Single-Molecule Protein Profiling Using Nanopores and Dimeric Aptamer-Modified DNA Carriers

Area of Science:

• Biotechnology
• Nanotechnology
• Biomarker Discovery

Background:

• Single-molecule protein profiling offers high sensitivity for rare biomarker detection, crucial for early disease diagnosis.
• Nanopore sensors provide sensitive, high-throughput protein detection in solution, suitable for clinical use.

Purpose of the Study:

• To develop an enhanced nanopore-based detection strategy for improved protein profiling sensitivity.
• To enable selective detection and characterization of specific proteins and protein interactions.

Main Methods:

• Utilized an aptamer-protein-aptamer sandwich structure with DNA carriers for enhanced protein transport and detection.
• Analyzed charge-based dimerization of DNA carriers and characteristic subpeaks in nanopore events for protein identification.
• Correlated subpeak height to protein size for quantitative analysis.

Main Results:

• Achieved selective detection of dimeric proteins, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), at sub-picomolar limits.
• Demonstrated adaptability for proteins with two aptamer-binding sites, exemplified by thrombin detection.
• Enabled real-time monitoring of ligand-induced receptor dimerization in complex biological fluids.

Conclusions:

• The developed nanopore strategy significantly enhances single-molecule protein detection sensitivity and selectivity.
• This method provides a powerful tool for potential single-molecule-based diagnostics and monitoring of protein interactions.
• The approach is versatile and adaptable for a wide range of protein targets and clinical applications.