Nanogel surface coatings for improved single-molecule imaging substrates

Abstract

Surfaces that resist protein adsorption are important for many bioanalytical applications. Bovine serum albumin (BSA) coatings and multi-arm poly(ethylene glycol) (PEG) coatings display low levels of non-specific protein adsorption and have enabled highly quantitative single-molecule (SM) protein studies. Recently, a method was developed for coating a glass with PEG-BSA nanogels, a promising hybrid of these two low-background coatings. We characterized the nanogel coating to determine its suitability for SM protein experiments. SM adsorption counting revealed that nanogel-coated surfaces exhibit lower protein adsorption than covalently coupled BSA surfaces and monolayers of multi-arm PEG, so this surface displays one of the lowest degrees of protein adsorption yet observed. Additionally, the nanogel coating was resistant to DNA adsorption, underscoring the utility of the coating across a variety of SM experiments. The nanogel coating was found to be compatible with surfactants, whereas the BSA coating was not. Finally, applying the coating to a real-world study, we found that single ligand molecules could be tethered to this surface and detected with high sensitivity and specificity by a digital immunoassay. These results suggest that PEG -BSA nanogel coatings will be highly useful for the SM analysis of proteins.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

“…0 thickness, MatTek Corporation) were used for all surface treatments. PEG-BSA nanogels were prepared using 8-arm PEG-vinyl sulfone and BSA, as described by Tessler et al [11]. Dishes were first cleaned with piranha solution and treated with APTES as described above.…”

“…We investigated 14 different dyes from 5 different suppliers: Alexa 488, Alexa546, Alexa555, Tetramethylrhodamine (TMR), Bodipy FL, and Rhodamine Red C2 (Molecular Probes -Invitrogen), Cy3 (GE Healthcare), CF488A, CF568, CF633 and CF640R (Biotium), Atto565 and Atto647N (AttoTec), and Fluorescein (Sigma Aldrich), and determined for each its brightness, bleaching and non-specific adhesion when conjugated to the Affibody, the latter via its effect on the apparent diffusion of molecules on the basal membrane of live T47D cells grown on PEG-BSA nanogel passivated glass [11]. Maleimide derivatives of the dyes were used to label a single cysteine in the anti-EGFR affibody; in some cases, specific dye isomers were used and this is specified in the Materials and Methods.…”

Ultralow Protein Adsorbing Coatings from Clickable PEG Nanogel Solutions: Benefits of Attachment under Salt-Induced Phase Separation Conditions and Comparison with PEG/Albumin Nanogel Coatings

“…Adsorption to most nanogel coatings was quantified using a 1:100 dilution of the physiological concentration (Figure 2A), while adsorption to several less resistant coatings was quantified using a 1:1000 dilution (Figure 2B). Previously, VS:BSA nanogel coatings were shown more resistant to IgG adsorption 12 and cell adhesion 8 than VS:Am nanogel coatings. Here, we confirmed the extremely low amount of fibrinogen adsorption on the VS:BSA nanogel coatings by TIRF and compared the resistance to the commonly used non-covalent coating PLL- g -PEG.…”

“…Strategies for incorporating PEG have evolved from covalent grafting of linear or star shaped chains 3, 4 and adsorption of block copolymers 5 to surface initiated polymerization of patterned brush coatings 6 or microscopically thin hydrogels, 7 and deposition of preformed hydrogel particles. 8–11 However, advances have not kept pace with the demands of implantable biomaterials as well as in vitro diagnostics 9, 12 both of which remain limited by the lowest levels of protein adsorption achievable. 13, 14 …”

“…12, 23 Interestingly, a hybrid nanogel coating of PEG crosslinked with BSA, outperformed a 100% PEG nanogel coating, although a monolayer of eight-arm PEG was shown to be more repellent than a monolayer of BSA. 12 While the mechanism behind this phenomenon remains unknown, elimination of the albumin component remains desirable for implantable biomaterials due to possible immunogenic responses 24 and gradual degradation of the protein. Here we illustrate methods for the attachment of clickable 100% PEG nanogel solutions that significantly improve the coating’s resistance to protein adsorption, as measured by single molecule counting.…”