Abstract 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.

Single-molecule counting is the most accurate and precise method for determining the concentration of a biomarker in solution and is leading to the emergence of digital diagnostic platforms enabling precision medicine. In principle, solid-state nanopores-fully electronic sensors with single-molecule sensitivity-are well suited to the task. Here we present a digital immunoassay scheme capable of reliably quantifying the concentration of a target protein in complex biofluids that overcomes specificity, sensitivity, and consistency challenges associated with the use of solid-state nanopores for protein sensing. This is achieved by employing easily-identifiable DNA nanostructures as proxies for the presence ("1") or absence ("0") of the target protein captured via a magnetic bead-based sandwich immunoassay. As a proof-of-concept, we demonstrate quantification of the concentration of thyroid-stimulating hormone from human serum samples down to the high femtomolar range. Further optimization to the method will push sensitivity and dynamic range, allowing for development of precision diagnostic tools compatible with point-of-care format.
© 2021. The Author(s).