Giant magnetoresistive biosensors for real-time quantitative detection of protease activity

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Proteases are enzymes that cleave proteins and are crucial to physiological processes such as digestion, blood clotting, and wound healing. Unregulated protease activity is a biomarker of several human diseases. Synthetic peptides that are selectively hydrolyzed by a protease of interest can be used as reporter substrates of unregulated protease activity. We developed an activity-based protease sensor by immobilizing magnetic nanoparticles (MNP) to the surface of a giant magnetoresistive spin-valve (GMR SV) sensor using peptides. Cleavage of these peptides by a protease, releases the magnetic nanoparticles resulting in a time-dependent change in the local magnetic field. Using this approach, we detected a significant release of MNPs after 3.5 minutes incubation using just 4 nM of the cysteine protease, papain. In addition, we show that proteases in healthy human urine do not release the MNPs, however addition of 20 nM of papain to the urine samples resulted in a time-dependent change in magnetoresistance. This study lays the foundation for using GMR SV sensors as a platform for real-time quantitative detection of protease activity in biological fluids.</jats:p>

A GMR enzymatic assay for quantifying nuclease and peptidase activity

“…A previous attempt to develop a GMR-based peptidase assay utilized a thioether linkage between a cysteine residue on the substrate and a maleimide-coated surface (Adem et al, 2020). However, the thioether bond was insufficiently stable in aqueous solutions and, therefore, not useful for point-of-care assays that require storage of the assembled substrate on the GMR sensor.…”