Biomedical Engineering Reference
In-Depth Information
Fig. 7 Schematic representation of capture probes as used in a standard direct hybridization [
25
]
fluorescence resonance energy transfer (FRET) assay. Capture probes are immobilized (5
0
? 3
0
) via
C6 carbon or hexaethylenglycol linker to the microbead surface by either covalent cross-linking via
amide bonds or streptavidin-biotin binding, respectively. The capture probes contain a
poly(dT)
10-20
region which is used as spacer, and for the relative quantification and characterization
of non-labeled capture probes. The gene-specific region (15-35 nt) is complementary to the target
sequence. For FRET assay, capture probes carry a reporter dye at the 3
0
position
The recovery rate was determined as the ratio of immobilized microbeads to the
number of microbeads after seeding (Fig.
6
(b)). A coating concentration of 14 ng
mL
-1
PLL is sufficient to obtain a microbead recovery rate of at least 80%.
3.2 Immobilization of Biomolecules on the Surface
of Microbeads
VideoScan predominantly utilizes thermo-tolerant color-coded carboxylated
PMMA microbeads (PolyAn GmbH, Germany) with a diameter of 9-18 lm. This
simple approach allows rapid developmental cycles with comparatively low
technical demands. Biomolecules are immobilized by three different procedures:
passive adsorption, cross-linking and immobilization of biotinylated biomolecules
on streptavidin-coated microbeads. In the following sections we will refer to
protocols for the covalent and highly thermostable immobilization of DNA capture
probes via amide bonds according to Rödiger et al. [
3
] or Armstrong et al. [
24
]
(Fig.
7
). The immobilization of biotinylated oligonucleotides was done via biotin-
streptavidin interaction according to Rödiger et al. [
3
].
A set of 18 microbead populations was used to evaluate the success of capture
probe immobilization and target accessibility. We independently tested two fluo-
rescence-labeled (Atto 647N) capture probes (MLC-2v-cap, aCS-cap, Table
8
)
containing a poly(dT)
20
and target-specific region (Fig.
7
). Microbeads were loaded