Single-molecule detection of amino acid phosphorylation using electron tunnelling currents: toward neurodegenerative disease diagnosis

Protein phosphorylation is one of the most prevalent post-translational modifications regulating biological functions, and its dysregulation is closely associated with neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Despite extensive studies, direct detection of phosphorylation at the single-molecule level remains challenging because conventional mass spectrometry and antibody-based assays require complex pretreatments and cannot comprehensively resolve site-specific modifications. To address this challenge, we employed mechanically controllable break junction (MCBJ) measurements to probe the single-molecule conductance of serine (Ser), threonine (Thr), tyrosine (Tyr), and their phosphorylated counterparts. Distinct conductance trends were observed depending on the amino acid species, reflecting the influence of phosphate substitution on the electronic states. Density functional theory (DFT) calculations revealed that phosphorylation-induced shifts in HOMO levels and changes in π-conjugation account for the observed conductance variations. Furthermore, statistical analysis combined with machine-learning-based classification enabled discrimination between phosphorylated and non-phosphorylated amino acids with high accuracy, demonstrating that single-molecule electrical signals contain sufficient molecular information for identifying phosphorylation. This study establishes an electronic readout approach for amino acid phosphorylation and provides a proof of concept for extending single-molecule electrical techniques toward quantitative and sequencing-oriented analyses of protein modifications.