Two-Color Digital Immunoassay for Simultaneous Detection of Low-Abundance Neurodegenerative Disease Protein Biomarkers

We developed a novel two-color digital immunoassay platform for the simultaneous detection of neurodegenerative disease protein biomarkers (neurofilament light chain (NfL) and interleukin-6 (IL-6)) in a homogeneous, wash-free format. This multiplex approach employs two antibody pairs: capture antibodies conjugated with porphyrin-metal organic framework (pMOF) as singlet oxygen (¹O₂) nanogenerators, and detection antibodies functionalized with spectrally distinct upconversion nanoparticles (UCNPs) serving as nanoreporters─specifically BHQ2-modified Er³⁺-doped UCNPs for NfL detection and dabsyl-modified Tm³⁺-doped UCNPs for IL-6 detection, both with luminescence caged by ¹O₂-cleavable quenchers. Antigen binding formed immunocomplexes between nanoprobe pairs, triggering specific luminescence activation in distinct channels: green emission at 540 nm for NfL and blue emission at 475 nm for IL-6. The spatially restricted diffusion radius of pMOF-generated ¹O₂ ensured the selective cleavage of proximal quenchers, enabling digital quantification via single-molecule fluorescence imaging. The platform demonstrated exceptional sensitivity with cross-talk-free performance, achieving detection limits of 25 aM for NfL and 11 aM for IL-6. Serum analysis confirmed the biosensor's potential for comprehensive neurological biomarker detection, establishing its utility for large-scale neurodegenerative disease screening.