title: Ultrasensitive and specific measurement of protease activity using functionalized photonic crystals.
abstract: Herein is presented a microsensor technology as a diagnostic tool for detecting specific matrix metalloproteinases (MMPs) at very low concentrations. MMP-2 and MMP-9 are detected using label free porous silicon (PSi) photonic crystals that have been made selective for a given MMP by filling the nanopores with synthetic polymeric substrates containing a peptide sequence for that MMP. Proteolytic cleavage of the peptide sequence results in a shift in wavelength of the main peak in the reflectivity spectrum of the PSi device, which is dependent on the amount of MMP present. The ability to detect picogram amounts of MMP-2 and MMP-9 released by primary retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells stimulated with lipopolysaccharide (LPS) is demonstrated. It was found that both cell types secrete higher amounts of MMP-2 than MMP-9 in their stimulated state, with RPE cells producing higher amounts of MMPs than IPE cells. The microsensor performance was compared to conventional protease detection systems, including gelatin zymography and enzyme linked immunosorbent assay (ELISA). It was found that the PSi microsensors were more sensitive than gelatin zymography; PSi microsensors detected the presence of both MMP-2 and MMP-9 while zymography could only detect MMP-2. The MMP-2 and MMP-9 quantification correlated well with the ELISA. This new method of detecting protease activity shows superior performance to conventional protease assays and has the potential for translation to high-throughput multiplexed analysis.
authors: School of Chemistry, The Australian Centre for NanoMedicine and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, UNSW Australia , Sydney, New South Wales 2052, Australia.
authors: Bakul
authors: B
authors: Gupta
authors: School of Medical Sciences, UNSW Australia , Sydney, New South Wales 2052, Australia.
authors: Kelly
authors: K
authors: Mai
authors: School of Chemistry, The Australian Centre for NanoMedicine and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, UNSW Australia , Sydney, New South Wales 2052, Australia.
authors: Stuart B
authors: SB
authors: Lowe
authors: School of Medical Sciences, UNSW Australia , Sydney, New South Wales 2052, Australia.
authors: Denis
authors: D
authors: Wakefield
authors: School of Medical Sciences, UNSW Australia , Sydney, New South Wales 2052, Australia.
authors: Nick
authors: N
authors: Di Girolamo
authors: EMBL Australia Node in Single Molecule Science, School of Medical Sciences and ARC Centre of Excellence in Advanced Molecular Imaging, UNSW Australia , Sydney, New South Wales 2052, Australia.
authors: Katharina
authors: K
authors: Gaus
authors: School of Physics, UNSW Australia , Sydney, New South Wales 2052, Australia.
authors: Peter J
authors: PJ
authors: Reece
authors: School of Chemistry, The Australian Centre for NanoMedicine and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, UNSW Australia , Sydney, New South Wales 2052, Australia.
authors: J Justin
authors: JJ
authors: Gooding
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003460:Crystallization; D057075:Enzyme Assays; D006801:Humans; D007498:Iris / Q000166:cytology / Q000201:enzymology; D057230:Limit of Detection; D020778:Matrix Metalloproteinase 2 / Q000032:analysis* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000032:analysis* / Q000378:metabolism; D058608:Nanopores / Q000648:ultrastructure; D055095:Optics and Photonics / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D016062:Porosity; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000201:enzymology; D012825:Silicon / Q000737:chemistry*
chemical_list: D010455:Peptides; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D012825:Silicon