Molekularbiologische Affinitätsstudien : neue Anwendungen elektrophoretischer, miniaturisierter Trennsysteme

Molecular biological affinity studies is a field with an abundance of interesting
analytical methods. The following work summarises the current methods in a short
overview before focussing on the electrophoretic measurements. They belong to an
active area of research, which is suitable for a large spectrum of applications. The
possibility to transfer affinity studies from capillary to a miniaturised system – the chip
– is the main aim of this work. In the age of high throughput screening, fast new
analytical methods are required and miniaturised systems, such as the
electrophoretic chip, become a common demand.
Up-to-now the application of EMSA (electrophoretic mobility shift assay) was only
used on capillary. Here it was successfully transferred for the first time to a planar
miniaturised system. In order to characterise the microchip, known systems
(neurotransmitters and cyclodextrins) have been measured. As for the binding
parameters, the measurements proofed that chips deliver the same results as the
capillary, however in a shorter time and with less material consumption.
Apart from measurements with neurotransmitters, studies with artificial receptors and
peptides, with single-stranded DNA and metal cations have been accomplished. The
results of the binding affinities have been compared to calorimetric measurements
and confirmed the quality of the ACE (affinity capillary electrophoresis)
measurements on chip. By choosing certain oligonucleotide sequences and suited
buffer systems, the binding selectivity of metal ions for tetranucleotides could be
shown. Considering the building of binary and tertiary complexes, which depends on
the kind of buffer molecules, the binding constants and the amount of binding
partners were calculated. Although not the same number of measurements has been
compiled for the microchip as for the capillary, in principal the suitability of the chip to
examine non-covalent bindings to oligonucleotides and DNA strands was shown.
Methodical specialities as well as qualitative differences between
measurements on the capillary and on the chip have been observed and
documented. In some areas of research, important for electrophoretic ACE
measurements, chips are superior to capillary. The smaller amount of probes and
buffer and the shorter separation time, which allows the measurement of instable
systems, all speak in favour of the chip. Due to the significantly shorter separation
times, the phenomenon of diffusion has a distinctly smaller impact on the peak
broadening. This improves the separation efficiency. High separation efficiency is
important for ACE analytics, especially so when the separation is done in seconds.
The worse sensitivity of the chip could only be put down to technical peculiarities and
less efficient detection systems of the chips used here. Although capillaries with
normal separation lengths deliver qualitatively better results at the moment, the
microchip will be superior to the classical capillary electrophoresis as soon as reliable
injection methods are available and the steering of probe and separation media are
solved, additional to its present advantages.