Multi-confocal fluorescence correlation spectroscopy.

We report a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique that combines a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM, calculated by using the spherical wave approximation, makes it possible to produce several diffraction limited laser spots. The fastest acquisition mode leads to a time resolution of 100 microseconds. By using solutions of sulforhodamine G we demonstrated that the observation volumes are similar to that of a standard confocal set-up. mFCS experiments have also been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock.

Long-range fluorescence quenching by gold nanoparticles in a sandwich immunoassay for cardiac troponin T.

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.