Abstract

Single-molecule proteomic studies are critically important for understanding the molecular origins of cellular phenotypes. However, no currently available technology can achieve both the single-molecule sensitivity and high dynamic range required to comprehensively analyze the complex mixtures of proteins in biological samples. One approach to achieve high sensitivity across a wide dynamic range would be to create a protein array that arranges billions of single molecules with regular spacing on a patterned surface. However, creating such a protein array has remained an unsolved challenge for the field. Here, we present a highly scalable method for fabricating dense single-molecule protein arrays using a specially designed DNA origami structure, protein click-conjugation, photolithography and surface functionalization. The origami-structure is enhanced via terminal deoxynucleotidyl transferase-extension, which generates brush-like projections, increasing the effective size of the origami from 88 nm to greater than 200 nm. These particles are large enough to enable super-Poisson deposition of individual protein molecules on a nano-patterned chip (>98% occupancy with only 1% of sites occupied with multiple protein molecules). This approach allowed for single-molecule protein display of 600 million protein molecules per microscope slide-sized chip with the potential to scale further with denser feature spacing. We hypothesize that this technology will ultimately enable the development of highly scalable proteomic analysis platforms that address the currently unmet need for protein measurements at single-molecule sensitivity across an exceptionally wide dynamic range of protein concentrations.