A highly sensitive fluorescent immunoassay based on avidin-labeled nanocrystals

Nanocrystals of the fluorogenic precursor fluorescein diacetate (FDA) were applied as labels in order to improve on the assay sensitivity achieved in our previous studies. Each FDA nanocrystal can be converted into ∼2.6×10⁶ fluorescein molecules, which is useful for improving immunoassay sensitivity and limits of detection. NeutrAvidin was simply adsorbed onto the surface of the FDA nanocrystals, which were coated with distearoylglycerophosphoethanolamine (DSPE) modified with amino(poly(ethylene glycol))(PEG(2000)-Amine) as an interface for coupling biomolecules. This can be applied to detect different kinds of analytes that are captured by corresponding biotinylated biomolecules in different bioanalytical applications. The applicability of the NeutrAvidin-labeled nanocrystals was demonstrated in an immunoassay using the labeled avidin–biotin technique. Biotinylated antibody and FDA-labeled avidin were applied to the assay sequentially. The performance was compared with the traditional sandwich-type assay for mouse immunoglobulin G detection. Following the immunoreaction, the nanocrystals were released by hydrolysis and dissolution instigated by adding a large volume of organic solvent/sodium hydroxide mixture. The limit of detection was lower (by a factor of 2.5–21) and the sensitivity was (3.5–30-fold) higher than immunoassays using commercial labeling systems (FITC and peroxidase). This study shows that using fluorescent nanocrystals in combination with the avidin–biotin technique can enhance assay sensitivity and provide a lower limit of detection without requiring long incubation times as in enzyme-based labels.