Multi-analyte homogenous immunoassay based on quenching of quantum dots by functionalized graphene

We propose a homogenous multi-analyte immunoassay based on the quenching of quantum dot (QD) fluorescence by means of graphene. Two QDs with emission maxima at 636 and 607 nm were bound to antibodies selective for mouse or chicken immunoglobulins, respectively, and graphene functionalized with carboxylic moieties was employed to covalently link the respective antigen. The antibody-antigen interaction led graphene close enough to QDs to quench the QD fluorescence by resonance energy transfer. The addition of free antigens that competed with those linked to graphene acted as a "turn-on" effect on QD fluorescence. Fluorescence emitted by the two QDs could be recorded simultaneously since the QDs emitted light at different wavelengths while being excited at the same wavelength and proved to be linearly correlated with free antigen concentration. The developed assay allows measuring both antigens over 2-3 orders of magnitude and showed estimated limits of detection in the nanomolar range. This approach is thus a promising universal strategy to develop homogenous immunoassays for diverse antigens (cells, proteins, low-molecular-mass analytes) in a multi-analyte configuration.