Nucleic Acid Amplification Boosts Electrochemically Addressable Probe Networks for Ultrasensitive Plate-Based Electrochemiluminescence Immunoassays

Electrochemiluminescence immunoassays (ECLIAs) offer low-background and highly sensitive protein biomarker detection, but their performance is often severely limited by the poor electrochemical accessibility of luminophore labels in bulky immunocomplexes. Herein an electrochemically addressable probe network-based ECL immunoassay (EAPN-ECLIA) was developed, in which rolling circle amplification (RCA) converts target recognition into a densely labeled ruthenium probe network at the carbon electrode interface. The RCA-generated DNA scaffold recruits biotinylated probes and Ru-labeled streptavidin, increasing Ru-complex loading while positioning a portion of Ru-labels sufficiently close to the electrode surface for direct electrooxidation and efficient ECL generation. In addition, a constraint-aware scoring and ranking algorithm was proposed to guide the selection of carbon-based electrode materials for ECLIAs. Compared with conventional plate-based ECLIA, EAPN-ECLIA improved detection sensitivity by approximately 5000-fold. For plasma phosphorylated Tau181, a key Alzheimer’s disease biomarker, the platform achieved a limit of detection of 85.81 fg/mL, reliable plasma recovery, and effective discrimination of patients with AD from healthy controls with an area under the curve of 0.9650. This strategy highlights electrochemical addressability, rather than luminophore loading alone, as a key design principle for ultrasensitive ECLIAs.