Biomedical Engineering Reference
In-Depth Information
Fig. 17 Homogeneous microliter-volume VideoScan hybridization assay detection limit.
Varying concentrations of VIM-cap (d) and aCS-cap (r) were quantified in 1 lL on spots of
Advalytix
slides. We used the direct hybridization assay (Fig.
7
) for aCS-cap (r) and the
microbead hybridization probe assay (Fig.
16
) for VIM-cap (d). GAPDH-cap (m) was used as
negative control for non-specific quenching and to provide a threshold (—) calculated by the
three-sigma rule. Above the threshold, signals were considered significant. H, hybridization
efficiency (Eq.
3
)
It was possible to combine the VideoScan system with AmpliGrid slides for the
quantitative detection of analytes in a sample volume of 1 lL (Fig.
17
). GAPDH-
cap was used as negative control for non-specific quenching and for estimating a
threshold value by the three-sigma rule. Signals above threshold were considered
significant for VIM-cap and aCS-cap. As determined by graphical analysis, the
detection limit is about 9 nM of oligonucleotides in 1 lL reaction volume.
Interestingly, this was similar for both the homogeneous multiplex direct
hybridization assay (Fig.
7
) and the microbead hybridization probe assay (Fig.
16
).
The VideoScan system has been adopted for direct hybridization and FRET
hybridization assays. It offers a platform for parallelized reactions for various
nucleic acid applications. This design enables customized assays in small reaction
volumes to save reagents, samples and processing times.
6 VideoScan PCR: Analysis and Application
To date, the simple hybridization of nucleic acids has been analyzed using
VideoScan. The extension of the hardware with a heating/cooling unit (HCU)
(Fig.
2
b) dramatically increases the application range enabling dynamic heating,
e.g., PCR conditions. Melting curves and hybridization temperature optimization
are possible now—both are important to design and performance well performing
PCR assays.
