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 (Mnps) 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. Proteases play an important role in various physiological activities such as food digestion 1 , wound healing 2 , immune function 3 , and intracellular protein turnover 4. These enzymes cleave between amino acids in proteins and peptides and are the largest class of post-translational modifying enzymes in the human proteome 5. In cancer and neurodegeneration, unregulated proteolysis can occur when excess proteases are present at the site of disease or alternatively when the endogenous inhibitors are lacking 6,7. Detection and quantitation of proteases in biofluids can provide a greater understanding of diagnosis and staging of these diseases 8,9. For example, increased levels of the prostate-specific antigen (PSA) protease in blood is correlated with prostate cancer. This protease is quantified by an enzyme-linked-immunosorbent assay (ELISA) 10. However, since proteases are catalytically active, there is considerable interest in quantifying enzyme activity rather than protein levels 11,12. Recently, Ivry and colleagues showed that activity from two aspartic acid proteases, gastricin and cathepsin E, is significantly increased in pre-malignant pancreatic cyst fluid when compared to benign cyst fluid 13. Development of an assay to screen patient-derived cyst fluid for these protease activities has the potential to stratify patients for surgical intervention or surveillance. In addition, high levels of protease activity occurs in sputum of patients with chronic obstructive pulmonary disease 14,15 , cystic fibrosis 16-18 , and in non-healing wounds 19,20. Many notable studies have been carried out to detect cathepsin-B and matrix metalloproteinases (MMPs), both involved in cancer and tumor metastasis, using nanoparticles 21,22 , fluorescent tomographic imaging 23 , and droplet microfluidics 24. A nanoparticle-based approach 25 was also used to detect the serine protea...
