Biotinylated Fluorescent Polymeric Nanoparticles for Enhanced Immunostaining

Abstract

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.

Brightness of fluorescent organic nanomaterials

“…alginate or polyvinyl alcohol). Negatively charged NPs can be readily immobilized on glass surfaces pre-treated with cationic polymers, such as poly(ethylene imine); 24,33 whereas for biotinylated NPs (including commercially available QDs), 34,35 the glass surface is pre-treated with BSA-biotin and then neutravidin, while for DNA-functionalized NPs, biotinylated complementary strands can be deposited on the glass surface pre-treated with BSA-biotin and then neutravidin. 35 Care has to be taken to achieve sufficient separation of the NPs and to avoid formation of aggregates during adsorption to ensure that actual single particles are imaged.…”

White Light Emissive Tetraphenylethene Molecular Cage-Based Hybrid Nanoparticles for Intracellular Long-Term Imaging

“…Fluorescence bioimaging is one of the main driving forces in determining the dynamics and functions of bioprocesses in live cells, due to its advantages of low toxicity, high spatiotemporal resolution, and utilization of an inexpensive instrument. − To date, many types of fluorescent probes, such as fluorescent organics, − fluorescent proteins, , inorganic nanoparticles, , and semiconductor polymer nanoparticles, have been developed and widely used in bioimaging. Comparatively, among these fluorescent probes, fluorescent organic probes, including the small organic probes − and fluorescent organic nanoparticles, − attracted more attention because of their controllable synthesis, stable luminescence, good biocompatibility, sensitive responsiveness, and high signal-to-noise ratio. Small molecule organic fluorophores are characterized by their compact size, enabling efficient penetration into biological tissues, rapid response to environmental changes, and flexibility in design, providing valuable advantages in bioimaging applications.…”

Label-Free and Real-Time Optical Detection of Affinity Binding of the Antibody on Adherent Live Cells

“…With intact cells as detection probes, on the other hand, the most utilized configuration for biorecognition detection relies on labeled reporters, e.g., using the fluorescent dye-labeled antibody coupled with fluorescent microscopy to report the affinity binding events. , This necessitates costly and tedious labeling of the biomolecules. The detection specificity may also be damaged.…”