Advancing Early Alzheimer’s Disease Detection Through Pan-Phosphorylation Binders and Single-Molecule Nanopore Technology

Nanopore single-molecule technology, with its high sensitivity and long-read capability, offers powerful opportunities for protein sequencing and early disease detection. To realize this potential, four steps are essential: protein capture, amino acid and post-translational modification discrimination, controlled translocation, and accurate signal interpretation. Controlled translocation is especially critical for producing clear, distinguishable signals, while robust analysis requires a spectral reference library. This project addresses uncontrolled protein movement by introducing binders that specifically recognize phosphorylation, acting as a “molecular brake” to establish a pan-phosphorylation-binder-assisted nanopore platform. Using this approach, we will map phosphorylated Tau and APP—key Alzheimer’s disease biomarkers—laying the foundation for early diagnostic applications. The platform will also expand nanopore capabilities for detecting diverse post-translational modifications and multiple phosphorylated proteins. Our objectives are: (1) to generate a pan-phosphorylation binder pool using protein engineering and a modified PANCS-binder screening system; (2) to build a spectral reference library for phosphorylation types based on dwell-time profiles; and (3) to create single-molecule nanopore profiles for early Alzheimer’s detection.