Genetically functionalized ferritin nanoparticles with a high-affinity protein binder for immunoassay and imaging

- Human IgG-specific repebody was genetically fused to N-terminus of ferritin subunit.
- The RbF-NPs exhibited 1000-fold higher binding affinity for human IgG than free repebody due to multivalency.
- The Cy3-laleled RbF-NPs generated stronger fluorescent signals than Cy3-labeled free repebody in immunoassays and imaging.
- The RbF-NPs can be effectively used in various types of immunoassays and imaging.

Molecular detection of target molecules with high sensitivity and specificity is of great significance in bio and medical sciences. Here, we present genetically functionalized ferritin nanoparticles with a high-affinity protein binder, and their utility as a signal generator in a variety of immunoassays and imaging. As a high-affinity protein binder, human IgG-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was used. The repebody was genetically fused to the N-terminal heavy-chain ferritin, and the resulting subunits were self-assembled to the repebody-ferritin nanoparticles composed of 24 subunits. The repebody-ferritin nanoparticles were shown to have a three-order of magnitude higher binding affinity toward human IgG than free repebody mainly owing to a decreased dissociation rate constant. The repebody-ferritin nanoparticles were conjugated with fluorescent dyes, and the resulting nanoparticles were used for western blotting, cell imaging, and flow cytometric analysis. The dye-labeled repebody-ferritin nanoparticles were shown to generate about 3-fold stronger fluorescent signals in immunoassays than monovalent repebody. The repebody-functionalized ferritin nanoparticles can be effectively used for sensitive and specific immunoassays and imaging in many areas.

312