Labeled Antimicrobial Peptides for Detection of Microorganisms
ARMY NATICK SOLDIER RESEARCH DEVELOPMENT AND ENGINEERING CENTER MA
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Antibodies have traditionally been used for pathogen detection but often lack stability and sensitivity. Current biosensor technologies using fluorescently labeled antibodies are generally capable of detection of 10exp 3 to 10exp 44 bacterial cellsml. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Fluorescently labeled antimicrobial peptides AMPs were evaluated as a potential replacement of labeled antibodies in a sandwich assay. Due to their small size relative to antibodies peptides can bind to cell surfaces with greater density, yielding increased optical signal and improving sensitivity. The AMPs cecropin PI, SMAP29, and PGQ were synthesized with a C-terminal cysteine to which the fluorescent dye Cy5 was attached via maleimide linker chemistry. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. Preliminary screening against E. coli 0157H7 using a whole-cell solution binding assay revealed that Cy5 cecropin PI enhanced the detection 10-fold relative to a Cy5 labeled anti-E. coli 0157H7 antibody. Detection sensitivity of labeled antibody and peptide with a prototype immuno-magnetic bead biosensor revealed that Cy5 cecropin PI again resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli 0157H7 indicated that antimicrobial activity was not predictive of the sensitivity of the fluorescent assay. Labeled peptides also exhibit binding to spores and other bacteria, demonstrating potential utility for detection of other microorganisms.