Development of Sphere-Polymer Brush Hierarchical Nanostructure Substrates for Fabricating Microarrays with High Performance

In this work, a sphere-polymer brush hierarchical nanostructure-modified glass slide has been developed for fabricating high-performance microarrays. The substrate consists of a uniform 160 nm silica particle-self-assembled monolayer on a glass slide with a postcoated poly­(glycidyl methacrylate) (PGMA) brush layer (termed PGMA@3D(160) substrate), which can provide three-dimensional (3D) polymer brushes containing abundant epoxy groups for directly immobilizing various biomolecules. As a typical example, the interactions of three monosaccharides (4-aminophenyl β-d-galactopyranoside, 4-aminophenyl β-d-glucopyranoside, and 4-aminophenyl α-d-mannopyranoside) with two lectins (biotinylated ricinus communis agglutinin 120 and biotinylated concanavalin A from Canavalia ensiformis) have been assessed by PGMA@3D(160) substrate-based carbohydrate microarrays. The carbohydrate microarrays show good selectivity, strong multivalent interaction, and low limit of detection (LOD) in the picomolar range without any signal amplification. Furthermore, the proposed sphere-polymer brush hierarchical nanostructure substrates can be easily extended to fabricate other types of microarrays for DNA and protein detection. PGMA@3D(160) substrate-based microarrays exhibit higher reaction efficiencies and lower LODs (by at least 1 order of magnitude) in comparison to those of two-dimensional microarrays, which are fabricated on planar epoxy substrates, making it a promising platform for bioanalytical and biomedical applications

Sensitive Detection of Protein Kinase A Activity in Cell Lysates by Peptide Microarray-Based Assay

In the present work, the activities of protein kinase A (PKA) in cell lysates have been detected by a peptide microarray-based resonance light scattering assay with gold nanoparticle probes. Highly sensitive detection of PKA activity in 0.1 μg total cell proteins of SHG-44 cell lysate (corresponding to 200 cells) is achieved by a selected peptide substrate. The experimental results also demonstrate that the assay can be employed to evaluate expression levels of PKA activity in different cell lines and chemical (e.g., Forskolin )-mediated PKA activity fluctuation in living cells. In addition, PKA inhibition by the inhibitor (H89) is shown, suggesting the potential for screening PKA inhibitors at the living cell level

Poly(glycidyl methacrylate-<i>co</i>-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality

We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly­(glycidyl methacrylate-<i>co</i>-2-hydroxyethyl methacrylate) (P­(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P­(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P­(GMA-HEMA) brush-based microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL^–1 for MMP-2 and 5.7 pg mL^–1 for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P­(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained