You can manage bookmarks using lists, please log in to your user account for this.
Media type:
E-Book
Title:
Microfluidic device for the generation and replication of DNA microarrays and the label-free detection of biomolecular interactions on these arrays
University thesis:
Dissertation, Universität Freiburg, 2020
Footnote:
Description:
Abstract: The development of a microfluidic to generate and replicate DNA microarrays on standard microscope slides via solid phase polymerase chain reaction (spPCR) and the subsequent label-free measurement of biomolecule interactions on these microarrays is discussed in this thesis. Former works showed, that it is basically possible to generate DNA microarrays via solid phase PCR from NGS (next generation sequencing) chips, but neither replications of this DNA arrays could be generated nor where the used surfaces suitable for high- throughput label-free screening.<br>To generate multiple replicates in combination with label-free measurements, first a PDMS based, durable, robust and easy to handle microfluidic chip with thousands of small reaction chambers has been developed and a digital solid phase PCR protocol and workflow have been established. A batch-wise process which meets even high demand for molding of these chips is introduced. DNA test pools, containing two different DNA sequences were distributed in the small reaction chambers in a digital manner so that typically each cavity contains one single DNA strand or none. The microchambers are then sealed with a microscope glass slide. The flexible PDMS chip provides strong but reversible sealing during the polymerase chain reaction. PCR primer precoated glass slides as well as PDMS chips provide the possibility to immobilize the DNA strands via spPCR. Therefore, a DNA microarray is generated on the glass substrate, ready for label-free screening whereas the PDMS chip can be reused to make several replicates of this initial array onto more glass slides. As only one side of the DNA strand is covalently bound during the PCR reaction, it can be freely decided to use the DNA array as double strand DNA or single strand DNA array. Typically, microarrays are screened via fluorescence endpoint or ratiometric measurements. As such kinetic data are unavailable. Furthermore, the fluorescent labeling is expensive, time consuming and bears high risk of artefact generation. This disadvantages can be prevented with label-free detection, which normally needs an expensive high-tech device. Therefore, a novel implementation approach of a known label-free measurement system (single colour reflectometry = SCORE) was realized within a standard inverse fluorescent microscope. This approach allows to switch on demand from classical fluorescence to label-free kinetic measurement of molecular binding events.<br>The first milestone was to design different microfluidic cavity chips and to develop a method which allows the rapid production of the chips that are based on a PDMS / Aluminum composite. These chips have the advantage to be durable enough to withstand the harsh conditions of PCR cycling but are also able to seal the reaction chambers and prevent leakage in a reversible manner. The chips are cost- and material-e