Biotinylated Fluorescent Polymeric Nanoparticles for Enhanced Immunostaining.
Fiche publication
Date publication
février 2023
Journal
Small methods
Auteurs
Membres identifiés du Cancéropôle Est :
Pr LEHMANN Maxime, Dr KLYMCHENKO Andrey
Tous les auteurs :
Yudhistira T, Da Silva EC, Combes A, Lehmann M, Reisch A, Klymchenko AS
Lien Pubmed
Résumé
The performance of fluorescence immunostaining is physically limited by the brightness of organic dyes, whereas fluorescence labeling with multiple dyes per antibody can lead to dye self-quenching. The present work reports a methodology of antibody labeling by biotinylated zwitterionic dye-loaded polymeric nanoparticles (NPs). A rationally designed hydrophobic polymer, poly(ethyl methacrylate) bearing charged, zwitterionic and biotin groups (PEMA-ZI-biotin), enables preparation of small (14 nm) and bright fluorescent biotinylated NPs loaded with large quantities of cationic rhodamine dye with bulky hydrophobic counterion (fluorinated tetraphenylborate). The biotin exposure at the particle surface is confirmed by Förster resonance energy transfer with dye-streptavidin conjugate. Single-particle microscopy validates specific binding to biotinylated surfaces, with particle brightness 21-fold higher than quantum dot-585 (QD-585) at 550 nm excitation. The nanoimmunostaining method, which couples biotinylated antibody (cetuximab) with bright biotinylated zwitterionic NPs through streptavidin, significantly improves fluorescence imaging of target epidermal growth factor receptors (EGFR) on the cell surface compared to a dye-based labeling. Importantly, cetuximab labeled with PEMA-ZI-biotin NPs can differentiate cells with distinct expression levels of EGFR cancer marker. The developed nanoprobes can greatly amplify the signal from labeled antibodies, and thus become a useful tool in the high-sensitivity detection of disease biomarkers.
Mots clés
EGF receptors, antibody conjugation, dye-loaded polymeric nanoparticles, fluorescence imaging, fluorescent nanoparticles
Référence
Small Methods. 2023 02 19;:e2201452