Interferometric scattering microscopy supports real-time mass-resolved label-free single-molecule immunoassay

Area of Science:

• Biophysics
• Immunology
• Analytical Chemistry

Background:

• Protein-protein interactions are crucial for biological processes and therapeutic development.
• Current assays indirectly measure these interactions, obscuring native dynamics.
• A need exists for direct, real-time measurement of single-molecule binding events.

Purpose of the Study:

• To introduce a novel label-free, real-time, mass-resolved single-molecule immunoassay.
• To directly observe and quantify individual protein-protein binding events.
• To provide a general framework for quantitative single-molecule immunoassays.

Main Methods:

• Utilizing interferometric scattering microscopy (iSCAT) to detect light scattered from single proteins.
• Developing an antibody-functionalized surface for capturing binding events.
• Analyzing binding and unbinding kinetics from single-molecule dwell times.

Main Results:

• Demonstrated real-time detection of individual antibody-antigen interactions with single-molecule sensitivity.
• Measured association rates that scale linearly with concentration over three orders of magnitude.
• Obtained quantitative IgM concentrations in human serum agreeing with ELISA, and determined dissociation rates.

Conclusions:

• The bioaffinity iSCAT platform offers label-free, real-time, single-molecule sensitivity for immunoassays.
• This method provides direct measurement of binding kinetics and concentrations.
• It represents a significant advancement for understanding biomolecular interactions and therapeutic discovery.