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

Our article entitled “Breaking the low concentration barrier of single-molecule fluorescence quantitation to the sub-picomolar range” has recently been published in Small Methods.

Until now, the concentration range for effective single-molecule fluorescence detection was largely restricted to the concentrations above 50 pM, overlooking the immense potential of the single-molecule fluorescence for biosensing applications at sub-picomolar sensitivity.

In our work, we present a simple and effective modification to a confocal microscope setup that pushes the detection limit of fluorescence lifetime correlation spectroscopy (FLCS) down to 0.1 pM, overcoming the so-called low concentration barrier without the need for complex instrumentation or preconcentration techniques.

Main elements of significance:

• Adding a diaphragm on the laser excitation beam achieves a large detection volume together with a high fluorescence brightness per molecule. This is the key to push the sensitivity towards sub-picomolar concentrations.
• For the first time, we clearly discuss the physical parameters setting the lower the limit of quantification in FLCS and we introduce a universal figure of merit allowing to compare between experimental configurations.
• We demonstrate the effectiveness of our approach by monitoring the interaction dynamics of biotin-streptavidin binding, a highly relevant biochemical system yet challenging to measure on conventional systems due to its exceptionally high affinity.

These results open new avenues for applying single-molecule fluorescence detection to biosensing at sub-picomolar concentrations, with significant implications for biological and medical diagnostics.

Open access at Small Methods and available on the repository HAL 05043006.