Immobilization and orientation-dependent activity of a naturally occurring antimicrobial peptide.

A naturally occurring antimicrobial peptide, SMAP-29, was synthesized with an n-terminal or c-terminal cysteine, termed c_SMAP and SMAP_c, respectively, for site-directed immobilization to superparamagnetic beads. Immobilized SMAP orientation-dependent activity was probed against multiple bacteria of clinical interest including Acinetobacter baumannii, Pseudomonas aeruginosa, Bacillus anthracis sterne and Staphylococcus aureus. A kinetic microplate assay was employed to reveal both concentration and time-dependent activity for elucidation of minimum bactericidal concentration (MBC) and sub-lethal effects.

Kinetic microplate assay for determining immobilized antimicrobial peptide activity.

Antimicrobial peptide immobilization onto surfaces is of great interest, although characterization of activity can be problematic. The kinetic microplate method described here determines the minimum bactericidal concentration (MBC) of immobilized antimicrobial peptides through a combination and modification of traditional solution assays, overcoming the difficulties of working with a solid substrate. The technique enables rapid, accurate evaluation of immobilized peptide lytic behavior, elucidating both dose- and time-dependent activity at multiple concentrations.

Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli.

The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time-dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 microM.

Antimicrobial peptide preferential binding of E. coli O157:H7.

The studies presented here explore antimicrobial peptide preferential binding behavior for a target pathogen, Escherichia coli O157:H7. A modified immunoassay and surface plasmon resonance were employed to evaluate immobilized peptide binding of whole bacterial cells. The knowledge gained may guide the rational design of peptides with enhanced species binding selectivity.

Aptamer-based sensor arrays for the detection and quantitation of proteins.

Aptamer biosensors have been immobilized on beads, introduced into micromachined chips on the electronic tongue sensor array, and used for the detection and quantitation of proteins. Aptamer chips could detect proteins in both capture and sandwich assay formats. Unlike most protein-based arrays, the aptamer chips could be stripped and reused multiple times.

DNA hybridization and discrimination of single-nucleotide mismatches using chip-based microbead arrays.

The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers.

Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing.

Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S2-ODNs) with sulfurs replacing one or both of the non-bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one-bead one-S-ODN and one-bead one-S2-ODN libraries.