Chaotropic Surface Chemistry for Protein Profiling with Solid-State Micropores

Area of Science:

• Biochemistry
• Nanotechnology
• Analytical Chemistry

Background:

• Proteins' biological functions are determined by their amino acid sequences, with variations linked to diseases and treatment outcomes.
• Conventional nanopore analysis of proteins faces limitations due to issues like stochastic capture and rapid translocation.
• Precise control over pore dimensions is crucial for traditional nanopore-based protein analysis.

Purpose of the Study:

• To develop a scalable method for sequence-sensitive protein analysis that bypasses the need for sub-nanometer pore control.
• To convert subtle, sequence-dependent protein surface chemistry into amplified, particle-scale transport signatures.
• To enable accurate discrimination of protein variants using readily fabricated micro-scale pores.

Main Methods:

• Immobilizing monoclonal antibody variants with distinct amino acid sequences on polymeric microparticles.
• Measuring particle-protein complex passage through silicon nitride (SiN_x) micropores.
• Utilizing chaotropic chemistry (guanidine hydrochloride) to modulate surface charge and reshape the electrokinetic landscape.
• Analyzing resistive pulse waveforms generated during particle translocation using machine learning.

Main Results:

• Chaotropic perturbation effectively modulated the electrokinetic properties around particle-protein complexes in a variant-dependent manner.
• Distinct resistive pulse waveforms were generated, allowing for near-perfect discrimination of antibody variants.
• The method demonstrated sequence sensitivity by differentiating antibody variants with minor sequence variations.
• Machine learning algorithms successfully classified the antibody variants based on their unique transport signatures.

Conclusions:

• This novel approach shifts protein sensing from geometrical constraints in nanopores to chemical amplification in micrometer-scale pores.
• The method offers a scalable and accessible route for sequence-sensitive biomarker profiling.
• It eliminates the requirement for advanced nanofabrication, making protein analysis more widely applicable.