Here we present a procedure for quantifying single protein molecules affixed to a surface by counting bound antibodies. We systematically investigate many of the parameters that have prevented the robust single-molecule detection of surface-immobilized proteins. We find that a chemically adsorbed bovine serum albumin surface facilitates the efficient detection of single target molecules with fluorescent antibodies, and we show that these antibodies bind for lengths of time sufficient for imaging billions of individual protein molecules. This surface displays a low level of nonspecific protein adsorption so that bound antibodies can be directly counted without employing two-color coincidence detection. We accurately quantify protein abundance by counting bound antibody molecules and perform this robustly in real-world serum samples. The number of antibody molecules we quantify relates linearly to the number of immobilized protein molecules (R(2) = 0.98), and our precision (1-5% CV) facilitates the reliable detection of small changes in abundance (7%). Thus, our procedure allows for single, surface-immobilized protein molecules to be detected with high sensitivity and accurately quantified by counting bound antibody molecules. Promisingly, we can probe flow cells multiple times with antibodies, suggesting that in the future it should be possible to perform multiplexed single-molecule immunoassays.

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.

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Single-molecule protein analysis provides sensitive protein quantitation with a digital read-out and is promising for studying biological systems and detecting biomarkers clinically. However, current single-molecule platforms rely on the quantification of one protein at a time. Conventional antibody microarrays are scalable to detect many proteins simultaneously, but they rely on less-sensitive and less quantitative quantification by the ensemble averaging of fluorescent molecules. Here we demonstrate a single-molecule protein assay in a microarray format enabled by an ultra-low background surface and single-molecule imaging. The digital read-out provides a highly sensitive, low femtomolar limit of detection and 4 orders of magnitude of dynamic range through the use of hybrid digital-analog quantification. From crude cell lysate, we measured levels of p53 and MDM2 in parallel, proving the concept of a digital antibody microarray for use in proteomic profiling. We also applied the single-molecule microarray to detect the p53-MDM2 protein complex in cell lysate. Our study is promising for development and application of single-molecule protein methods because it represents a technological bridge between single-plex and highly multiplex studies.