Graft copolymer-templated mesoporous TiO₂ films micropatterned with poly(ethylene glycol) hydrogel: Novel platform for highly sensitive protein microarrays

In this study, we describe the use of organized mesoporous titaniumoxide (TiO₂) films as three-dimensional templates for protein microarrays with enhanced protein loading capacity and detection sensitivity. Multilayered mesoporous TiO₂ films with high porosity and good connectivity were synthesized using a graft copolymer consisting of a poly(vinyl chloride) (PVC) backbone and poly(oxyethylene methacrylate) (POEM) side chains as a structure-directing template. The average pore size and thickness of the TiO₂ films were 50-70 nm and 1.5 μm, respectively. Proteins were covalently immobilized onto mesoporous TiO₂ film via 3-aminopropyltriethoxysilane (APTES), and protein loading onto TiO₂ films was about four times greater than on planar glass substrates, which consequently improved the protein activity. Micropatterned mesoporous TiO ₂ substrates were prepared by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures on TiO₂ films using photolithography. Because of nonadhesiveness of PEG hydrogel towards proteins, proteins were selectively immobilized onto surface-modified mesoporous TiO₂ region, creating protein microarray. Specific binding assay between streptavidin/biotin and between PSA/anti-PSA demonstrated that the mesoporous TiO₂-based protein microarrays yielded higher fluorescence signals and were more sensitive with lower detection limits than microarrays based on planar glass slides.