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

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.