Activity-based assay of matrix metalloproteinase on nonbiofouling surfaces using time-of-flight secondary ion mass spectrometry.
A label-free, activity-based assay of matrix metalloproteinase (MMP) and its inhibition was demonstrated on peptide-conjugated gold nanoparticles (AuNPs) with nonbiofouling poly(oligo(ethylene glycol) methacrylate) (pOEGMA) films using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Following surface-initiated atom-transfer radical polymerization of OEGMA on a Si/SiO2 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 (IC50) 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.
Kim
Young-Pil
YP
Department of Biological Sciences, KAIST, Daejeon 305-701, Korea.
Lee
Bong Soo
BS
Kim
Eunkyung
E
Choi
Insung S
IS
Moon
Dae Won
DW
Lee
Tae Geol
TG
Kim
Hak-Sung
HS
D004797:Enzyme-Linked Immunosorbent Assay; D006046:Gold / Q000737:chemistry; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D020783:Matrix Metalloproteinase 7 / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D053768:Metal Nanoparticles / Q000737:chemistry; D008689:Methacrylates / Q000737:chemistry; D008911:Minocycline / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D012825:Silicon / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods; D013499:Surface Properties
D061965:Matrix Metalloproteinase Inhibitors; D008689:Methacrylates; D010455:Peptides; C524499:polyethylene glycol methacrylate; D011092:Polyethylene Glycols; D006046:Gold; D012822:Silicon Dioxide; D020783:Matrix Metalloproteinase 7; D020778:Matrix Metalloproteinase 2; D008911:Minocycline; D012825:Silicon