Nanopore Fingerprinting of Neurodegenerative Proteins and Phosphoproteins within a Minute

Reliable identification of proteins and their post-translationally modified variants remains a formidable analytical challenge due to charge heterogeneity, sequence similarity, and comparable molecular weights. In this study, we demonstrate distinctive nanopore current fingerprints for clear identification of neurodegenerative disease-associated Tau protein and its phosphorylated variants using an asymmetric-electrolyte sensing system composed of different salts. The asymmetric configuration facilitates simultaneous detection of positively, neutrally, and negatively charged peptide fragments, resulting in 3.2-16-fold higher capture frequencies and 2.1-5.3-fold longer event durations, thereby yielding information-rich fingerprints that enhance protein recognition. Protein profiling was achieved within 1 min through integration with a droplet nanopore platform, which reduces sample consumption to the nanogram level while increasing throughput to >1800 events per minute. This work advances the nanopore fingerprinting approach for rapid, high-throughput, and low-sample protein biomarker detection, offering strong potential for clinical proteomics and early disease diagnosis.