Development of a Separation and Detection System for Bacillus anthracis Spores Based on Peptide Conjugates Identified from Peptide Library

Abstract

A simple, specific and rapid identification system against Bacillus anthracis spores was developed using specific capture peptides conjugated with a bead-based biosensor, which was tightly and specifically bound onto the spore surface of B. anthracis. We successfully detected and separated B. anthracis spores from the spores of B. thuringiensis and B. cereus using this peptide-magnetic bead conjugates by fluorescence and anthrax-specific analyses. For more convenient mediation of high-throughput detection against B. anthracis spores, streptavidin-biotin interactions of spore-peptide and spore-peptide-magnetic bead conjugates were performed, and we demonstrated the separation and detection of B. anthracis spores using this method. In the presence of mixed condition of several types of Bacillus spores, the B. anthracis spores were easily separated using the oligopeptide-conjugated magnetic beads, thus allowing the clear detection of B. anthracis spores from B. cereus, B. subtilis, and B. thuringiensis spores. This oligopeptidebased strategy was rapidly and unambiguously identified as little as one viable B. anthracis spore in less than 1 h with a simple binding assay format. When assessed for its effectiveness for specifically and selectively detection of environmental spores phylogenetically similar to B. anthracis spores, such as B. thuringiensis and B. cereus spores, the system was free of false-positive signals. In this study, we developed a high-throughput detection system for B. anthracis spores with oligopeptides using fluorescence, magnetic beads, and positional scanning-synthetic peptide combinatorial libraries (PS-SPCLs). Specific capture oligopeptides were developed to specifically bind against the surface of B. anthracis spores. Several peptide ligands were conjugated with Fluorescein Isothiocyanate (FITC) to detect fluorescent signals, and magnetic beads were used to facilitate rapid isolation by magnetic polarity conjugated with streptavidin to bind with biotin-conjugated oligopeptides. The resulting system was capable of the detection of specific spores with high sensitivity in less than 1 h. We also optimized the system to maximize its binding affinity to spores of two B. anthracis strains, namely, B. anthracis ΔSterne Finally, to further improve the performance of the capture peptides, we newly designed their amino acid sequences using PS-SPCLs that can bind B. anthracis spores with greater specificity. The detection system based on these specific capture peptides is a high-throughput fluorescence-based assay that employs quantum dots. These specific binding systems can be employed to rapidly, specifically and selectively detect and separate B. anthracis spores in mixed conditions. Development of a Separation and Detection Materials and Methods MaterialsStreptavidin-conjugated magnetic beads (~ 1-2 μm in diameter) and a portable handheld magnetic separator were purchased from PureBiotech (San Diego, CA). The capture peptides used in this study (Table 1) were synthesized by Peptron (Korea)....