Peptide-functionalization Of Poly(Methacrylic Acid) Brushes For Highly Sensitive Protease Assays

Proteases exist ubiquitously in all living forms and play important roles in regulating numerous biological and physiological processes(e.g.food digestion,blood clotting,cell apoptosis and disease development)by cleaving peptide bonds in proteins or peptides.Many diseases such as cardiovascular disease,Alzheimer disease and cancers are correlated with dysfunction or overactivity of proteases.Currently,the protease assay mainly relies on high-performance liquid chromatography(HPLC)and mass spectrometry(MS).However,these methods require bulky and expensive instrumentations and trained personnel.Therefore,developing of novel materials for fast and sensitive protease assay is in demand for timely and point-of-care diagnosis of diseases.In the first part of this thesis,poly(methacrylic acid)(PMAA)brushes was synthesized via surface-initiated atom transfer radical polymerization(SI-ATRP)on the surface of silica nanoparticles.Next,the PMAA brushes was functionalized with series of proteolytically cleavable fluorescein-labeled peptides for highly sensitive protease assay.Upon the proteolytic cleavage of the peptides,a short peptide fragment with fluorescent tag(GGK-FITC)is released to the solution,which can be easily detected with a bench-top fluorescence microscope.The grafting densities of PMAA brushes and peptides can be readily tuned by controlling the monomer concentrations of sodium methacrylate in the ATRP reaction.Owing to the three dimensional architecture of PMAA brushes,the loading surface density of peptides can reach 22.4 peptides/nm2,which is much higher than direct immobilization on silica nanoparticles without polymer brushes.Because of the high loading density of peptides,the limit of detection(LOD)of trypsin can reach 1.4 pM in buffer solution or 2.6 nM in non-diluted serum.By rational design of peptide substrates,the peptide-functionalized PMAA brushes can be readily expanded to detect other proteases,such as matrix metalloproteinase-2(MMP-2),a virtual biomarker for many cancers,with an LOD of 1.1 pM.The proteolytically cleavable peptide-functionalized PMAA brushes offer a starting point for fast and sensitive protease assay.Despite the advances of the peptide-functionalized PMAA brushes as mentioned above,the protease assay requires multiple centrifugation and spin-filtration steps,which hinders the application of such materials for point-of-care tests of proteases.To address this issue,a gel pad array chip was fabricated to trap the released peptide fragments.In this way,the LOD for detecting trypsin in Tris buffer solution(pH 8.0)and non-diluted serum were 157 pM and 243 nM,respectively.The LOD for detecting MMP-2 and MMP-9 in Tris buffer solution(pH 8.0)were 1.67 nM and 1.22 nM,respectively.To achieve lower LOD,an adamantane(Ad)motif was tethered to the side chain of the peptide,and the P-cyclodextrin(?-CD)was copolymerized with polyacrylamide to fabricate the gel pad array.Thererfore,the released peptide fragments modified with Ad can be easily captured by the ?-CD in the polymer network of the gel pad array,and consequently resulted in improved sensitivity.The LOD for detecting trypsin was 13.2 pM.Collectively,we synthesized peptide-functionalized PMAA brushes on silica nanoparticles to serve as scaffold for rapid and sensitive protease assay,and the gel pad array significantly simplified the procedures for the signal readout.The above studies lay the groundwork for the design of novel protease assays and promote the developing of point-of-care diagnostic devices.