Surface passivation and functionalisation for mass photometry

Abstract

Interferometric scattering (iSCAT) microscopy enables the label‐free observation of biomolecules. Consequently, single‐particle imaging and tracking with the iSCAT‐based method known as mass photometry (MP) is a growing area of study. However, establishing reliable cover glass passivation and functionalisation methods is crucial to reduce nonspecific binding and prepare surfaces for in vitro single‐molecule binding experiments. Existing protocols for fluorescence microscopy can contain strongly scattering or mobile components, which make them impractical for MP‐based microscopy. In this study, we characterise several different surface coatings using MP. We present approaches for cover glass passivation using 3‐aminopropyltriethoxysilane (APTES) and polyethylene glycol (PEG, 2k) along with functionalisation via a maleimide‐thiol linker. These coatings are compatible with water or salt buffers, and show low background scattering; thus, we are able to measure proteins as small as 60 kDa. In this technical note, we offer a surface preparation suitable for in vitro experiments with MP.