Protein Detection in Blood with Single-Molecule Imaging

Abstract

The ability to accurately identify and characterize proteins in blood samples would facilitate disease diagnosis and monitoring. Current blood tests, including ultra-sensitive assays, depend on ensemble measurements which are hampered by background detection errors. Single-molecule imaging approaches, by contrast, directly inspect individual target proteins, offering much greater reliability than ensemble methods. However, the detection limit of single-molecule imaging has thus far been confined to the picomolar (10−12 M) range, and these approaches have not been previously translatable to clinical samples. Here we describe Single-Molecule Augmented Capture (SMAC), a single-molecule imaging technique to visualize and quantify individual proteins-of-interest down to the sub-femtomolar (<10−15 M) range with minimal detection errors, even in complex fluids such as blood. We demonstrate SMAC in a wide variety of applications with human blood samples, including the analysis of secreted proteins (prostate-specific antigen and anti-p53 autoantibodies), membrane proteins (programmed death-ligand 1), and rare intracellular proteins (mutant p53). SMAC opens the door to the use of single-molecule imaging in non-invasive disease profiling. This platform can be adapted to multiplex or high-throughput formats to characterize heterogeneous biochemical and structural features of circulating proteins-of-interest.