Rapid and improved surface passivation method for Single-Molecule experiments.

Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein-protein interactions, and protein-nucleic acid interactions. Biomolecules are commonly immobilized on the microscope surface to extend the observation time. However, non-specific interactions between biomolecules and the surface present a major challenge. The first critical step in these experiments is preparing the surface using polyethylene glycol (PEG) coated slides, which facilitate biomolecule immobilization while minimizing non-specific interactions. The surface treatment typically uses PEG-SVA (Succinimidyl Valerate) coated slides, and the protocol for the treatment is lengthy and time-consuming. To overcome this issue, we have developed a process that uses PEG-Silane to improve efficiency while maintaining reproducibility. Here, we present a one-step, rapid PEGylation methodology that can be completed in minutes rather than hours. We demonstrate its validity and feasibility through single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule photobleaching experiments across various biological samples.