Highly Fluorescent Magnetic Nanobeads with a Remarkable Stokes Shift as Labels for Enhanced Detection in Immunoassays

Fluorescence immunoassays are popular for achieving high sensitivity, but they display limitations in biological samples due to strong absorption of light, background fluorescence from matrix components, or light scattering by the biomacromolecules. A powerful strategy to overcome these problems is introduced here by using fluorescent magnetic nanobeads doped with two boron-dipyrromethane dyes displaying intense emission in the visible and near-infrared regions, respectively. Careful matching of the emission and absorption features of the dopants leads to a virtual Stokes shift larger than 150 nm achieved by an intraparticle Forster resonance energy transfer (FRET) process between the donor and the acceptor dyes. Additionally, the magnetic properties of the fluorescent beads allow preconcentration of the sample. To illustrate the usefulness of this approach to increase the sensitivity of fluorescence immunoassays, the novel nanoparticles are employed as labels for quantification of the widely used Tacrolimus (FK506) immunosuppressive drug. The FRET-based competitive inhibition immunoassay yields a limit of detection (LOD) of 0.08 ng mL(-1), with a dynamic range (DR) of 0.15-2.0 ng mL(-1), compared to a LOD of 2.7 ng mL(-1) and a DR between 4.1 and 130 ng mL(-1) for the immunoassay carried out with direct excitation of the acceptor dye.