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

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.