Digital immunoassay for biomarker concentration quantification using solid-state nanopores

Abstract

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.

Efficient Simulation of Arbitrary Multicomponent First-Order Binding Kinetics for Improved Assay Design and Molecular Assembly

“…The simulation setup for this model consists of four primary components: capture antibodies bound to magnetic beads (A), a target protein (T), a detector antibody (D), and a labeling molecule (L), such as an enzyme 6 or a DNA strand. 16 Assuming no cross-reactivity between species and no nonspecific background molecules, these four components can eventually bind together into an ATDL complex, forming every permutation of subcomplexes along the way. Our idealized assay consists of the 10 possible species representing the set of subcomponents: four base components (A, T, D, and L), three 2-component subcomplexes (AT, TD, and DL), two 3-component subcomplexes (ATD and TDL), and the full ATDL complex.…”

“…As an illustration of the capabilities of this software, we first approximate a generic bead-based immunoassay with a workflow similar to that which is used in the SiMoA technology 6 , 10 or to the nanopore electrical readout, which we conducted recently. 16 Since accurate values of the on- and off- rates are not always available for the capture antibody and detector antibody pairings in those digital assays, we instead make some approximations. To do so while still gaining useful physical insights, we normalize parameters by the K D value of the capture-antibody pairing.…”

“…Results are consistent with the intuitive conclusion that fewer wash steps will always be better from the perspective of the total available labeled target at the end of the assay, but it should be noted carefully that the details of the readout scheme may affect what is optimal from a signal-to-background ratio perspective. For example, in the nanopore readout case, 16 because any free-floating label that remains after washing was the result of dissociation from a complex involving the capture antibody and target, it still reports accurately on the target concentration even if it is no longer complexified during readout.…”

“… (a) Comparison between an idealized simulation (blue triangles) and experimental results from He et al 16 (black squares). (b) Illustration of the effects of nonbinding misassembled probes and of the effects of probes that can bind in the absence of a linker strand.…”

All on size-coded single bead set: a modular enrich-amplify-amplify strategy for attomolar level multi-immunoassay

“…Traditionally, due to the lack of a self-replication mechanism for proteins, the detection of related substances is mainly based on enzyme-linked immunosorbent assays (ELISA). [6][7][8] Despite the convenience that ELISA and its variants have brought to us, clinical applications still urgently require further advances in quantitative protein analysis in sensitivity, dynamic range and multiplex sensing ability with low sample consumption related to limited precious samples. [9][10][11] First, in terms of sensitivity, most ELISA-based assays oen consume about 100 mL of sample for well-based adsorption and subsequent detection, with the limit of detection (LOD) generally around the pM level.…”