Entwicklung eines miniaturisierten elektrophoretischen Analysensystems auf Keramikbasis zur Bestimmung von Polyphenolen.

A substantial goal of this work was the development of a LTCC (low temperature co-fired ceramic) microchip as a capillary electrophoresis separation system for „lab on a chip“- applications. A device could be constructed with its assistance electroactive substances and can be amperometric detected after capillary electrophoretic separation in LTCC microchips. It could be shown that mixtures up to three polyphenols are separable due to their migration times and electro-chemically detection. Compared to a published LTCC system (Henry et. al, 1999) the standard deviation of the calculated migration times and effective electrophoretic mobilities were about 7 to 10% higher. A further disadvantage were the large peak widths. These disadvantages can possess numerous causes: 1) Due to laminating the working electrode cannot be positioned exactly before the capillary exit. Contraction effects increase this aspect. If the position of the electrode is too close to the exit, fluctuation in the separation voltage and reduction in the sensitivity can be observed. If the distance is too large diffusion effects appear, which contribute to band broadening. A new sinter technology might be an improvement. The contraction possesses a small influence on three-dimensional structures. Another option would be the integration of the electrochemical measure unit. To decouple the electrical field a appropriate method would be the integration of a platinum decoupler into the channel. 2) Another reason for the smaller separation efficiency might be the size of the sample zone. The use of µTAS (micro total analysis systems) applications will need a adapted high voltage device. Based on this the electrokinetic injection had been modified, so that only few nano-litres of the sample solution were needed. Nevertheless, band broadening and a small resolution of the peaks were observed. With the pinched injection a slow change of the potentials can lead to a large sample zone. Then the sample is not separated in discrete, exactly defined zones. Probably it dependeds on construction effects, which cause a running after of the sample solution from the side capillaries. Microscopic photographs showed that the edges were strongly off rounded in the crossing zone of the capillaries. An accurate potential separation is no longer possible thereby between the individual capillaries. By mechanical stamping of the capillaries in particular it comes to uneven wall structures after laminating and sintering in the crossing zone. This can lead also to an influence of the electroosmotic flow. By the use of new laser technology (NeodymYAB laser) these inhomogeneities should be excluded. The samples were injected by an electrokinetic method and detected amperometric. Depending on the used polyphenol the detection limit was between 12,5 µmol and 100 mM. Band broadening leads to a decrease in separation efficiency. Although, a mixture of dopamine, pyrogallol and gallic acid could be clearly separated, amperometrically detected in LTCC microchips. Migration time and mobility were comparable with the individual measurements. As well as the height and width of the peaks. For the detection of polyphenols in matrix-rich samples a higher specificity is necessary. In order to reach this, attempts were accomplished for the integration of phenol oxidizing enzymes into the microfluidic separation system. Model systems were compiled, which serve for a later immobilization on a gold electrode. The functionality of immobilized enzymes were tested photometrically with suitable substrates. Depending on the used enzyme different surfaces were suitable to immobilize these without influencing the substrate binding side. During the immobilization on not functionalised gold prisms the highest absorptions were partly measured. However the stability of the connection, in particular the use in a flow system is to be considered. The photometric measurements of the enzyme activity with the help of a color based assay was applicable with all immobilization methods. LTCC is suitable as material for the development of „lab on a chip “- applications. The properties of the material are comparable to glass but the production is less easier and expensive. Thus, this low sintering ceramic is an ideal substitution of existing microchip applications. The tests which were carried out are very promising. Changes in the production and construction of LTCC microchips should minimize the mentioned disadvantages. The sales market in the medical and pharmaceutical industry is enormous and the need of small, transportable analytical devices with simple operation will continue to increase in the next few years.