Label-Free Profiling of Immunoglobulin Isotypes Using Solid-State Nanopores.

Area of Science:

• Biophysics
• Analytical Chemistry
• Immunology

Background:

• Accurate antibody isotype profiling is crucial for immune monitoring, disease diagnosis, and therapy guidance.
• Current immunoassays often lack the molecular precision to capture antibody conformational dynamics.
• Existing nanopore sensing methods typically require fragmentation or functionalization, limiting analysis of intact isotypes.

Purpose of the Study:

• To develop a label-free nanopore-based assay for direct discrimination of intact antibody isotypes (IgG, IgA, IgM).
• To resolve intrinsic molecular signatures of antibody isotypes in real time under non-denaturing conditions.
• To establish a scalable framework for direct, multiplexed immunoprofiling using nanopore technology.

Main Methods:

• Utilized glass nanopipettes in a lithium chloride electrolyte to create an electroosmotic-flow-dominated regime.
• Employed flow-driven transport and nanoscale confinement to generate distinct ionic fingerprints for each isotype.
• Incorporated affinity-based signal modulation using protein G to enhance isotype separability while preserving native structure.

Main Results:

• Successfully discriminated full-length IgG, monomeric IgA, and IgM in real time based on distinct ionic fingerprints.
• Demonstrated robust separation of isotypes in binary and ternary mixtures with high accuracy (p < 0.0001).
• Confirmed distinguishability of isotype-specific fingerprints within a diluted human serum background.

Conclusions:

• The developed electroosmotic flow-governed nanopore assay enables direct, label-free profiling of intact antibody isotypes.
• This method offers a scalable framework for multiplexed immunoprofiling and has potential applications beyond antibodies for proteomic targets.
• The assay preserves native antibody structure and binding functionality, advancing diagnostic and therapeutic monitoring capabilities.