Labeling of Amine-Modified Material Surfaces with PFB-Fluorescein

Kaja Jaskot,1 Angelika Maria Mielcarek,2 Łukasz Popenda,1 Ahmet Kertmen,3 Olena Ivashchenko,1 Bartosz F Grześkowiak,1 Agnieszka Fedoruk-Wyszomirska,4 Kamilla Bąkowska-Żywicka,5 Agata Tyczewska,5 Jan Barciszewski,1,5 Patrick M Perrigue1 1NanoBioMedical Centre, Adam Mickiewicz University, Poznań, Poland; 2Faculty of Chemistry, Adam Mickiewicz University, Poznań, Poland; 3Faculty of Physics and Astronomy, Adam Mickiewicz University, Poznań, Poland; 4Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland; 5Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, PolandCorrespondence: Patrick M Perrigue, NanoBioMedical Centre, Adam Mickiewicz University, Wszechnicy Piastowskiej 3, Poznań, 61-614, Poland, Email [email protected]: Fluorescent labeling is a widely used method for visualizing and tracking materials. 5-(Pentafluorobenzoylamino) fluorescein (PFB-F) is a dye in which the pentafluorobenzoylamino group substituent functions as a reactive linker. While PFB-F has been used for labeling biomolecules, its application in labeling surfaces has not been explored yet. Here, we report straightforward method for attaching PFB-F to amine-modified material surfaces requiring only dimethyl sulfoxide (DMSO) solvent and room temperature (RT).Methods: We conducted nuclear magnetic resonance (NMR) and thin layer chromatography (TLC) analysis on a mock reaction involving PFB-F and N-butylamine to evaluate the process of attachment via the PFB linker. PFB-F was also reacted with poly-L-lysine to evaluate conjugation to polymers bearing primary amine groups. Silica nanoparticles (SiNPs) labeled with PFB-F were administered to nematode worms, followed by confocal fluorescence microscopy.Results: NMR and TLC analysis confirmed covalent bonding between PFB-F and primary amines via nucleophilic aromatic substitution (SNAr) involving displacement of at least one fluorine atom. PFB-F was successfully conjugated to poly-L-lysine, demonstrating efficient reaction with polymers containing primary amine groups. SiNPs labeled with PFB-F were ingested by nematode worms and successfully tracked in vivo.Discussion: These results demonstrate that PFB-F is a versatile and effective reagent for stable surface labeling. This study paves the way for broader applications of PFB-F in material design.Keywords: fluorescein, PFB-F, nanoparticles, material surface, conjugate chemistry, C. elegans