Biomedical Engineering Reference
In-Depth Information
that the only components imparting assay specificity are the immobilized AMP-
capture reagents. One potential solution is through use of a nonspecific, whole cell
stain [
62
]; in our laboratory, however, we have observed significant staining of the
AMP-modified surfaces in the absence of any microbial targets. Potentially, AMP-
based tracers can also be used to detect bound microbes, as shown by Arcidiano
[
36
,
37
]. However, detection of bound targets is dependent on the specificity of
the AMP moiety of the tracer
as well as
the AMP-capture species, potentially
confounding efforts to deconvolute the binding patterns for classification purposes.
Smith's group at Notre Dame has developed a novel compound that could likewise
be used as a tracer in AMP-based assays. They have shown that a Zn-dipicolinic
acid-squaraine rotaxane conjugate stains a wide variety of bacterial species and
apoptotic mammalian cells, but does not appear to bind to healthy mammalian cells
[
63
]; further, when used as an imaging agent, this compound was able to discrimi-
nate between bacterial infections and sterile inflammations in mice [
64
].
Integration of a series of peptide “beacons” as capture reagents would
completely eliminate the need for a labeled tracer species, much as molecular
beacons have become more commonplace for reagentless detection of nucleic
acids [
65
]. While simple in concept, there have been only a handful of studies
demonstrating specific detection using peptide beacons [
66
,
67
]. The dearth of well-
characterized beacons based on native peptides is likely an indication of the
challenges in creating beacon-type structures while accounting for the inherent
complexities in peptide structure/function relationships.
A significant challenge remains in the ability to transfer AMP-based assays from
platform to platform. It has been well recognized within the antimicrobial coatings
community that the method of AMP immobilization affects their biocidal activities
(reviewed recently by Costa et al. [
47
,
48
]). It stands to reason that similar effects
will be encountered in AMP surfaces used for detection. Indeed, we and others have
observed significant differences in affinities and specificities by AMPs immobilized
onto different platforms through presumably analogous chemical linkages [
51
,
52
,
68
,
69
]. This poses a significant challenge if each detection platform has its own
specific chemistry required for attachment. In our own laboratory, we have devel-
oped a process for attaching AMPs to organic polymers such that patterns of
binding to different AMPs are similar to those observed on silanized silicate
surfaces [
52
,
68
], but this process may not be applicable to all sensor substrates.
Moreover, although binding patterns were similar, they were not identical. Until a
universal methodology to allow identical AMP density and presentation on all
surfaces is developed, we anticipate that deconvolution of binding patterns—used
to classify detected microbes—will require re-optimization on each new platform.
Clearly, AMP-based detection is still a work in progress. The groundwork of
putting AMPs into biosensors and detecting microbes and microbial markers has
been shown; in a limited number of cases, the potential benefits of using AMPs
(such as stability in a broad range of pHs and salt concentrations) have been
demonstrated. Although solution-phase systems have aptly demonstrated the prom-
ise for AMP-based detection in clinical samples, significant effort is still required to
provide evidence that biosensor-based tests are useful with complex samples such
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