Nanocrystal biolabels with releasable fluorophores for immunoassays.

Abstract

A novel signal amplification technology based on a new class of biofunctional fluorescent nanocrystals holds promise to improve the sensitivity and the limits of detection of immunoassays. A two-step approach without layer-by-layer techniques is described to encapsulate the fluorogenic precursor fluorescein diacetate (FDA) nanocrystals (107-nm average size) followed by conjugation of the antibody. Distearoylphosphatidylethanolamine (DSPE) modified with amino(poly(ethylene glycol)) (PEG(2000)Amine) is coated on the surface of the FDA nanocrystals to provide a interface for the antibody coupling. Anti-mouse antibodies are attached to the nanocrystalline FDA biolabels by adsorption. A high molar ratio of fluorescent molecules to biomolecules (2.8 x 10(4)) is achieved in this nanocrystal biolabel system. The analytical performance of the nanocrystal-based label system is evaluated in a model sandwich immunoassay for the detection of mouse IgG. After separation of the nonbound antibody nanocrystal labels, fluorophores are released by hydrolysis and dissolution of the nanocrystalline FDA. Due to the release of the fluorophores (fluoresceins) into a large volume of organic solvent/sodium hydroxide mixture, self-quenching is suppressed. The FDA[DSPE-PEG(2000)Amine]-modified biolabels have a highly stable colloidal suspension with minimized nonspecific interactions. The limit of detection was lowered by a factor of 5-28, and the sensitivity was 400-2700-fold higher compared with a state-of-the-art immunoassay using directly fluorescent-labeled antibodies. Our approach provides high sensitivity and low limits of detection without the need for long incubation times, making it an interesting alternative in biolabel technology.