Biomedical Engineering Reference
In-Depth Information
acid on QCM-D and SPR surfaces [
46
]. They found that the PMB was essential to
achieve high binding of LPS. Two excellent reviews have recently been published on
covalent immobilization or tether protocols of AMPs onto solid surfaces, with
emphasis on the development of antimicrobial materials [
47
,
48
].
Mor's group has also investigated the activity of a synthetic “AMPs”—
oligoacyllysines (OAKs)—immobilized on an SPR chip [
49
]. OAKs are synthetic
copolymers with repeats of acyllysines as a mimic for naturally occurring AMPs.
A resin-linked OAK bead was able to capture about 3,000 bacterial cells for use as a
concentration step. Captured
E. coli
was confirmed as still viable with real-time
PCR. Mor and coworkers were able to regenerate the beads multiple times with an
ethanol wash.
3.3 High-Throughput Screening Platforms
High-throughput screening (HTS) capabilities are important and necessary for the
rapid detection of multiple agents in a single reaction with minimal sample
processing. Here we discuss two current efforts to transition AMP-based detection
assays, previously demonstrated in low-density slide arrays, to commercial micro-
titer plate- and bead-based HTS platforms.
3.3.1 Microtiter Plates
Microtiter plates are effective solid-phase platforms for multiplexed, HTS and
analysis. The multiwell format, available in 96, 384, 1,536, and even 3,456 or
9,600-well plates, provides ease of automation, high capacity for paralleled data
collection, and versatile application of technologies. The most commonly used
approach for bioimmobilization to microtiter plates is noncovalent adsorption.
However, given the aforementioned importance of structure and presentation of
AMPs for cell capture, it is imperative that surface attachment strategies take into
consideration native conformation and molecular orientation to ensure functionally
active peptide display. Mello and coworkers first used amine-directed peptide
immobilization on maleic anhydride-activated microtiter plates to demonstrate
the ability of cecropin P1 to capture and immobilize bacterial cells on solid surfaces
[
50
]. Subsequently, AMPs were synthesized with C-terminal cysteine residues for
site-directed immobilization to maleimide-activated microtiter plates. The surface-
bound AMPs demonstrated preferential binding behavior for Gram-negative
E. coli
O157:H7 [
51
]. Mello's group has continued to focus on thiol-targeted chemistry for
AMP immobilization, which has been highly productive for large-scale AMP
screening for biodetection of pathogens.
The usefulness of AMPs for detection and diagnostic purposes relies heavily on
the ability to immobilize them on the surface of a detection platform in a reliable
and predictable manner that supports target capture. Therefore, we investigated the
Search WWH ::
Custom Search