Biomedical Engineering Reference
In-Depth Information
capture [
32
]. While
Escherichia coli
binding to immobilized PMB was detected at
10
7
colony-forming units (cfu) per mL using an antibody tracer, Cy5-PMB failed to
detect their surface-bound
E. coli
even at tenfold higher concentrations. Our own
studies have shown that PMB and other AMPs are often modified on
multiple
side
chains even when using a 1:1 molar ratio of amine-specific labels [
33
]. Presumably,
the loss of
multiple
positive charges impacts both the affinity and specificity of the
modified AMPs [
22
,
34
,
35
].
To circumvent the pitfalls associated with amine-targeted modification, Mello's
group has utilized AMPs synthesized with a C-terminal cysteine [
36
,
37
]; this
unique thiol can be targeted by thiol-specific fluorophores without affecting overall
peptide charge or presumably structure. Indeed, in assays utilizing an antibody for
capture and fluorescent cysteinyl-AMPs as tracers, an improvement in assay sensi-
tivity was observed with Cy5-cysteine-cecropin P1, albeit not with all cysteinyl-
labeled AMPs tested. In contrast to Lim's observations [
32
], no decrement in assay
sensitivity was apparent for any of these labeled AMPs.
3.2 Biosensor-Based Detection Using Immobilized AMPs
Employing AMPs as the capture molecule in biosensors for the detection of bacteria
is another application. They can be used to bind broad categories of bacteria.
Both competitive assays and sandwich immunoassays have been demonstrated as
sensors for the presence of bacteria or their components (i.e., LPS). The main issue
in this area is to immobilize the AMP to a solid support such as fibers or planar
waveguides without losing their ability to bind to bacteria. As with the solution-
phase AMPs used as tracers, the chief requirement is the AMP's ability to bind to
the bacteria, not its biocidal ability.
The first example of an immobilized AMP for detection as a capture molecule in
a biosensor was described by James et al. at the Naval Research Laboratory; the
system used was an evanescent wave-based biosensor, where fluorescent signals are
read only from fluorescent molecules bound to the surface [
38
]. In this study, PMB
was covalently immobilized on the surface of a tapered optical fiber using
mercaptopropyl silane and an amine-reactive cross-linker. A known quantity of
fluorophore-labeled LPS competed with unlabeled LPS for binding to PMB. As the
concentration of unlabeled LPS increased, the fluorescence signal decreased. LPS
was used as the target as this is a major component of Gram-negative bacteria and
has been proposed as being part of the AMP-bacteria interaction. In this competi-
tive fluorescence-based assay, unlabeled LPS was detected at 12.5 ng/mL in buffer
and 25 ng/mL in plasma. Though the assay was run for 2 min, binding was
completed within 30 s. Optimization of the AMP concentration used for immobili-
zation was examined; the authors found that 10 mg/mL worked best, with higher
concentrations as well as the lower concentrations reducing sensitivity.
This work has since been expanded at the Naval Research Laboratory by
increasing the number of AMPs immobilized on a surface and developing
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