Activity-Based Assay of Matrix Metalloproteinase on Nonbiofouling Surfaces Using Time-of-Flight Secondary Ion Mass Spectrometry

摘要

A label-free, activity-based assay of matrix metalloproteinase (MMP) and its inhibition was demonstrated on peptide-conjugated gold nanoparticles (AuNPs) with non-biofouling poly(oligo(ethylene glycol) methacrylate) (pOEG-MA) films using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Following surface-initiated atomtransfer radical polymerization of OEGMA on a Si/SiO_(2) substrate, the MMP activity was determined by analyzing the cleaved peptide fragments using TOF-SIMS on the peptide-conjugated AuNPs. The use of nonbiofouling pOEGMA films in conjunction with AuNPs synergistically enhanced the sensitivity of assays for MMP activity and its inhibition in human serum. The detection sensitivity of MMP-7 in serum was as low as 20 ng mL~(-1) (1 pmol mL~(-1)), and the half-maximal inhibitory concentration (IC_(50)) of minocycline, which is a MMP-7 inhibitor, was estimated to be 450 nM. It is anticipated that the developed system will be broadly useful for conducting activity-based assays of serum proteases, as well as for screening of their inhibitors, with high sensitivity in a high-throughput manner.