Biomedical Engineering Reference
In-Depth Information
multiplexed assays using these AMP arrays. The system used in these later studies
is also an evanescent wave-based biosensor where fluorescent signals obtained are
limited to those molecules/cells captured on the sensor substrate. As mentioned
earlier, different AMPs have different affinities for different bacteria. Based on
their previous work with antibodies, Kulagina and coworkers took advantage of the
ability to immobilize different capture molecules in specific spots on an optical
waveguide and immobilized several different AMPs (magainin I, cecropin, poly-
myxin, parasin) via their primary amines in stripes across the slide [
8
,
39
,
40
].
Various bacterial solutions were run through channels perpendicular to the pat-
terned AMPs, followed by fluorescently labeled antibodies. When laser light was
launched into the waveguide forming an evanescent wave region, only the areas in
the evanescent wave with the full complex of AMP-bacteria-fluorescent antibody
fluoresced. Dose-response curves were generated and compared to those obtained
with immobilized antibodies on the same slides. The limits of detection were
similar for
E. coli
,
Salmonella
, and
Bacillus
spp., but a few showed improved
detection limits (killed
Francisella tularensis
, killed
Brucella
, and killed
Yersinia
pestis
). Importantly, while distinguishable binding patterns were observed for each
AMP and each species tested, similar species (e.g., alpha Proteobacteria, gamma
Proteobacteria, Firmicutes) produced similar, but not identical, patterns. These
observations support the hypothesis that AMP arrays can be used to classify
detected microbes into their appropriate phylum, class, order, and possibly family
and genus, based on their patterns of binding. In addition to both Gram-positive
and -negative bacteria, viruses were also detected; interestingly, the patterns of
AMP binding for the two viruses tested were virtually identical, again supporting
the potential for detection and classification based on AMP arrays.
Recently, Manoor and coworkers developed a microcapacitive electrode biosen-
sor for the detection of
E. coli
and
Salmonella
for water monitoring and pharma-
ceutical use [
41
]. This system incorporated a single peptide —magainin I —which
was modified such that it contained a cysteine on either the C- or N-terminus
for immobilization to gold electrodes. They demonstrated detection down to
10
3
bacteria/mL with selectivity for Gram-negative and pathogenic organisms.
They were able to demonstrate bacterial selectivity between Gram-positive and
Gram-negative bacteria, as well as between
E. coli
and
Salmonella
.
A major requirement for AMP integration on these and other biosensors is the
retention of activity after immobilization. Though not pursuing microbial detection
per se, Mello and coworkers have immobilized AMPs onto surfaces by various
means and assessed for activity, structure, and presentation/orientation; surface
plasmon resonance (SPR), sum frequency generation vibrational spectroscopy,
and quartz crystal microbalance with dissipation monitoring (QCM-D) were used
[
42
-
45
]. Their work published to date has been on the AMPs that have been
cysteine-modified at the C-terminus for immobilization. This work can be used to
further develop biosensors that employ gold surfaces for transduction. They
demonstrated that the immobilized cecropin was bound to the surface and
maintained its biocidal abilities. To demonstrate binding of LPS to surfaces,
Ansorena's group immobilized PMB via mercaptoundecanoic or mercaptopropanoic
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