Strategies for Overcoming the Single-Molecule Concentration Barrier

Abstract

Fluorescence-based single-molecule approaches have helped revolutionize our understanding of chemical and biological mechanisms. Unfortunately, these methods are only suitable at low concentrations of fluorescent molecules so that single fluorescent species of interest can be successfully resolved beyond background signal. The application of these techniques has therefore been limited to high-affinity interactions despite most biological and chemical processes occurring at much higher reactant concentrations. Fortunately, recent methodological advances have demonstrated that this concentration barrier can indeed be broken, with techniques reaching concentrations as high as 1 mM. The goal of this Review is to discuss the challenges in performing single-molecule fluorescence techniques at high-concentration, offer applications in both biology and chemistry, and highlight the major milestones that shatter the concentration barrier. We also hope to inspire the widespread use of these techniques so we can begin exploring the new physical phenomena lying beyond this barrier.

Ultraviolet Resonant Nanogap Antennas with Rhodium Nanocube Dimers for Enhancing Protein Intrinsic Autofluorescence

“…Altogether, our work significantly advances the field of nanotechnology and biosensing by demonstrating the success of ultraviolet nanogap antennas for label-free protein detection. Extending the practical application of plasmonic nanoantennas into the deep UV range broadens the capabilities to investigate individual proteins in their native state under physiological concentrations. − The robustness of the achieved gap sizes further enhances the practical applicability of our approach, positioning it as a promising technique in this domain. Beyond label-free protein autofluorescence detection, resonant UV nanoantennas are highly relevant to advance several other plasmonic applications, including resonant Raman spectroscopy, , circular dichroism spectroscopy, photodetectors, and photocatalysis …”

“…While the autofluorescence quantum yield of tryptophan in most proteins is typically on the order of a few percent, there is a compelling interest in utilizing UV nanoantennas to significantly amplify the autofluorescence signal from single proteins, rendering it easily detectable. Leveraging UV-FCS experiments, we unlock powerful perspectives for local measurements of concentration, mobility, brightness, and stoichiometry of label-free proteins. − …”

“…Our UV resonant nanogap antennas provide enhancement factors up to 120-fold for the autofluorescence brightness of single proteins. The UV light is concentrated into 40 zL detection volumes, which in turn enables UV autofluorescence correlation spectroscopy (UV-FCS) at concentrations exceeding 50 μM. , We also investigate the modification of different fluorescence photokinetic rates by rhodium nanoantennas and demonstrate excellent agreement between our experiments and numerical simulations. Overall, our study expands the applicability of plasmonic nanoantennas down to the deep UV range, , broadening the capabilities to interrogate single proteins in their native state at physiological concentrations. − …”

Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells

“…However, weak biomolecular interactions ( K d > 1 μM) that require high concentrations cannot be studied at the nano- to picomolar concentrations that are typically employed in in vitro single-molecule experiments. Moreover, studying biomolecules in their natural habitat, the crowded environment of live cells, is also very challenging, as protein concentrations in cells are often in the high nanomolar to micromolar range, which is incompatible with single-molecule observations . Fundamentally, the concentration limit for single-molecule observation is bounded by the size of the observation volume, which can be minimized using common optical sectioning methods such as confocal microscopy, total internal reflection microscopy (TIRF), or light-sheet microscopy .…”

“…Fundamentally, the concentration limit for single-molecule observation is bounded by the size of the observation volume, which can be minimized using common optical sectioning methods such as confocal microscopy, total internal reflection microscopy (TIRF), or light-sheet microscopy . Despite such improvements, the volumes remain on the order of femtoliters, which puts the concentration limit for isolating single molecules at ≈1 nM. , …”