Biomedical Engineering Reference
In-Depth Information
examine large numbers of samples for up to 500 targets simultaneously. Each bead
set can be separately coated with a different recognition molecule (e.g., AMPs) and
then mixed together to create user-tailored, multiplexed assays. Bead arrays have
been widely used for clinical diagnostics, transcriptional and expression profiling,
autoantibody screening, and food, water, and environmental monitoring [
54
-
61
].
We have recently demonstrated that AMPs could be immobilized onto
carboxylated polystyrene beads and used to detect
E. coli
,
Salmonella
,
Bacillus
,
and
Listeria
in Luminex assays [
61
]. Detection limits generally ranged from 10
4
to
10
5
cfu/mL when attaching AMPs using amine-targeted linkages. Sensitivities were
generally tenfold poorer when AMPs were immobilized via C-terminal cysteines
using thiol-targeted chemistry. Initial studies looking at amine-directed linking
showed similar effects to those observed on other substrates and in other systems.
Effectiveness of target capture was highly dependent on the nature of the surface to
which the AMPs were attached (e.g., hydrophobic/hydrophilic, passivated/
unpassivated with protein scaffolds, basic/acidic surfaces). Surprisingly, linkers
with long flexible “tether” regions were not the most effective for retention of binding
activity, but AMP density was not measured or accounted for in these studies.
More recently, we have adopted a more systematic approach for discriminating
between the relative effects of initial surface characteristics, linker “tether” regions,
and AMP density. Using commercial heterobifunctional linkers terminated with
maleimide (thiol-reactive) and
N
-hydroxysuccinimidyl ester (amine-reactive)
groups, density of both functional groups and immobilized AMPs could be accu-
rately determined; moreover, the individual effects of various “tether” characteri-
stics (e.g., hydrophobicity, flexibility, length) on AMP affinity and selectivity could
be separately quantified. Interestingly, when comparing linkers with similar tether
properties, we observed that increased freedom of movement (increased length,
flexibility) often resulted in lower binding activities. These differences were
striking when comparing binding activities of two helix-hinge-helix peptides,
cecropin A and melittin, for the closely related species,
E. coli
and
Salmonella
.
Both melittin and cecropin A exhibited similar patterns of binding when
immobilized by linkers incorporating aliphatic tethers. However, by simply
integrating a phenyl or cyclohexane moiety in the linker, cecropin A became highly
specific for
Salmonella
(versus
E. coli
); this effect could be reversed by increasing
the chain length of the hydrophobic linker, again yielding binding at similar levels
as
E. coli
(Fig.
5
). These intriguing results point to the potential of custom-tailoring
surfaces to suit the user's desired specificity for detection by simply altering the
tether used to link each AMP to the surface.
4
Issues and Challenges
The ability to detect a broad variety of microbial targets is desired for many
applications: diagnostics, environmental monitoring, industrial process monitoring,
food quality and safety assessments, and biodefense. As such, the more robust,
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