UC San Diego

We are developing a point-of-care test for detection of proteases. Proteases are enzymes that cleave proteins and play an important role in many physiological processes. However, their unregulated activity can trigger onset of many diseases like cancer, respiratory diseases, and other infections. Some proteases are highly specific and only cleave substrates with a specific peptide sequence which allow them to be used as biomarkers for detection of specific diseases. We have demonstrated proof-of-concept optical test that starts with immobilizing a cleavable peptide on the surface of a 96-well plate. This peptide contains a specific cleavable sequence for papain, a commercially available protease that is related to several disease-associated proteases. Fluorescent tags were attached to the peptide through streptavidin-biotin chemistry. The total florescence directly correlates with the amount of peptide immobilized to the surface. Addition of papain was predicted to cleave the peptide and cause a time-dependent reduction in the fluorescent signal. The assay was then translated to the magnetic biosensors via GMR sensors and streptavidin coated magneto nanoparticles (MNPs). This test works by immobilizing magnetic nanoparticles onto surface of the GMR sensor via a BSA-peptide conjugate. Once peptide cleavage by papain occurs, the MNPs are released away from the sensor surface. This change in the magnetic field of the GMR sensors is detected in real-time. We hope to further optimize this test by improving the signal reduction rate to background and testing other diseases such as cystic fibrosis by detecting specific proteases in cystic fibrosis patient sputum samples.