Rational Design of Peptide-Functionalized Poly(Methacrylic Acid) Brushes for On-Chip Detection of Protease Biomarkers

There is an increasing demand for developing new materials and approaches for rapid and sensitive detection of protease biomarkers. Herein, the poly(methacrylic acid) (PMAA) brushes were synthesized from silica nanoparticles via surface-initiated atom transfer radical polymerization (ATRP), and flexibly functionalized with different fluorescein-labeled peptides, serving as the substrates for protease assay. To facilitate the point-of-care detection of protease, polyacrylamide gel pad arrays were fabricated to allow permeation of fluorescein-labeled peptide fragments cleaved from the PMAA brushes. This experimental setup enables an on-chip protease assay with an adequate limit of detection (LOD) for detecting trypsin in a buffer solution (3.9 pM) or in serum (1.4 nM) and good specificity for differentiation of trypsin and chymotrypsin. By using this experimental setup, matrix metalloproteinase-2 and matrix metalloproteinase-9 can be detected with LODs of 2.5 nM and 3.3 nM, respectively. Moreover, by introducing an adamantine (Ad) motif to the side-chain of the peptide fragment and β-cyclodextrin (β-CD) groups to the gel pad matrix, a 2.2-fold lower LOD was achieved for the detection of trypsin (1.8 pM) due to the supramolecular self-assembly of Ad and β-CD. Given the advances in the ease of sample handling, this rational design of peptide-functionalized PMAA brushes could be useful for on-chip detection of protease biomarkers or the screening of potential protease inhibitors.