Application of Micro- and Nanotechnologies in Cytogenetics
Chromosome abnormalities, such as translocations may cause various genetic disorders and are also associated with heametological malignancies. Translocation is a rearrangement between two chromosome arms that results in two derivative chromosomes. The current detection methods such as karyotyping and FISH require a use of expensive reagents and can only be performed in specialized laboratories. This PhD project aims at developing new strategies for point-of-care detection of chromosome translocations by applying micro- and nanotechnologies to increase the sensitivity. The project started with development of a microfluidic device for controlled chromosome spreading. The device, made in Topas®, was used to facilitate the evaporation of the fixative solution to achieve proper spreading. In the device we obtained a comparable spreading efficiency to the traditional methods but with reduced reagents volume. To propose a new strategy for chromosome translocation detection we developed a double hybridisation assay. To detect the translocation it is necessary to determine that the two DNA sequences forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The first example of the translocation detection was presented on lab-on-a-disc using uorescently labeled DNA fragments, representing the derivative chromosome. It allows for sorting of the DNA chromosomal fragments into separate compartments followed by translocation detection. To allow for cheaper detection an electrical label-free approach has been investigated using silicon nanowires BioFETs, metallic and conductive polymer electrodes. We present here our findings regarding the DNA hybridisation sensing using these sensors. They showed an improved sensitivity and are all label-free, which makes them inexpensive candidates for a novel cytogenetic analysis as a replacement of standard techniques. The metallic electrodes as the most reliable were selected for further development of a complete device for translocation detection. We developed a setup enabling electrochemical measurements on a spinning lab-on-a-disc platform. An electrical swivel was used to provide reliable connections between the electrodes on a disc and a potentiostat. We have demonstrated the applicability of the setup to standard electrochemical techniques. Lab-on-a-chip devices are being constantly developed to improve the analysis at a reduced cost and time. The presented devices that were developed using microand nanotechnologies are label-free and due to their sensitivity show a potential to be applied to chromosome translocation analysis with an improved detection efficiency.