Breaking the Low Concentration Barrier of Single-Molecule Fluorescence Quantification to the Sub-Picomolar Range

Single-molecule fluorescence techniques provide exceptional sensitivity to probe biomolecular interactions. However, their application to accurately quantify analytes at the picomolar concentrations relevant for biosensing remains challenged by a severe degradation in the signal-to-background ratio. This so-called 'low concentration barrier' is a major factor hindering the broad application of single-molecule fluorescence to biosensing. Here we break into the low concentration limit while keeping intact the confocal microscope architecture and without requiring complex microfluidics or preconcentration stages. Using fluorescence lifetime correlation spectroscopy (FLCS) and adding a diaphragm to the laser excitation beam, we achieve a limit of quantitation (LOQ) down to 0.1 pM, significantly below the state-of-the-art. We identify the physical parameters setting the LOQ and introduce a broadly applicable figure of merit (FoM) that determines the LOQ and allows for a clear comparison between experimental configurations. Our approach preserves the ability to monitor dynamic interactions, diffusion times, and distinguish species in complex mixtures. We illustrate this feature by measuring the biotin-streptavidin association rate constant which is highly challenging to assess quantitatively due to the strong affinity of the biotin-streptavidin interaction. These findings push the boundaries of single-molecule fluorescence detection for biosensing applications at sub-picomolar concentrations with high accuracy and simplified systems.