Digital Immunoassay Protocol for Solid-State Nanopore Sensing

The ability to digitally count single molecules enables accurate and precise determination of the concentration of a disease biomarker. Owing to their intrinsic single-molecule sensitivity and fully electronic detection capability, solid-state nanopores show great promise for this task toward point-of-care diagnostic applications. Here, we describe the protocols for implementing a magnetic bead-based immunoassay strategy coupled with digital detection and downstream solid-state nanopore electrical readout. The digital scheme employs DNA nanostructures, as proxy labels for the presence (“1”) or absence (“0”) of the target protein. We provide step-by-step protocols for assembling and purifying DNA nanostructures; Preparing magnetic beads decorated with capture antibodies; Conjugating secondary detection antibodies to bind the DNA label; Functionalizing gold nanoparticles; and Running the full assay where thyroid stimulating hormone (TSH) from human serum samples is quantified down to the femtomolar range. The protocols and assay scheme presented herein are easily generalized to the quantification of a wide range of target proteins, by selecting the appropriate antibody pair.