Laser based functionalization for graded immobilization of biomolecules on biocompatible polymer surfaces

Surface modification is a powerful tool for the development of cell guiding structures for tissue engineering as well as for the development of in vitro assays for molecular screening. In both applications the biomolecule concentration linked to the surface is an important functionalization property. While cell guiding structures need continuous linear gradients of either increasing or decreasing signaling molecule concentration, assays for molecular screening need distinct areas of functionalization for testing of various molecule concentrations. Therefore a laser based method for local surface activation is under investigation. Laser irradiation is a powerful tool, which provides a high temporal and spatial resolution for the generation of structures in the micrometer range. In the current research we investigated the influence of UV-Laser irradiation with the wavelengths of 193nm and 248nm on the activation of poly(methyl metacrylate) (PMMA) and poly-ε-caprolactone (PCL). We demonstrate that PMMA and PCL can be locally activated. In a subsequent wet chemical step the activated groups can be functionalized with amine groups. These amines can be used for immobilization of bioactive compounds like RGD (Arginine- Glycine-Aspartate)-peptides or lactose. RGD-peptides will serve as cell recognition domain and enhance cell adhesion, while lactose serves as a model molecule for further surface modification. We found out that by defined pulse accumulation the activation dose can be increased. This leads to an increase of the surface activation and therefore the increase of the functionalization density. The successful activation and amination of the surfaces was shown by staining with an amino-specific fluorescent dye and XPS-analysis. The process window for PMMA and PCL activation with irradiation of 193nm differ, but the increasing fluorescence signal corresponds to the increasing irradiation dose. Activation with irradiation of 248nm leads to a weak surface activation The RGD-peptide functionalized surfaces have been seeded with neuronal B35-cells which differentiate under serum deprived conditions. We could successfully demonstrate that cells favor areas with higher concentration of RGD-peptides. Finally we showed that lactose can be successfully linked to the amines. The specific binding of lactose could be demonstrated by staining with a FITC labeled Erythrina cristagally Lectin