High-Sensitivity Magnetic Particle Spectroscopy Platform for Precise, Specific, and Rapid Detection of Infectious...

Accurate biosensing using magnetic particle spectroscopy (MPS) depends on the interplay between biomolecular binding and the fidelity of the measurement system. MPS enables wash-free, label-free biosensing by monitoring Brownian relaxation-induced changes in magnetic nanoparticle (MNP) dynamics. However, its practical performance is often limited by a weak signal-to-noise ratio, excitation feedthrough, environmental noise, slow acquisition, and insufficient mass sensitivity. Here, a gradiometer-based MPS system incorporating noise suppression, signal integration, and harmonic detection was developed to monitor relaxation changes upon biomolecular binding. Single-core SHP-25 MNPs were conjugated with recombinant human angiotensin-converting enzyme 2 (ACE2) protein, antinucleoprotein (anti_NP) antibodies, and AS1411 aptamer to target the SARS-CoV-2 receptor-binding domain (RBD), influenza A hemagglutinin type-1, neuraminidase type-1 (H1N1) nucleoprotein, and breast cancer biomarker nucleolin, respectively. Conjugation was validated by FT-IR spectroscopy and DLS to confirm chemical attachment and hydrodynamic size modulation, while ICP-MS was used for iron-mass normalization. Relaxation changes from conjugation and target binding were recorded by MPS. The third-harmonic showed the highest sensitivity to binding-induced relaxation delays, with detection limits of 1 nM (100 fmol) for ACE2 and anti_NP and 12 nM for AS1411. Target-binding yielded statistically significant responses at concentrations as low as 5 nM for RBD, H1N1, and nucleolin. The harmonic ratio R₅₃ (fifth/third) increased monotonically with binding, providing a mass-normalized recognition metric with reduced dependence on nanoparticle concentration. The system achieved an iron-mass sensitivity of 1 ng Fe. Comparative analysis shows that monoclonal antibody-conjugated MNPs provide greater specificity and quantitative robustness in MPS measurements than polyclonal antibodies. The high sensitivity of the developed MPS, combined with the superior specificity of monoclonal antibodies, enables precise biosensing with potential clinical relevance.