{
  "abstract": "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.",
  "abstract_shown": true,
  "affiliations": [
    "Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA; Department of Systems Biology, Harvard Medical School, Boston,"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 20,
  "authors": [
    {
      "name": "F. Xuan"
    },
    {
      "name": "T. W. Fan"
    },
    {
      "name": "X. Chen"
    },
    {
      "name": "Y. Wang"
    },
    {
      "name": "W. Kim"
    },
    {
      "name": "W. Jiang"
    },
    {
      "name": "J. Tan"
    },
    {
      "name": "X. Jiang"
    },
    {
      "name": "Y. Zhang"
    },
    {
      "name": "P. Kivisakk"
    },
    {
      "name": "B. L. Hammerschlag"
    },
    {
      "name": "O. L. Lopez"
    },
    {
      "name": "A. D. Cohen"
    },
    {
      "name": "X. Zeng"
    },
    {
      "name": "T. E. Atri"
    },
    {
      "name": "A. Singh"
    },
    {
      "name": "N. J. Ashton"
    },
    {
      "name": "S. E. Arnold"
    },
    {
      "name": "T. K. Karikari"
    },
    {
      "affs": [
        0
      ],
      "name": "P. Yin"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/e4py7gmxq2t42.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.05.756571",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756571v1.full",
  "id": "e4py7gmxq2t42",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756571v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "bioengineering"
    ],
    [
      "openalex",
      "1312|Advanced biosensing and bioanalysis techniques"
    ]
  ],
  "openalex": "W7220864745",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756571v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-05",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/e4py7gmxq2t42.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:26:22Z",
      "native_id": "10.64898/2026.10.05.756571",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.05.756571v1"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:bioengineering",
      "subfield": "systems-synthetic"
    }
  ],
  "title": "Achieving near single-molecule sensitivity in separation-free protein detection using engineered kinetic proofreading",
  "title_text": "Achieving near single-molecule sensitivity in separation-free protein detection using engineered kinetic proofreading",
  "type": "preprint",
  "updated_at": "2026-10-30T12:26:22Z",
  "url": "https://research.draconitic.com/paper/e4py7gmxq2t42",
  "venue": "bioRxiv",
  "versions": []
}