Water‐Soluble Fluorescent Polymer Dyes with Tunable Emission Spectra for Flow Cytometry Applications

• Published in: Angewandte Chemie International Edition 2024-05, Vol.63 (20), p.e202402616-n/a
• Main Authors: Berndt, Daniel, Glaap, Dorina, Jennings, Travis, Dose, Christian, Werz, Daniel B., Reckert, Dirk N. H.
• Format: Article
• Language: English
• Subjects: Antibodies; Biological analysis; Dyes; dyes/pigments; Emission spectra; Emissions; Energy transfer; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fluorescent dyes; Fluorescent Dyes - chemistry; Fluorescent indicators; fluorescent probes; Humans; Molecular Structure; Polymers; Polymers - chemistry; Populations; Reagents; Solubility; Water - chemistry
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202402616

The application of spectrally unique, bright, and water‐soluble fluorescent dyes is indispensable for the analysis of biological systems. Multiparameter flow cytometry is a powerful tool for characterization of mixed cell populations. To discriminate the different cell populations, they are typically stained by a set of fluorescent reagents, e.g., antibody‐fluorophore conjugates. The number of parameters which can be studied simultaneously strongly depends on the availability of reagents which can be differentiated by their spectral properties. In this study a series of fluorescent polymer dyes was developed, that can be excited with a single violet laser (405 nm) but distinguished by their unique emission spectra. The polyfluorene‐based polymers can be used on their own, or in combination with covalently bound small‐molecule dyes to generate energy transfer constructs to red‐shift the emission wavelength based on Förster resonance energy transfer (FRET). The polymer dyes were utilized in a biological flow cytometry assay by conjugating several of them to antibodies, demonstrating their effectiveness as reagents. This report represents the first systematic investigation of structure–property relationships for this type of fluorescent dyes. A series of water‐soluble fluorescent polymer dyes was developed. All π‐conjugated polymers are excited by a 405 nm laser, but are distinguishable by their emission spectra. The emission can be tuned by covalently binding of small‐molecule dyes (Förster resonance energy transfer). The dyes were bioconjugated to antibodies and used as reagents for multiparameter flow cytometric analysis.