We observe an enhancement of fluorescence from a single fluorescent sphere, which is sandwiched between two individual gold nanoparticles, forming a hot spot of strong field enhancement. The fluorescence enhancing hot spot is custom-designed by the deliberate assembly of gold nanoparticles with an atomic force microscope cantilever. The fluorescence intensity is monitored while the separation between the two gold nanoparticles is reduced by gradually pushing the gold nanoparticles closer to the fluorescent sphere. The fluorescence enhancement is maximal when the distance between the two gold nanoparticles is smallest, when the excitation polarization is parallel to the axis of the sandwich, and when the fluorescent sphere is positioned exactly on the axis connecting the two gold nanoparticles.
We report the first homogeneous sandwich immunoassay with gold nanoparticles (AuNPs) as fluorescence quenchers. The sandwich assay is designed for the detection of the protein cardiac troponin T (cTnT) by its simultaneous interaction with two different antibodies, one attached to AuNPs and the other labeled with fluorescent dyes. We demonstrate the working principle of the assay and using time-resolved fluorescence spectroscopy, we determine the quenching efficiency of the gold nanoparticles. In spite of the relatively large separation distance between dye molecules and AuNPs, ranging from 3 to 22 nm, the AuNPs quench the fluorescence with efficiencies as high as 95%. A limit of detection of 0.02 nM (0.7 ng/mL) was obtained for cTnT, which is the lowest value reported for a homogeneous sandwich assay for cTnT. These results illustrate the use of metallic nanoparticles as fluorescence quenchers in immunoassays where the large biomolecules involved impose distances for which energy transfer between fluorophores would be inefficient.
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