Biomedical Engineering Reference
In-Depth Information
Fig. 6 Principle of microbead immobilization on PLL-coated wells. a (1) PLL is used to coat the
bottom of the well. After 2 h, coating is finished and remaining PLL solution is removed. (2) A
suspension of capture probe-loaded microbeads is added to the well. Gravitational force leads to a
rapid sedimentation (*10 min) of microbeads which adhere to the PLL. (3) The evaporation of
suspension supernatant leads to permanent immobilization of the microbeads. b Effect of PLL
concentration on microbead recovery. Two different microbead populations were loaded with
capture probes fyuA or ompA and subsequently immobilized on PLL-coated NucleoLink
stripes.
Coating wells with 14 ng mL
-1
PLL resulted in a recovery rate above 80% of microbeads applied
and this concentration was used throughout further experiments
with dynamic temperature profiles at elevated temperatures suffer from intense
microbead motion, due to convection and Brownian motion. We identified the
polycationic polymer poly-
L
-lysine (PLL) as an excellent substrate for immobili-
zation capture of molecule-loaded microbeads on standard plastic wells. PLL
was previously reported for the immobilization of cells and different biomolecules
on glass and plastic materials [
18
,
19
]. This simple procedure fulfilled all
requirements: (a) to keep various polydispersed microbead populations randomly
mixable, (b) to introduce no adverse effect into the optical well properties, (c) no
non-specific binding of reagents, (d) general applicability for various biomole-
cules, (e) no adverse effect on the ligand value and (f) equal distribution of
single microbeads as a densely packed monolayer on the well bottom. It therefore
made obsolete complicated methods developed for microbead immobilization, like
the use of etched surface structures (e.g., Illumina BeadArray systems), mechan-
ical retention or microbead embedding in hydrogel-coated slides [
11
,
13
,
20
-
23
].
To determine optimal conditions for the immobilization of microbeads in
multiplex assays, we started with two microbead populations. Oligonucleotide
capture probes encoding for fyuA or ompA (Table
3
) were coupled onto the
microbead surface (see
Sect. 3.2
). PLL hydrobromide (Sigma-Aldrich, MW
30,000-70,000) was resolved in phosphate-buffered saline (PBS) in concentrations
ranging from 0 to 14 ng mL
-1
. The NucleoLink
modules were incubated with 40
lL of the PLL solutions for 2 h at 40 C (Fig.
6
a(1)). The supernatant was removed
and the PLL-coated modules were washed with 100 lL of 1:100 PBS for 5 min at
room temperature. Afterwards, microbeads suspended in 1:100 PBS were added to
the PLL-coated modules (Fig.
6
a(2)). The microbead suspension was left to dry
(Fig.
6
a(3)). Residues of buffer substances were removed by washing in ddH
2
O.
