{
  "abstract": "Backscattering interferometry (BSI) enables label-free, immobilization-free detection of molecular interactions in free solution, but published studies report conflicting outcomes, including saturable, nonsaturating, and poorly reproducible responses. Here, we develop a signal-additivity framework that distinguishes interaction detection from determination of the thermodynamic equilibrium dissociation constant, K d , and provides a basis for reconciling these reports. For a two-state system at fixed total target concentration, quantitative K d analysis requires the final referenced and control-subtracted response to be an affine function of equilibrium complex concentration over the fitted range. A general refractive-index expansion shows that nonlinear self-and crosscomponent contributions can survive control subtraction and generate composition-dependent residual signals. Illustrative simulations with a known input value, K d,true , show that such residuals can produce smooth, apparently saturable curves with biased apparent dissociation constants, K d,app , or strong concentration-range dependence of K d,app . Concentration-design analysis further illustrates, for a specified ligand-dependent residual model, a trade-off between upper-isotherm coverage and high-concentration residuals. Reinterpretation of representative BSI studies indicates that specificity, reproducibility, and apparent saturation support interaction detection but do not independently establish signal additivity. We therefore propose a workflow based on matched controls, concentration-range analysis, residual inspection, parameter-stability assessment, and binding-model evaluation, with measurements at different target concentrations when feasible. Although developed here using BSI, the underlying criterion applies more broadly to affinity studies in which a processed response is assumed to report equilibrium binding populations. BSI-derived values should be reported as K d,app unless signal additivity, adequate isotherm coverage, concentration accuracy, and binding-model adequacy are independently supported.",
  "abstract_shown": true,
  "affiliations": [
    "Department of Chemistry",
    "York University",
    "Centre for Research on Biomolecular Interactions"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 6,
  "authors": [
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Xingzhi Che"
    },
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Alexander MacLellan"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Ayesha Maqsood"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Elyaa AlSafadi"
    },
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Tong Ye Wang"
    },
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Sergey N. Krylov",
      "orcid": "0000-0003-3270-2130"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/fs0f36kqswbzx.bib",
  "cited_by": 0,
  "doi": "10.26434/chemrxiv.15009536/v1",
  "fulltext_url": null,
  "id": "fs0f36kqswbzx",
  "issns": [],
  "landing_url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009536/v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1312|Advanced Biosensing Techniques and Applications"
    ]
  ],
  "openalex": "W7214587965",
  "pdf_url": "https://chemrxiv.org/doi/pdf/10.26434/chemrxiv.15009536/v1",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-28",
  "publisher": "American Chemical Society (ACS)",
  "ris": "https://research.draconitic.com/paper/fs0f36kqswbzx.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:21Z",
      "native_id": "10.26434/chemrxiv.15009536",
      "source": "chemrxiv",
      "url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009536/v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Advanced Biosensing Techniques and Applications",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Signal Additivity as a Practical Criterion for Validating Kd Determination: Reconciling Conflicting Backscattering Interferometry Results",
  "title_text": "Signal Additivity as a Practical Criterion for Validating Kd Determination: Reconciling Conflicting Backscattering Interferometry Results",
  "type": "preprint",
  "updated_at": "2026-10-30T12:17:21Z",
  "url": "https://research.draconitic.com/paper/fs0f36kqswbzx",
  "venue": "ChemRxiv",
  "versions": []
}