Microfluidics engineering: a lab-on-chip bioassay for in vivo nematode testing
Carr, John Anthony (2010) Microfluidics engineering: a lab-on-chip bioassay for in vivo nematode testing. Masters thesis, Iowa State University.
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The nematode is a microscopic worm belonging to the phylum Nematoda. With over 2200 described genera in about 250 families, the nematode is among the most diverse of all pseudocelomates. These worms are the most ubiquitous multicellular organisms on earth and are crucial for maintaining soil nutrients and overall symbiotic relationships between plants and certain organisms. However, as many as 33% of the estimated 40,000 nematode species have been classified as parasitic. Of particular interest to the farming community, nematode parasites can infect plants (e.g. corn, soybean and wheat), animals (e.g. pigs, sheep, goats and cows) and even humans, causing illness and severe agricultural loss. Conventional control methods based on chemotherapy face a major challenge as nematodes are developing resistances to the known nematocides. As new resistant isolates emerge and new drugs are developed to control them, there is a great need for improved methods of screening resistance and determining dose response. In this thesis, a microfluidic platform for screening drugs and their dose response on the locomotive behavior of parasitic nematodes is presented. The system offers reduced experimental time, higher sensitivity, and, for the first time, real-time observation of drug effects at a single worm resolution. The presented lab-on-chip bioassay can be reliably used to identify changes in multiple locomotion parameters and to determine exposure effects as a function of time. Existing nematode motility and migration assays do not offer such a level of sophistication. The device comprises two principal components: (i) microchannels to study nematode motility during the pre- and post-exposure periods of the experiment and (ii) a drug well for administering the dose and studying drug effects at different exposure times. The drug screening experiment can be described by three main phases: (i) ‘pre-exposure study’ - worms are inserted into the microchannels and their locomotion is characterized, (ii) ‘dose exposure’ – worms are guided from the microchannels into the drug well and exposed to a dose for a predefined time and (iii) ‘post-exposure study’ – worms are guided back into the microchannels where their locomotion is characterized and compared to pre-exposure motility. We demonstrate the workability of the microfluidic platform on the parasitic Oesophagotomum dentatum (levamisole sensitive, SENS and levamisole resistant, LEVR) using levamisole as the test drug. The proposed scheme of drug screening on a microfluidic device is expected to significantly improve the resolution, sensitivity and throughput of in vivo nematode testing, while offering new details on the real-time exposure effects of new and existing anthelmintics. A second project, ‘the electrotactic nematode gate’, is presented as a byproduct of the aforementioned lab-on-chip bioassay. Current microfluidic methods for gating (i.e. opening or closing a certain pathway to) nematode movement use a pinch type or polymer membrane valve. Although effective, these valves are generally large static structures, lack the potential for automation and, in some cases, require a multistep molding process. The electrical gate presented in this thesis advantageously uses the electrotactic response of nematodes to generate a dynamic, microscale gate that can be easily programmed and integrated into an automated bioassay. The gate requires only two electrode ports that have a separation larger than the nematodes’ penetration depth (10-300 μm depending on species) and can therefore be fabricated as a single-mold microfluidic device. It is expected that the presented device will help to streamline new and existing bioassays, especially in the Caenorhabditis elegans community.
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