A novel ratiometric fluorescent immunoassay for human α-fetoprotein based on carbon nanodot-doped silica nanoparticles and FITC

The application of fluorescent carbon nanodots (C-dots or CD), non-toxic particulate organic labels, to disease biomarker detection is still in its earliest stage of development. In the effort described here, a novel ratiometric immunoassay was developed to target a model protein disease biomarker, alpha-fetoprotein (AFP), using C-dot doped silica nanoparticles (CD-SNPs) and fluorescein isothiocyanate (FITC) as signaling agents. Highly fluorescent C-dots were hydrothermally synthesized from citric acid and ethylene diamine. The C-dots were then encapsulated in silicate shells to yield 45 nm nanoparticles using a novel reverse microemulsion method, enabling convenient handling (centrifugation and washing) and straightforward surface chemistry modification to facilitate development of bioassays. Capture antibody capped CD-SNPs (Ab₁-CD-SNPs), together with FITC labeled antibodies (Ab₂-FITC) constituted a new ratiometric immunosensor for AFP, in which CD-SNPs functioned as both solid supports for washing and separation and as built-in reference signaling agents to correct for inconsistent environmental effects. A calibration curve was established between the ratiometric signal (the ratio of fluorescence signals of FITC and C-dots, F/C) and AFP concentration in the broad range of 0.317-280 μg dL^-1, exhibiting a useful linear range (0.317-35 μg dL^-1, R² = 0.9977), low detection limit (0.317 μg dL^-1) and acceptable recovery (105-120%). This assay format can be applied to a wide range of immunoassay targets. Our demonstration of encapsulating low-cost, easily synthesized, highly-fluorescent C-dots into silica nanoparticles for use in immunoassays will be useful in expanding future applications of these carbon nanomaterials to areas such as in vivo cellular imaging, drug delivery, and in vitro cell labeling and biomolecule sensing.