Fluorescent ferritin nanoparticles and application to the aptamer sensor.

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Biology

Background:

• Fluorescent proteins like enhanced green fluorescent protein (eGFP) are crucial for biological imaging and assays.
• Ferritin nanoparticles offer a stable and versatile platform for protein display.
• Developing highly sensitive detection probes is essential for early disease diagnosis.

Purpose of the Study:

• To synthesize and characterize novel fluorescent ferritin nanoparticles (FFNPs) with enhanced properties.
• To conjugate DNA aptamers to FFNPs for use as reporter probes.
• To evaluate the performance of aptamer-FFNP probes in a cancer marker detection assay.

Main Methods:

• Bacterial expression of a hybrid gene encoding human ferritin heavy chain (hFTN-H), a spacer, and fluorescent proteins (eGFP or DsRed).
• Self-assembly of hFTN-H to form 12 nm nanoparticles displaying fluorescent proteins on the surface.
• Site-directed mutagenesis to introduce cysteine residues for DNA aptamer conjugation.
• Aptamer-based "sandwich" assay for detecting platelet-derived growth factor B-chain homodimer (PDGF-BB).

Main Results:

• Successfully synthesized green and red fluorescent ferritin nanoparticles (gFFNPs/rFFNPs) with amplified fluorescence intensity and enhanced stability compared to free fluorescent proteins.
• DNA aptamers were successfully conjugated to gFFNPs via engineered cysteine residues.
• The aptamer-conjugated gFFNPs demonstrated significantly enhanced detection signals, affinity, and assay sensitivity for PDGF-BB detection, achieving a limit of detection as low as 100 fM.
• The assay performed effectively in both phosphate-buffered saline and diluted human serum.

Conclusions:

• Fluorescent ferritin nanoparticles (FFNPs) represent a superior platform for fluorescent labeling compared to traditional fluorescent proteins.
• FFNPs conjugated with DNA aptamers serve as highly sensitive and stable reporter probes for aptamer-based sandwich assays.
• The developed FFNPs have broad applicability for detecting various biomarkers in biological samples, offering a promising tool for diagnostics.