Sangphil Park

Protein-conjugated, glucose-sensitive surface using fluorescent dendrimer porphyrin

A multi-functional dendrimer-coated surface has been prepared for the effective protein immobilization and detection of protein activity. The silicon surface was first modified with positively-charged amino groups using 3-aminopropyltriethoxysilane (APTES), and subsequently coated with ionic dendrimer porphyrin (DP) by electrostatic interaction. Fluorescence and atomic force microscopy (AFM) studies showed that the dendrimer was homogeneously coated on the APTES-modified silicon surface as dome-shaped features that protruded 1.0–2.5 nm above the surface and had diameters ranging from 50 nm to 100 nm. The dendrimer-modified surface showed a higher capacity to covalently bind proteins, compared to the control surfaces, and the protein activity was higher by a factor of two. Using the fluorescent property of the porphyrin core, the relative amounts of dendrimer and the enzyme-catalyzed reactions on the dendrimer-coated surface were examined by fluorescence microscopy. Glucose oxidase (GOX)-mediated glucose oxidation quenched fluorescence emission from the focal porphyrin core through a peroxidase-coupled system and from the quantitative relationship between quenching and glucose concentration, the GOX-catalyzed reaction could be characterized.

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.

Mesoporous TiO2 as a nanostructured substrate for cell culture and cell patterning

We investigated the behaviors of mammalian cells such as adhesion, morphology and proliferation on mesoporous TiO2-coated glass slides using NIH-3T3 fibroblasts as a model cell. A sol–gel process using amphiphilic poly(vinyl chloride) (PVC)-g-poly(oxyethylene methacrylate) (POEM) graft copolymer as a template produced defect or crack-free and homogeneous mesoporous TiO2 films over a large area with high porosity and good connectivity. Cells grown on the mesoporous TiO2 surfaces exhibited less spreading and had more filopodia than cells on the flat glass slides. The nanotopographical cues from the mesoporous TiO2 resulted in the formation of more focal adhesions, promoting cell adhesion and proliferation without decreasing cell viability and functionality. We also demonstrated the capability of controlling spatial placement of cells onto chemically and topologically structured templates by fabricating poly(ethylene glycol) (PEG) hydrogel micropatterns on mesoporous TiO2 films. Because a hydrogel precursor solution could infiltrate and become crosslinked within the multilayered mesoporous TiO2 films, the resultant hydrogel micropatterns were firmly anchored on the substrate without the use of adhesion-promoting monolayers. While nanoscale topographic cues from mesoporous structure contributed to enhancement of cellular behaviors, different chemistry between cell-repelling PEG hydrogel and cell-adhesive mesoporous region facilitated confinement of cells on the micrometer scale. This study suggests that developed mesoporous TiO2 films hold high potential for bioapplications showing high biocompatibility as surface coating materials for implants or as biomimetic platforms for cell patterning.