Achieving near single-molecule sensitivity in separation-free protein detection using engineered kinetic proofreading

Unlike nucleic acids, proteins cannot be directly amplified. Ultrasensitive detection must therefore couple efficient analyte-to-signal conversion with strong suppression of target-independent background. Consistently detecting ~100 molecules (below which Poisson sampling noise alone causes CV to exceed 10%) across protein targets in a format as scalable and accessible as PCR remains unsolved. Separation-based ultrasensitive immunoassays require target binding stable enough to survive stringent washing and are therefore constrained by wash-induced signal loss and, more fundamentally, the relative scarcity of exceptionally tight binders. Performance consequently varies across targets; detection limits below 1,000 molecules remain rare. Conventional separation-free proximity assays avoid washing but incur background from random probe collisions, with reported detection limits frequently exceeding 100,000 molecules. Here we introduce Successive Proximity Extension Amplification Reaction (SPEAR), a separation-free architecture in which autonomous engineered DNA nanodevices emulate biological kinetic proofreading. Two successive irreversible proximity-extension steps compound discrimination between analyte-dependent and background reactions, suppressing spurious signals while preserving efficient analyte-to-signal conversion. This design reduces reliance on high-affinity binders and enables sub-fM detection from 1 uL samples through simple 3-step mixing followed by standard qPCR readout. Across 11 biomarkers tested, SPEAR consistently achieved sub-100-molecule detection limits. Where compared, it outperformed ultrasensitive separation-based platforms, analytically and clinically, including for low-abundance Alzheimer's disease biomarkers. SPEAR also precisely quantified SARS-CoV-2 neutralizing antibody titers from finger-prick dried blood spots, concordant with the cell-based reference assay on venous blood. It detected a distinct mixed phosphorylation state of Tau, and targeting this state resolved previously observed capillary-venous discordance and improved amyloid-status discrimination. These results establish engineered autonomous successive molecular discrimination as a practical strategy for near-single-molecule protein detection, extending the sensitivity, scalability and accessibility of PCR-based testing to non-amplifiable analytes.