Iron Oxide Nanoclusters as In Vitro Biosensors of Proteolytic Activity

Abstract

We demonstrate a flexible, tunable scheme for synthesizing multifunctional, ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) and its application to the area of magnetic relaxation switches. USPIO cores (10nm) were functionalized with a poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-PEG) copolymer, yielding ~40nm micelles. PPS-PEG-ssDNA conjugates and fluorophore-conjugated PPS-PEG are incorporated into the micelle synthesis process, yielding ssDNA-coated magnetofluorescent particles. To form magnetic relaxation switches, we generated USPIO populations that display complementary ssDNA sequences. Mixing of complementary USPIOs leads to clustering, resulting in a significant increase in R2 magnetic relaxation. Treatment of the DNA-crosslinked USPIO clusters with restriction enzymes specific for the crosslinking sequence results in an irreversible return of R2 relaxation to baseline levels. The constructs demonstrate their utility as nanoscale sensors of restriction enzyme activity. The presented functionalization scheme can be extended to the generation of biosensors for other sources of proteolytic activity, for diagnostics and imaging applications for cancer and atherosclerosis.