The development of a microbead array for the detection and amplification of nucleic acids
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The focus of this doctoral thesis is on the development of a chip-based sensor array, composed of individually addressable agarose micro-beads, that is suitable for the real-time detection of DNA oligonucleotides. This research is consistent with recent trends in disease diagnostics following the miniaturization and integration of sample preparation and measurement steps towards portable devices capable of point of care analysis. Thus, the power and utility of this microbead array methodology for DNA detection is demonstrated here for the analysis of fluids containing a variety of similar short oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10,000 and limit of detection values of ~10-13 M are obtained readily with this microbead array system. These analytical characteristics, here exhibited are competitive with some of the best direct DNA detection methodologies before reported. As an extension of this work, an integrated self quenching based sensing system within the bead format has shown clear efficacy for the detection of HIV gag isolates and Bacillus anthracis (Sterne) purified strains and allows for the rapid detection of 100bp sequences with sensitivities in the subnanomolar range. Additionally, due to the tailored immobilization of specific sequences on each sensor element, the multiplexed detection of various sequences utilizing diverse strategies has been demonstrated. Use of the micro-bead array in tandem with the hybridization capabilities of molecular beacons, constitutes a powerful tool for the heterogeneous elucidation of specific sequences. Concomitantly, successful collaboration with the Chen group on the development of a miniaturized enzyme based nucleic acid amplification device has been reported. Purified strains of Bacillus anthracis (Sterne) have been successfully amplified by the miniaturized polymerase chain reaction (PCR) chip as seen by gel electrophoresis. One of the long term aims of this general area of research will be to couple the glass micro chip-based PCR amplification of oligonucleotides with the real-time detection capabilities of a bead based array. These efforts serve to establish some precedent for the bead-based microfluidics approach to be implemented in the context of genomics testing for the next generation of health care.