Enhanced sensitivity detection via orientational labelling of SPA-functionalized magnetic nanoparticles in lateral-flow immunoassays

Mycoplasma pneumonia, a primary aetiological agent of atypical pneumonia, necessitates the implementation of rapid point-of-care diagnostics. Lateral flow immunoassays (LFIAs) hold promise for point-of-care testing (POCT), yet their sensitivity levels are frequently constrained by probe affinity and matrix interference. We introduce an orientational labelling strategy that employs magnetic nanoparticles (MNPs) functionalized with staphylococcal protein A (SPA) to simultaneously enhance antibody orientation and facilitate magnetic enrichment. The SPA-MNPs were synthesized via aggregation‒precipitation crosslinking, utilizing the Fc-binding specificity of SPA to enable oriented antibody conjugation and reduce steric hindrance. The oriented probe demonstrated robust stability, exceptional specificity, and enhanced binding affinity, which were considered crucial for the detection of low-abundance targets. In concentration gradient tests, the orientational labelling method resulted in the lowest visible colorimetric signals at 10⁵ CFU·mL^-1, whereas conventional random probes (BSA-MNPs) resulted in detectable coloration at concentrations as low as 10⁶ CFU·mL^-1. The color sensitivity and LOD of the orientational labelling approach were further increased to 10⁴ CFU/mL and 0.668 × 10⁴ CFU/mL, respectively, through magnetic enrichment treatment. The capture binding kinetics analysis indicated that the enrichment efficiency depended on the binding effect of the antibody and the antigen concentration, showing a clear correlation with the dissociation constant (K_D). A clinical evaluation of 87 patient samples showed 88.5% agreement with the results of the quantitative fluorescence qPCR, which validated the diagnostic accuracy of the method. This study presents an orientational labelling platform for LFIA that combines Fc-directed antibody alignment with magnetic nanoparticle enrichment to overcome sensitivity barriers in the detection of low-abundance pathogens. This strategy comprises a universal design principle for achieving high-performance point-of-care testing (POCT) for various infectious diseases.