Breaking the Concentration Barrier for Single‐Molecule Fluorescence Measurements

Abstract

Fluorescence-based single-molecule techniques have become widely used tools to reveal dynamic processes of biomolecules and elucidate their molecular mechanisms. However, the concentration upper limit of labeled species that can be used in single-molecule fluorescence measurements is at the low nm range, which is below the Michaelis constants of many enzymatic reactions and physiological concentrations of many biomolecules. Such discrepancy limits the application of single-molecule fluorescence tools. Several techniques have been developed to break the concentration barrier. In this Concept, we focus on reviewing fundamental principles of these techniques and wish to inspire development of new and better tools to achieve this goal.

Mixed metal zero-mode guides (ZMWs) for tunable fluorescence enhancement

“…While confocal and total internal reection (TIRF) modalities are limited to a concentration barrier (<10 nM) to detect a single molecule event, 6 nanoapertures allow imaging of biological interactions at single molecule levels in micromolar concentrations. [7][8][9][10][11][12][13][14][15] As a result, ZMWs have been widely used for genomic sequencing, [16][17][18] protein-protein interaction, [19][20][21][22] ligand-receptor binding, [23][24][25][26][27][28] membrane bound diffusion events 29,30 and the study of membrane proteins at single molecule levels. 31,32 This capability is due to the aperture dimensions of the ZMWs, which result in cut-off wavelengths for the transmission of light smaller than the wavelength of excitation light.…”

Bleaching-resistant, near-continuous single-molecule fluorescence and FRET based on fluorogenic and transient DNA binding

“…A standard way to reduce the fluorescence background is through the use of an evanescent excitation field in a total-internal-reflection fluorescence (TIRF) microscope; however, this mode of microscope still cannot allow detection of single immobilised molecules in the presence of >50 nM of unbound label. [20][21][22][23] To further suppress the fluorescence background, we considered a strategy for quenching the fluorescence of unbound labels. This strategy uses a short ssDNA labelled with two ATTO647N fluorophores (one on either end) that exhibit contact-mediated quenching in solution; when bound to the target, the state of quenching is lifted, leading to the appearance of fluorescence corresponding to two ATTO647N fluorophores.…”

“…In principle, the dark interval between the binding of two transient labels to the same target can be decreased by increasing the rate of binding, which in turn can be achieved either by increasing the transient label concentration, or by changing the properties of the transient label to increase the on-rate constant or both. However, the concentration of fluorescent transient labels cannot be increased much above 30 nΜ, as unbound labels contribute to the fluorescence background and degrade the SNR of the measurement [20][21][22][23] , and this concentration will not allow for a high enough on-rate of short ssDNAs for continuous fluorescence traces.…”

Gold Ion Beam Milled Gold Zero-Mode Waveguides

“…As a new approach in SMS, the characterization of ZMW related to size and shape [ 21 , 22 , 23 , 24 , 25 ], metal and layering compositions [ 18 , 21 , 22 , 26 , 27 , 28 , 29 ], excitation wavelength [ 19 ], spatial position of fluorophores relative to the metal structures [ 30 , 31 , 32 ], and spectral overlap of the surface plasmon resonance and excitation/emission of fluorophores [ 33 , 34 , 35 , 36 , 37 ] have been highly active areas of research. The geometry and opacity of the ZMW enables measurements at physiological concentrations (∼1–100 μM) in SMS [ 30 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. As a result, ZMWs have found wide application in studies of genomic sequencing [ 47 , 48 , 49 , 50 ], protein–protein interaction [ 51 , 52 , 53 , 54 ], ligand–receptor binding [ 55 , 56 , 57 , 58 , 59 , 60 , 61 ...…”