Abstract
Single nanometric apertures in a metallic film are used to develop a simple and robust setup for dual-color fluorescence cross-correlation spectroscopy (FCCS) at high concentrations. If the nanoaperture concept has already proven to be useful for single-species analysis, its extension to the dual-color case brings new interesting specificities. The alignment and overlap of the two excitation beams are greatly simplified. No confocal pinhole is used, relaxing the requirement for accurate correction of chromatic aberrations. Compared to two-photon excitation, nanoapertures have the advantage to work with standard fluorophore constructions having high absorption cross-section and well-known absorption/emission spectra. Thanks to the ultra-low volume analysed within one single aperture, fluorescence correlation analysis can be performed with single molecule resolution at micromolar concentrations, resulting in 3 orders of magnitude gain compared to conventional setups. As applications of this technique, we follow the kinetics of an enzymatic cleavage reaction at 2 muM DNA oligonucleotide concentration.We also demonstrate that FCCS in nanoaper-tures can be applied to the fast screening of a sample for dual-labeled species within 1 s acquisition time. This offers new possibilities for rapid screening applications in biotechnology at high concentrations.
Highlights
Since its proposal [1] and first experimental demonstration [2], dual-color confocal fluorescence cross-correlation spectroscopy (FCCS) has proven to be a versatile and valuable tool, accessing a wide range of molecular parameters such as local concentrations, mobility, association/dissociation kinetics and enzymatic activity
Thanks to the ultra-low volume analysed within one single aperture, fluorescence correlation analysis can be performed with single molecule resolution at micromolar concentrations, resulting in 3 orders of magnitude gain compared to conventional setups
We report that fluorescence crosscorrelation spectroscopy (FCCS) in nanoapertures can be applied to fast sample screening for dual-labeled species at high concentrations, offering new possibilities for biotechnology
Summary
Since its proposal [1] and first experimental demonstration [2], dual-color confocal fluorescence cross-correlation spectroscopy (FCCS) has proven to be a versatile and valuable tool, accessing a wide range of molecular parameters such as local concentrations, mobility, association/dissociation kinetics and enzymatic activity (for a review, see references [3] and [4]). In single molecule fluorescence spectroscopy, nanoapertures milled in a metallic film (so-called “zero-mode waveguides”) have been used to reduce the observation volume down to 10 zeptoliter (10−20 L), gaining up to 5 orders of magnitude from the volume commonly obtained in confocal microscopy and allowing single molecule resolution at 100 μM concentration [16]. This has stimulated many experimental work on single-color fluorescence correlation spectroscopy, either on solutions [17, 18, 19, 20] or on lipid membranes [21, 22, 23]. We report that FCCS in nanoapertures can be applied to fast sample screening for dual-labeled species at high concentrations, offering new possibilities for biotechnology
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