Preparation of photolithographically patterned inverse opal hydrogel microstructures and its application to protein patterning.

Protein pattern has played an important role in biosensors, bioMEMS, tissue engineering, fundamental studies of cell biology, and basic proteomics research. Here, we developed a straightforward and effective protein patterning technique using macroporous poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel micropatterns as a three-dimensional (3D) template for protein immobilization. Micropatterns of macroporous hydrogels with inverse opal structures were prepared on poly(ethylene glycol) (PEG)-coated silicon substrates by combining a colloidal crystal templating method with photopatterning. The resultant inverse opal hydrogel (IOH) micropatterns were modified with 3-aminopropyltriethoxysilane using the hydroxyl groups in PHEMA for the covalent immobilization of proteins. Proteins were selectively immobilized only on the hydrogel micropatterns, while the PEG regions served as an effective barrier to protein adsorption. Because of their highly ordered and interconnected 3D macroporous structures and large internal surface areas, protein loading in the IOH micropattern was about six times greater than that on a non-porous hydrogel micropattern, which consequently improved the protein activity. The porosity of the hydrogel micropatterns could be controlled using different sizes of colloidal nanoparticles, and using smaller nanoparticles produced hydrogel micropatterns with higher protein loading capacities and activities. To demonstrate the potential use of IOH micropatterns in biosensor systems, biotin was micropatterned on the hydrogels and the specific binding of streptavidin was successfully assayed using IOH micropatterns with better fluorescence signals and sensitivity than that of the corresponding non-porous hydrogel micropatterns.

Micropatterned assembly of silica nanoparticles for a protein microarray with enhanced detection sensitivity.

We used an assembly of silica nanoparticles (SNPs) as a three-dimensional template for protein immobilization to prepare a protein microarray with enhanced protein loading capacity and detection sensitivity. SNPs were first modified with 3-aminopropyltriethoxysilane (APTES) for covalent immobilization of protein and micropatterned on poly(ethylene glycol)(PEG)-coated glass slides using elastomeric membranes with an array of holes. Proteins were selectively immobilized only on the SNP region, while the PEG regions served as an effective barrier to protein adsorption. Because of multi-layered SNPs that had curved surface, protein loading in the SNP micropattern was about six times greater than on a planar surface, as observed by fluorescence microscopy, which consequently improved the protein activity and reaction rate. GOX-catalyzed glucose oxidation and the molecular recognition mediated, specific binding between biotin and streptavidin were both successfully assayed using SNP microarrays, with better fluorescence signal and sensitivity than corresponding planar microarrays.