Cyanine-saccharide conjugates for bacterial diagnostics: Towards multiplexed assays

Fluorescence-based enzymatic assay, utilizing an enzyme-specific bacterial substrate stained with a fluorogenic (“off-on”) dye, is a commonly used technique for bacterial detection and identification. However, the scope of fluorogenic dyes is limited to several classes of organic fluorophores, and these dyes often suffer from insufficient brightness, among some other drawbacks. Aiming to expand the variety of efficient tools for enzymatic bacterial identification assays, including high-throughput multiplexed analyses, we designed novel fluorescently labeled bacterial enzyme substrates where, for the first time, the conventional (non-fluorogenic) dyes of the Cy5 and Cy7 series are conjugated to an enzyme-specific saccharide via an orthoester bond. These conjugates provide a strong and stable fluorescence signal inside target bacteria, enabling their selective identification, which was proven in the example of Gram-positive and Gram-negative pathogens—Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. The proposed approach, consisting of the labeling of enzyme substrates with highly-bright dyes emitting at distinct wavelengths, paves the way for the creation of fluorescent arrays for multiplexed bacterial diagnostics.