Environmental Engineering Reference
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reference, PCR sensitivity level can be assumed as 100 copies of target gene/mL
(derived from 5 copies per 50 L volume in a PCR vial). PCR and real time PCR, of
course, can be optimized to detect 1 copy in a reaction volume but it is seldom achieved
in routine analysis, especially when reaction volumes are also small. Methods without
some type of amplification or concentration have much poorer detection limit. For
example, conventional glass slide based DNA array using Cyanine-3 labeling and
confocal laser scanning microscopy can only detect 5 pM which is approximately 3.00 ×
10
9
copies of target gene/mL (Taton et al., 2000). As illustrated in Table 13.2, the issue
of detection limit persists in most microarray-based approaches that do not use PCR as a
pre-amplification step. Certain nanoparticle-based techniques using indirect observation
of the target do report much better sensitivity, in the zeptomolar range. The bio bar-code
amplification technique reported by (Nam et al., 2004) is one such example.
Figure 13.4
(a) Transmission electron microscopy (TEM) image of octanethiol
functionalized gold NPs (Canu et al., 2008), and (b) TEM image of Rubpy dye-doped
silica NPs (Zhou et al., 2004). Copyright Elsevier 2004 and 2008, reproduced with
permission.
Use of nanoparticle-based approaches is emerging as a competing approach to
the use of dyes (e.g., used in microarrays). The large surface area of nanoparticles
serves as a compatible and versatile substrate for biomolecule immobilization
(Niemeyer, 2001). This facilitates the conjugation with signaling reporter groups and
thus renders each nanoparticle a high-density signaling probe. For example, many
fluorophore molecules can bind to one nanoparticle probe, and each binding site on the
target can thus conjugate more fluorophore molecules. In addition to the contribution to
high assay sensitivity, nanoparticle labels have not only extraordinary target recognition
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