A novel high-sensitivity liquid-phase multi-stage immunoassay method with strong anti-interference capability based on AC magnetisation response of superparamagnetic nanoparticles

In this study, we propose a multi-stage all-liquid-phase magnetically responsive immunoassay based on bio-functionalised superparamagnetic nanoparticles, with the aim of achieving highly sensitive detection of small-molecule proteins (eg myoglobin and troponin) in whole blood/serum. The proposed method enables the specific capture of target biomarkers via a dual-target sandwich strategy, with a biotin–streptavidin amplification system incorporated to enhance the reaction signal. Meanwhile, harnessing the interlayer effects of the multi-stage reagents effectively suppresses non-specific background interference, thereby reducing noise and enhancing detection sensitivity. In this work, myoglobin (MYO) was selected as the model analyte, and a series of concentrations were prepared in both PBT buffer and horse serum matrices for methodological validation. Experimental results show that, in both serum-free and serum-containing environments, the proposed method demonstrates excellent detection sensitivity for MYO at concentrations as low as 0.1 ng/mL, coupled with a linear range spanning a 1,000-fold concentration interval and requiring only 15 min for the entire assay. The experimental results demonstrate the efficacy of the proposed method, highlighting its high stability and excellent resistance to background noises. These findings underscore its strong potential for clinical application.