Magnetophoretic Refinement and Microdroplet Confinement for Magnetic Relaxation Biosensing

Magnetic relaxation biosensing (MRB) is inherently limited by relaxation noise arising from magnetic bead heterogeneity and diffusion-mediated averaging in bulk aqueous environments. Here, we present a microdroplet-confined magnetic relaxation biosensing (mc-MRB) strategy that enhances signal stability and measurement sensitivity by physically regulating both magnetic perturbers and their relaxation microenvironment. A refined magnetophoretic-inertial microfluidic module removes low-moment and aggregated beads, yielding a homogeneous immunomagnetic nanobead population. Subsequent confinement within picoliter droplets restricts proton diffusion and stabilizes magnetic-field perturbations, generating a low-noise relaxation landscape distinct from bulk conditions. Using low-field NMR, the mc-MRB system demonstrates quantitative detection of Salmonella from 5.2 × 10¹ to 5.2 × 10⁵ CFU/mL with a limit of detection of 28 CFU/mL. Robust specificity and reliable performance are further demonstrated in complex food matrices. This work highlights microenvironment engineering as a general physical strategy for stabilizing magnetic relaxation signals in MRB.