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Title: Flow-through microchannel DNA chips
Author: Benoit, Vincent
ISNI:       0000 0001 3460 197X
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
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This work represents a contribution to the rapidly expanding field of microarray technology, which consists in the pooling of a large number of miniaturised biorecognition elements on a single solid substrate so as to allow heterogeneous bioassays to be performed in a highly multiplexed fashion. Numerous applications of life science research and pharmaceutical development, such as diagnostics and molecular medicine, are expected to greatly benefit from the increased levels of throughput provided by the microarray approach. However, most microarray platforms developed as of this writing have merely been based on a two-dimensional configuration in which the biorecognition sites are distributed on the surface of a planar, unpenetrable substrate; this format does not adress the mass-transfer limitations inherent to heterogeneous ligand binding kinetics and thus does not provide optimal assay performance in terms of speed and sensitivity. In this thesis, a novel concept is presented that has been devised to increase the intrinsic analytical performance of microarrays. The approach is based on the use of three-dimensional, uniformly porous substrates featuring a regular array of discrete, ordered, high aspect ratio microchannels. The fabrication of such microstructures, with feature sizes in the micrometre range and aspect ratios in excess of 30:1, was achieved using two subtractive silicon processing techniques, electrochemical etching and deep dry etching, whose relative merits have been assessed. Commercially available microchannel glass substrates were also used. Nucleic acid molecules were immobilised as capture probes on the sidewalls of the microchannels, in a conformation allowing biorecognition through heterogeneous hybridisation. To that end, a chemical strategy was developed for the functional immobilisation of oligonucleotide probes onto silica-rich substrates such as glass and oxidised silicon. Nucleic acid hybridisation was chosen as the model ligand binding process due not only to the relative ease of handling of DNA (including the availability of chemically pure, synthetic oligonucleotides) but also to its relevance, through genomics, to a number of clinical, medical and pharmaceutical applications. The particular wetting properties of microchannel substrates were characterised and subsequently taken into account in the development of a microarraying procedure based on non-contact, ink-jet dispensing technology. In this respect, the physical-chemical processes that control the formation of spots on microchannel substrates, i.e. the wetting properties of the substrates, as well as the main parameters involved in the arraying process, such as the concentration of the capture probe solution and volume of the aliquots dispensed, have been investigated. Medium-density microchannel oligoarrays were produced that comprised as many as 256 probe spots over a 1 cm2 area. Since the effective surface area available for probe immobilisation was increased by expansion into the third dimension, a higher spatial density of probes could be achieved as compared to planar substrates with the same lateral dimensions. Biorecognition events between the immobilised capture probes and solution-borne target molecules were allowed to take place, in a dynamic mode, by convectively pumping sample through the microchannel array. As each microchannel acted as a miniature analytical chamber, probe-target hybridisation kinetics were enhanced by spatial confinement, while recirculation of the sample provided enhanced mass transfer. The feasibility of using electrokinetic transport of the targets was also assessed. Quantitative detection of heterogeneous hybridisation events taking place along the microchannel sidewalls was achieved through a CCD camera-based epifluorescence detection scheme, in a first instance, conventional low magnification microscope objective lenses were used as imaging optics whose depth-of-field characteristics matched the thickness of the microchannel substrate. The suitability of the approach was illustrated by the achievement of detection limits as low as a few attomoles of fluorescent dye per spot. This level of performance was made possible by the remarkable optical properties of the substrate, which were characterised both experimentally and through simulations based on ray-tracing procedures. Since the use of microchannel chips of increased thickness provided a way of increasing the number of capture probe molecules immobilised within each spot without increasing the lateral dimensions of the spots, a range of chip thicknesses were considered in a first step towards optimisation of the substrate geometry. Potential limitations to the benefits associated with an increase in chip thickness were discussed, in terms of the effectiveness of both the fluorescence detection scheme and the process of probe immobilisation on the microchannel sidewalls. Comparative hybridisation experiments were conducted which demonstrated the improved performance of the flow-through microchannel configuration over the conventional planar configuration, with an observed circa 5 times enhancement in hybridisation kinetics and a one to two orders of magnitude higher detectability. A few routes for further improving the performance of flow-through microchannel DNA chips are suggested, along with other possible bioanalytical applications of the microchannel biochip platform.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochips; DNA microarray technology