Spatially designed electrochemical sensor for simultaneous detection of total and phosphorylated neurofilament light chain in neurodegeneration.

Phosphorylation plays a pivotal role in neurodegenerative diseases, and the ratio of phosphorylated neurofilament light chains (pNfL) to total neurofilament light chains (tNfL) (pNfL/tNfL) has emerged as a promising biomarker. Detecting serum pNfL/tNfL remains challenging due to the extremely low abundance of pNfL. Effective detection requires spatial differentiation between protein capture sites, recognition sites, and post‑translational modification sites, together with precise spatial compatibility among multiple signal amplification sources to simultaneously identify proteins and their phosphorylation states. In this study, we systematically analyzed the structure of pNfL to identify optimal capture and recognition sites with distinct spatial localization. Systematic evaluation demonstrated that matching the size of signal amplification particles to the target protein significantly enhances sensor performance. By optimizing particle size, signals of pNfL and tNfL were amplified using 2 nm carbon dots loaded with Cu2+/Ti4+ and an antibody–horseradish peroxidase complex, respectively. Coupled with an interface–solution dual‑pathway amplification strategy, this approach enabled convenient one‑step dual-signal electrochemical quantification of pNfL/tNfL. The developed sensor exhibited excellent sensitivity, selectivity, and reproducibility, with dynamic ranges of 0.2–20 pg·mL− 1 for both tNfL and pNfL. Preliminary serum analysis suggested that the pNfL/tNfL ratio provide improved discriminatory capability between neurodegenerative conditions, non-neurodegenerative brain injury, and healthy controls. Notably, the pNfL/tNfL ratio showed improved specificity compared to individual tNfL or pNfL measurements in this exploratory cohort. This work introduces a novel spatial design strategy that integrates particle size optimization with dual-pathway amplification, representing the first one-step dual-signal electrochemical platform capable of simultaneously quantifying total and phosphorylated neurofilament light chains in serum.