Biomedical Engineering Reference
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Fig. 8 Quality control of DNA capture probe loading and probe accessibility to hybridization.
The quantities (refMFI) of two fluorescence-labeled (Atto 647N) DNA capture probes
(a) MLC-2v-cap or (b) aCS-cap, Table
8
) were determined simultaneously on 18 microbead
populations (eight replicates). Each colored symbol for ''d'' , ''j'' , o r ''m'' represents the mean
of one microbead population. Error bars are given as standard deviation. Microbeads with
different quantities of capture probes were prepared as described in Rödiger et al. [
3
]. The
poly(dT)
20
region (Fig.
7
) common to both probes was interrogated by direct hybridization with
fluorescent Alexa Fluor 750 (AF750)-labeled poly(dA)
20
showing that capture probes on all
microbead populations are highly accessible. Data were fitted using the RKWard integrated
development environment (Rödiger et al. [
27
]) using the lm() function. Pearson's r correlation
coefficient was calculated using the cor() function. (c) Ratios of ligand channels of AF750 and
Atto 647N (A647N, ordinate) and microbead populations (abscissa) shows a high agreement
throughout all microbead populations and capture probes (dMLC-2v-cap, jaCS-cap). This
suggests that both fluorescence channels can be used in bioassays
with different quantities of capture probes according to Rödiger et al. [
3
].
The fluorescence signal of Atto 647N (A647N) indicated a successful probe
immobilization and allowed an inference regarding the quantity. The hybridization
with Alexa Fluor 750 (AF750) labeled poly (dA)
20
was indicative of the quantity
(compare Day et al. [
26
]) and functionality of the immobilized capture probe.
We found a very strong correlation (C0.94, p \ 0.05) and a linear relationship
for immobilized probes interrogated with the AF750-labeled poly(dA)
20
(Fig.
8
).
Thus the use of labeled poly(dA)
20
is an elegant and cost-effective approach to
characterizing the quantity and functionality of surface-bound capture probes.
We also calculated the ratio of the ligand channel values of AF750 and Atto 647N
and found a general agreement independent of the capture probe used in all
microbead populations (Fig.
8
c). The two different channels for ligand value
measurement provide an important feature for assay development.
