Abstract
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.
Highlights
Proteases are enzymes that cleave proteins and are crucial to physiological processes such as digestion, blood clotting, and wound healing
To identify a peptide sequence that is efficiently cleaved by papain, the enzyme was previously combined with an equimolar mixture of synthetic tetradecapeptides and cleavage products were quantified by mass spectrometry[51]
We identified a tetradecapeptide substrate, KWLIHPTFSYnRWP, that was rapidly cleaved between S-Y and Y-n, where lowercase’n’ corresponds to the non-natural amino acid, norleucine
Summary
Proteases are enzymes that cleave proteins and are crucial to physiological processes such as digestion, blood clotting, and wound healing. 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. We developed a heterogeneous protease assay that uses giant magnetoresistive spin-valve (GMR SV) sensors These sensors transduce changes in the local magnetic field into electrical signals and have been used as the read head in hard disk drives[34,35], current sensors[36,37], magnetic memory[38] and biosensors[39,40]. The sensors continuously quantify the local magnetic field changes enabling real-time monitoring of the assay
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