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Related Experiment Video

Updated: Jan 29, 2026

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Kinetic Profiling in One-Step Digital Immunoassays Enables Multiplex Quantification across an Ultrabroad Dynamic

Abtin Saateh1, Rojina Allamehnejad1, Wenhong Yang1

  • 1Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne CH-1015, Switzerland.

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Summary

This study introduces a kinetic framework to overcome the hook effect in immunoassays by analyzing single-particle signals over time. This method quantizes high analyte concentrations and reduces cross-reactivity for accurate biomarker detection in serum.

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Area of Science:

  • Biosensing
  • Analytical Chemistry
  • Biophysics

Background:

  • One-step sandwich immunoassays can suffer from the hook effect at high analyte concentrations, leading to inaccurate results.
  • The hook effect occurs when excess analyte inhibits sandwich complex formation, complicating quantitative analysis.

Purpose of the Study:

  • To develop a kinetic framework for resolving ambiguity caused by the hook effect in immunoassays.
  • To enable accurate quantification of biomarkers across a wide dynamic range, including high concentrations.
  • To address and minimize cross-reactivity in multiplexed biosensing applications.

Main Methods:

  • Utilized time-resolved single-particle plasmonic signals from gold nanohole arrays with nanoparticle reporters.
  • Tracked individual binding events and analyzed response-time profiles using mass-transport- and reaction-limited models.
  • Developed a digital framework to classify and decouple cross-reactivity in multiplexed assays.

Main Results:

  • Identified the kinetic transition responsible for the hook effect by comparing fit residuals.
  • Converted the hook effect into a quantitative feature by understanding the underlying kinetics.
  • Successfully applied the approach to multiplexed detection of cytokines and C-reactive protein in unprocessed human serum.
  • Enabled simultaneous quantification of biomarkers spanning over 9 orders of magnitude without sample splitting or dilution.

Conclusions:

  • Established a generalizable kinetic, cross-reactivity-aware biosensing paradigm.
  • The mechanistic strategy overcomes limitations of traditional immunoassays for complex biological samples.
  • This approach enhances the accuracy and dynamic range of biomarker quantification in clinical diagnostics.