Magnetic nanoparticles (MNPs) have been extensively used as contrasts and tracers for bioimaging, heating sources for tumor therapy, carriers for controlled drug delivery, and labels for magnetic immunoassays. Here, we describe a MNP Brownian relaxation dynamics-based magnetic particle spectroscopy (MPS) method for the quantitative detection of molecular biomarkers. In MPS measurements, the harmonics of oscillating MNPs are recorded and used as a metric for the freedom of rotational motion, which indicates the bound states of the MNPs. These harmonics can be collected from microgram quantities of iron oxide nanoparticles within 10 s. As the harmonics are largely dependent on the quantity of the MNPs in the sample, the MPS bioassay results could be biased by the deviations of MNP quantities in each sample, especially for the very low-concentration biomarker detection scenarios. Herein, we report three MNP concentration/quantity-independent metrics for characterizing the bound states of MNPs in MPS. Using a streptavidin-biotin binding system as a model, we demonstrate the feasibility of using MPS and MNP concentration/quantity-independent metrics to sense these molecular interactions, showing that this method can achieve rapid, wash-free bioassays, and is suitable for future point-of-care, sensitive, and versatile diagnosis.
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