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

Single-molecule protein profiling offers unparalleled sensitivity for detecting rare biomarkers, with significant potential for early disease diagnosis and monitoring. Nanopore sensors enable the detection of proteins in solution and are well-suited for clinical applications due to their high sensitivity and throughput. Here, we introduce a nanopore-based detection strategy utilising an aptamer-protein-aptamer sandwich structure to enhance protein detection further. The aptamer probe is extended with a double-stranded DNA carrier to enhance protein transport and detection efficiency. Upon protein binding, dimerization of the DNA carriers occurs, allowing charge-based classification of bound versus unbound states. Additionally, the sandwich complex produces a characteristic subpeak within individual nanopore events, with subpeak height correlating to protein size. This approach enables selective detection of dimeric proteins, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), with sub-picomolar detection limits. In principle, it can be adapted for any protein with two distinct aptamer-binding sites, as demonstrated with thrombin. This method also facilitates real-time monitoring of ligand-induced receptor dimerization in complex biological fluids, offering a powerful tool for potential single-molecule-based diagnostics.