Magnetic microsphere-based methods to study the interaction of teicoplanin with peptides and bacteria

Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal d-Ala-d-Ala intermediates in the peptidoglycan of the cell wall of Gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two d-Ala-d-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled d-Ala-d-Ala-d-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 × 10⁻⁶ and 3.0 × 10⁻⁶ mol L⁻¹, respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of Gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide–antibiotic interaction.