Sensitive single-molecule protein quantification and protein complex detection in a microarray format.

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.

Nanogel surface coatings for improved single-molecule imaging substrates.

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.

Protein quantification in complex mixtures by solid phase single-molecule counting.

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.