Detection of Cancer-Specific Proteases Using Magnetic Relaxation of Peptide-Conjugated Nanoparticles in Biological Environment

Protease expression is closely linked to malignant phenotypes of different solid tumors; as such, their detection is promising for diagnosis and treatment of cancers, Alzheimers, and vascular diseases. Here, we describe a new method for detecting proteases by sensitively monitoring the magnetic relaxation of monodisperse iron oxide nanoparticles (IONPs) using magnetic particle spectrometer (MPS). In this assay, tailored peptides functioning as activatable nanosensors link magnetic nanoparticles and possess selective sites that are recognizeable and cleaveable by specific proteases. When these linker peptides, labeled with biotin at N- and C-terminals, are added to the neutravidin functionalized IONPs, nanoparticles aggregate, resulting in well-defined changes in the MPS signal. However, as designed, in the presence of proteases these peptides are cleaved at predetermined sites, redispersing IONPs, and returning the MPS signal(s) close to its preaggregation state. These changes observed in all aspects of the MPS signal (peak intensity, its position as a function of field amplitude, and full width at half-maximumwhen combined, these three also eliminate false positives), help to detect specific proteases, relying only on the magnetic relaxation characteristics of the functionalized nanoparticles. We demonstrate the general utility of this assay by detecting one each from the two general classes of proteases: trypsin (digestive serine protease, involved in various cancers, promoting proliferation, invasion, and metastasis) and matrix metalloproteinase (MMP-2, observed through metastasis and tumor angiogenesis). This MPS based protease-assay is rapid, reproducible, and highly sensitive and can form the basis of a feasible, high-throughput method for detection of various other proteases.