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

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 classifi

SUBMITTER: Saateh A 

PROVIDER: S-EPMC12903848 | biostudies-literature | 2026 Feb

REPOSITORIES: biostudies-literature