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

TL;DR

A new immunoassay method uses kinetic profiling to accurately measure multiple biomarkers across a very wide concentration range, avoiding common measurement errors.

Contribution

A novel kinetic framework decouples cross-reactivity and enables ultrabroad dynamic range quantification in one-step immunoassays.

Findings

Kinetic analysis of single-particle plasmonic signals resolves hook effect ambiguity.

Multiplex quantification of biomarkers in human serum spans nine orders of magnitude.

Framework distinguishes and mitigates cross-reactivity in complex assays.

Abstract

In one-step sandwich immunoassays, where all binding
components
coexist in solution, excessive analyte levels can inhibit sandwich
complex formation by competing with labeled detection antibodies,
producing the well-known “hook effect.” Here we establish
a kinetic framework that resolves this ambiguity by analyzing time-resolved
single-particle plasmonic signals. Using gold nanohole arrays with
nanoparticle reporters, we continuously track individual binding events
and fit their response-time profiles to both mass-transport- and reaction-limited
models. Comparison of fit residuals identifies the dominant mechanism
in each concentration regime, revealing the kinetic transition that
gives rise to the hook effect and converting it to a quantitative
feature. The digital framework also classifies and mathematically
decouples distinct types of cross-reactivity in multiplexed assays,
minimizing off-target interference. Applied to multiplexed detection
of cytokines and C-reactive protein in unprocessed human serum, our
approach enables simultaneous quantification of low- and high-abundance
biomarkers, ranging in total over 9 orders of magnitude, without sample
splitting or analyte-specific dilution. This mechanistic strategy
establishes a generalizable paradigm for kinetic, cross-reactivity-aware
biosensing.

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