Fluorescent ferritin nanoparticles and application to the aptamer sensor.

We synthesized fluorescent ferritin nanoparticles (FFNPs) through bacterial expression of the hybrid gene consisting of human ferritin heavy chain (hFTN-H), spacer (glycine-rich peptide), and enhanced green (or red) fluorescent protein [eGFP (or DsRed)] genes. The self-assembly activity of hFTN-H that leads to the formation of nanoparticles (12 nm in diameter), the conformational flexibility of the C-terminus of hFTN-H, and the glycine-rich spacer enabled eGFPs (or DsReds) to be well displayed on the surface of each ferritin nanoparticle, resulting in the construction of green (or red) FFNPs [gFFNPs (or rFFNPs)]. As compared to eGFP (or DsRed) alone, it is notable that the developed FFNPs showed significantly amplified fluorescence intensity and also enhanced stability. DNA aptamers were chemically conjugated to gFFNP via each eGFP's cysteine residue that was newly introduced through site-directed mutagenesis (Ser175Cys). The DNA-aptamer-conjugated gFFNPs were used as a fluorescent reporter probe in the aptamer-based "sandwich" assay of a cancer marker [i.e., platelet-derived growth factor B-chain homodimer (PDGF-BB)] in phosphate-buffered saline buffer or diluted human serum. This is a simple two-step assay without any additional steps for signal amplification, showing that compared to the same aptamer-based assays using eGFP alone or Cy3, the detection signals, affinity of the reporter probe to the cancer marker, and assay sensitivity were significantly enhanced; i.e., the limit of detection was lowered to the 100 fM level. Although the PDGF-BB assay is reported here as a proof-of-concept, the developed FFNPs can be applied in general to any aptamer-based sandwich assays.

Lyophilization and enhanced stability of fluorescent protein nanoparticles.

Protein nanoparticles (PNPs) that are nanostructured biomaterials with intrinsic biological function have been widely employed as three-dimensional nanobiomaterials for sensitive bioassays, MRI contrast, semiconductor devices, template for hybrid materials, etc., and stable and long-term maintenance of PNPs seems to be of crucial importance. We evaluated the stability of PNPs and the efficacy of lyophilization for the long-term stability of PNPs, especially using green fluorescent protein nanoparticles (gFPNPs) as a model PNP. Fluorescence intensities and TEM images of gFPNPs were analyzed to monitor their functional and structural stabilities. Unlike the green fluorescent protein monomers (eGFP) that were gradually inactivated in aqueous solution, gFPNP in the same aqueous solution retained the initial fluorescence activity and spherical nanoparticle structure even for 2 weeks at 4°C. To ensure stable and long-term maintenance of gFPNPs, gFPNPs in aqueous solution were converted to the dried solid forms through lyophilization. It is notable that fluorescence activity and nanoparticle structure of gFPNPs that were lyophilized with both Tween 80 and sucrose were very stably maintained even for 10weeks at various storage temperatures (-20°C, 4°C, 25°C, and 37°C). During the period of 10weeks, the fluorescence of gFPNP was always more than 80% level of initial fluorescence at a wide range of temperature. Although this stability study was focused on gFPNPs, the developed optimal lyophilization conditions for gFPNPs can be applied in general to stable and long-term maintenance of many other PNP-derived biomaterials.