Surface Passivation with a Perfluoroalkane Brush Improves the Precision of Single-Molecule Measurements

Abstract

Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous–aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces.

A new platform for single molecule measurements using the fluorous effect

“…We recently demonstrated the suitability of perfluoroalkane brushes to monitor adsorption and desorption of single proteins over a prolonged duration via iSCAT microscopy, and their potential to study thermodynamic equilibria between monomeric and multimeric protein components. 8 This work expands on our previous results and presents an optimised protocol for the formation of perfluorinated SAMs on cover glasses, which have been tested with a commercial protein mixture. We believe these results are just the first example of many experimental possibilities that can be unlocked using perfluorinated monolayers for single-molecule studies.…”

“…Protein 2 results in the most intense peak after 1 minute, which indicates it presents the largest concentration, followed by protein 1 and protein 3. Using glass substrates, these peaks tend to be less intense over time due to the irreversible adsorption of proteins onto the substrate, 8 which limits the time span of the measurements. However, since the perfluorinated SAM prevents irreversible adsorption events, we were able take measurements for several minutes.…”

“…7 Furthermore, the coiled structure of PEG polymers excludes them from being used for iSCAT microscopy, a technique that measures the interference of light scattered from subwavelength objects: the conformational mobility of the polymer chains creates too much background noise, preventing the observation of single molecules. 8 The development of iSCAT microscopy as a label-free tool to detect protein mass by Young et al 9 -referred to as mass photometry -shows great promise to study molecular dynamics of biological process, and therefore it is paramount to develop suitable substrates that will allow full exploitation of its potential.…”

Functionalisation of 20 nm citrate-coated gold nanoparticles using perfluorodecanethiol

“…5 The lack of intramolecular interactions between fluorous moieties and other species makes fluorous layers good anti-fouling platforms. 6 Furthermore, fluorous-fluorous interactions are non-covalent and highly specific, and can be employed as orthogonal recognition modes 7 or for reversible molecular patterning. 8 We aim to expand these functionalities to build new reversible systems.…”

A quantitative description for optical mass measurement of single biomolecules

“…Recent developments in ultrasensitive light microscopy 1,2 have enabled the quantification of biomolecular mass, charge and size at the single molecule level and in solution [3][4][5][6][7][8] . In mass photometry (MP) light scattered from a protein when it binds to or moves along a glass coverslip in solution is detected together with partially reflected light from the glass-water interface (Fig.…”