  Magnetic nanoparticles (MNPs) with proper surface functionalization have been
extensively applied as labels for magnetic immunoassays, carriers for
controlled drug/gene delivery, tracers and contrasts for magnetic imaging, etc.
Here, we introduce a new biosensing scheme based on magnetic particle
spectroscopy (MPS) and the self-assembly of MNPs to quantitatively detect H1N1
nucleoprotein molecules. MPS monitors the harmonics of oscillating MNPs as a
metric for the freedom of rotational motion, thus indicating the bound states
of MNPs. These harmonics can be readily collected from nanogram quantities of
iron oxide nanoparticles within 10 s. H1N1 nucleoprotein molecule hosts
multiple different epitopes that forms binding sites for many IgG polyclonal
antibodies. Anchoring IgG polyclonal antibodies onto MNPs triggers the
cross-linking between MNPs and H1N1 nucleoprotein molecules, thereby forming
MNP self-assemblies. Using MPS and the self-assembly of MNPs, we achieved the
sensitivity of 44 nM (442 pmole) for detecting H1N1 nucleoprotein. In addition,
the morphologies and the hydrodynamic sizes of the MNP self-assemblies are
characterized to verify the MPS results. Different MNP self-assembly models
such as classical cluster, open ring tetramer, chain model as well as multimers
(from dimer to pentamer) are proposed in this paper. Herein, we claim the
feasibility of using MPS and the self-assembly of MNPs as a new biosensing
scheme for detecting ultralow concentrations of target biomolecules, which can
be employed as rapid, sensitive, and wash-free magnetic immunoassays.
