Wide range detection of C-Reactive protein with a homogeneous immunofluorimetric assay based on cooperative fluorescence quenching assisted by gold nanoparticles

Homogeneous sandwich immunofluorimetric assays are valued for the rapid, low-cost and accurate detection of analytes in liquid phase. However, their exploitation with analytes covering a wide range of concentrations is limited by low sensitivity and the hook effect. Here, we describe a homogeneous immunofluorimetric system based on the quenching of fluorescence in a Förster resonance energy transfer (FRET) donor/acceptor couple of antibody functionalized with two different dyes, respectively fluorescein (donor) and eosin (acceptor), which form a sandwich multi-component assembly with antibody-functionalized gold nanoparticles (GNPs) in the presence of the analyte. The resulting cooperative fluorescence quenching is assisted by the GNPs scaffold through the nanomaterial-surface energy transfer (NSET) effect, which gives an extended linear response versus the antigen concentration that is not possible with the bi-component assays. This immunofluorimetric method allows accurate, reproducible and immediate detection of C-reactive protein (CRP) in the wide concentration range of clinical interest (over two orders of magnitude from 3.5 to 455 nM, 0.4–52 mg/L), without the hook effect. Moreover, the method does not require sample treatment or washing steps. The concept of this multi-component FRET/NSET fluorescence quenching system can be extended to any analyte amenable to the detection with homogeneous sandwich assays.