Lokale Laserfunktionalisierung mit Biomolekülen zur Herstellung von Konzentrationsgradienten
Aachen / Publikationsserver der RWTH Aachen University (2015) [Dissertation / PhD Thesis]
Page(s): V, 109 S. : Ill., graph. Darst.
In 1986 Srinivasan et al. investigated surface activation of PMMA for technical applications. Wet chemical functionalisation of PCL for implant surfaces is already state of the art. The goal of this study is to show that PMMA and PCL surfaces can be activated by Excimer-laser irradiation with wavelength 193 nm and 248 nm. Activated surfaces will be wet chemically functionalized with bioactive molecules in a following step. Process parameters (fluence, number of pulses and repetition rate) have to be defined which allow an intensive but non-destroying activation of the surface. It is expected, that due to the high temporal and spatial resolution of laser irradiation a various numbers of activation pattern with various number of activated groups can be realized. By laser irradiation discrete homogeneous structures as well as concentration gradients of peptides will be generated. To show the functionality of surface activation on the one hand the peptide RGD will be linked to the activated surface and on the other hand lactose as a model for glycoproteins will be linked.To allow a high activation without destroying of surfaces the ablation threshold and the process window for both materials and wavelengths is determined. For first activation fluency needs to reach a threshold. The experiments show that a higher irradiation dose activates higher numbers of reactive groups which will afterwards allow a higher density of functional groups. Above the ablation threshold materials become destroyed. Various products of PMMA and PCL occur after surface activation. While MMA occurs after PMMA irradiation, PCL shows no side products. In PCL the polymer chains are locally damaged but stay linked. Therefore the polymer surface enlarges with a higher number of active groups. In PMMA the surface is not enlarged. Therefore only a limited number of active groups can be achieved. Irradiation at a wavelength of 193 nm leads to ten times higher amount of active groups, which can be shown by XPS analysis and fluorescence staining experiments. Amines are covalently linked to surfaces activated with 193 nm irradiation, while those on surfaces activated with 248 nm are mainly non-covalently linked. Therefore these samples have to be washed with phosphate buffered saline (pH=7.4), while linking proteins to the surface.By irradiation of PMMA and PCL with 193 nm irradiation carboxylic groups occur. Both materials show a low optical penetration depth, and therefore less thermal effects. The penetration depth for 248 nm in PMMA is around 0.96 µm. This leads to thermal effects in PMMA and the formation of CO2-Groups, which leads to an unspecific activation. The wavelength 248 nm has a penetration depth of 25 µm in PCL which most probably induces thermal effects such as melting or cumulated photochemical effects.The activated and aminated surfaces can be used for functionalization with bioactive groups. RGD and Lactose can be linked to amine groups. Cell culture tests show that cells recognize the different regions of functionalization density. This shows that irradiation with laser is an efficient tool for production of tailored surfaces with controlled number of functional groups. Current methods use inflexible mask based irradiation systems. These systems can only structure linear patterns. The benefit of laser systems for surface activation is that whether the sample or the laser beam can be moved which give the opportunity of free choice of geometries. The process allows structuring of linear structures as well as dots with various functionalization density as well as free geometries in small areas. This allows the design of new miniaturized test beds which give the opportunity to test substances for new medicine in various numbers of concentrations. On the other hand free geometries for the functionalization of implant surfaces are possible to make them whether cell repellent or adhesive. The linkage of sugar in various concentrations could be a method to investigate the concentration dependent binding properties of glycoproteins. This could lead to new test systems for the recognition of disease markers.This study shows a method which allows the controlled local activation of PCL and PMMA by UV-Excimer irradiation. In future studies the process could be modified toward smaller machines based on smaller UV-Laser sources such as microchip laser. This could allow the development of new activation processes for the concentration dependent high-throughput testing.