quenched. Sha
et al. (2007) have used Nanobarcodes
™
(NBC) as the metal surface. They have developed
a SERS beacon assay for the detection of viral RNA by combining the sensitivity of SERS
technology and the multiplexing potential of NBC. The dimensions of NBC are about
300 nm
6-9
m
m. NBC has been synthesized by the sequential electrochemical deposition
of metal ions into templates with uniformly sized pores (
Nicewarner-Pena et al., 2001; Reiss
et al., 2002; Walton et al., 2002
).
The authors report that their dual-mode design uses a molecular beacon probe with a hairpin
structure.
This holds the dye at the 3
0
end close to the nanoparticle surface when the probe is
assembled to it by a 5
0
-thiol group. As the dye is close to the metal nanoparticle surface its
fluorescence is quenched (
Sha et al., 2005; Stoermer et al., 2006
). SERS spectra are observed
when the dye is held near the metal surface. They point out that as the probe hybridizes to the
target sequence
(a) The Raman label is forced to separate from the metal surface, and
(b) The SERS signal intensity drops.
This is because, as
Wolkow and Moskovits (1987), and Campion and Kambhampati (1998)
explain, the Raman enhancement is strongly dependent on the distance between the Raman
label and the metal nanoparticle. An inverse 12th order of dependence is exhibited here.
Sha et al. (2007) report that their technique permits them to detect a wide variety of oligonu-
cleotide analytes that include viral DNA, DNA biothreat agents, food borne pathogens, SNP
(single nuclear polymorphism) or mutant detection, gene expression products, and even bio-
marker proteins. Thus, their technique is quite flexible for detection purposes.
Sha et al. (2007) show that the HCV (hepatitis C virus) probe sequence is (CH
2
)6 gcggag
CAT AGT GGT CTG CGG AAD CGG TGActcgc (CH
2
)
7
Cy5-3
0
and the oligo sequence
is TCA CCG GTT CCG CAG ACC ACT ATG.
The nanobarcodes were made according to the procedure described previously (
Nicewarner-
Pena et al., 2001; Reiss et al., 2002; Walton et al., 2002
). Alternating layers of gold and silver
