Label-Free Chemiuminescent Assays For Protease Detection

Proteases, which hydrolyze protein or peptide into small pieces, play critical roles in many physiological processes. The perturbations of protease activity underlie multiple pathological conditions such as inflammation, cardiovascular diseases, and cancer. So it is of great significance to measure trypsin activity in clinical diagnosis. Here we developed novel, label-free chemiluminescent sensors for protease detection. The research in my assay consists of two parts:In the first part, we report a label-free streptavidin-modified magnetic beads(SA-MBs)-based sensing platform for turn-on chemiluminescent(CL) detection of protease using trypsin as model analyte. A biotinylated peptide containing an arginine and a terminal cysteine was used as the substrate of trypsin. Upon adding the peptide into a basic luminol-NaIO4 solution, the terminal cysteine induced a strong CL signal. Surprisingly a much lower CL was emitted when the peptide was immobilized on the surface of streptavidin-modified magnetic beads(SA-MBs). Based on this phenomenon, we designed a turn-on CL sensing system for protease using trypsin as model and its inhibitors screening. The simple method does not require washing or separating procedures. The linear range for trypsin detection was 0.01 nM-2 nM. Trypsin at a concentration as low as 10 pM can be assayed using this new CL sensing system. The IC50(the concentration at 50% inhibiting) for trypsin inhibitors SBTI, PMSF and EDTA was 1 nM, 0.01 mM and 3.28 mM, respectively. This new sensing strategy could be easily extended to assay other proteases by simply changing the peptide substrate.In the second part, a novel label-free chemiluminescent(CL) sensing platform for trypsin and its inhibitors screening was developed by taking advantage of luminol-NaIO4-(Cys-containing peptide, P) system and graphene oxide(GO). The assay was based on different catalytic activity of P and P-GO complex in luminol-NaIO4 CL reaction. A strong CL emission was obtained when P mixed with NaIO4 and basic luminol, while a much lower CL signal was observed when P-GO complex instead of P was injected into the mixture of NaIO4 and basic luminol. Based on the room-temperature electron paramagnetic resonance spectroscopy(EPR) study, we speculated that the catalytic activity of P in luminol-NaIO4 CL system may be inhibited when the peptides were adsorbed on GO surface. Based on the above observation, a label-free CL sensing platform for trypsin was developed. The linear range for trypsin detection was 0.01 nM-10 nM. The proposed method enabled the determination of trypsin with a detection limit of 7.3 pM, which can also be employed for screening of trypsin inhibitors. The IC50 for trypsin inhibitors SBTI and EDTA was 0.7 nM and 12 mM, respectively. The method can be easily generalized for monitoring the hydrolysis activity of other proteases by simply changing the peptide substrate sequence.