Date of Award
Doctor of Philosophy
Electrical and Computer Engineering
The concept of in vivo screening of whole animals that model diseased conditions, in contrast to in vitro tests on cell cultures and tissues that may not translate to clinical trials, is now gaining wide-spread acceptance. One model whole animal, Caenorhabditis elegans, has been extensively studied to understand molecular mechanisms of ageing, cell death, development, and neuronal signaling. Because of its small size, this worm is especially suited for microfluidic systems having microscale geometries, regulated fluid flow, and imaging options.
In this thesis, a survey of some microfluidic systems for C. elegans research is first presented (Chapter 1). Methods of capturing and restraining single worms, exposing them to chemical stimuli (e.g. gases, drugs, toxicants), and reading signals from the pharynx and neurons are discussed. The following chapters describe our work on understanding worm behavior in different microenvironments using a combination of microfluidics, real-time imaging, and computer-controlled data collection. The engineering tools developed in this work are aimed to be simple in operation/handling, reliable and robust, information rich, portable for easy transport, and requiring minimal human intervention.
We designed a series of sinusoidal microchannels with fixed wavelength and modulating amplitude to study the locomotion patterns of C. elegans (Chapter 2). The sinusoidal microchannels attempt to mimic the physical nature of soil and test the worms' ability to bend their bodies during navigation. The simple, passive locomotion assay is able to differentiate the wild-type worms from mutants showing uncoordinated or quasi-uncoordinated movement.
The natural worm movement changes upon exposure to chemicals or toxins (e.g. hydrogen cyanide). Exposure to gaseous hydrogen cyanide kills wild-type C. elegans; however, deletion of specific genes confers varying levels of resistance to the worms. We designed a microfluidic device to characterize the toxicity of aqueous potassium cyanide and conferred resistance in mutants lacking specific genes (Chapter 3). Results from microfluidic experiments were consistent with those from gas assay experiments.
The above platform was employed to test the effectiveness of four commercially available drugs known to paralyze the worm's neuromuscular system (Chapter 4). We observed interesting phenotypic differences in each drug environment, suggesting that an optimal combination of the four drugs may be more effective than individual drugs, even at lower dosage. We used an algorithmic search method, provided by Dr. Chih-Ming Ho, to find this winning drug cocktail through searching 32 combinations. The idea of creating superior-performing cocktails from existing drugs using algorithmic search, in contrast to discovering new drugs using biologically-driven hypotheses, is compelling for the pharmaceutical industry.
Besides pharmacology, the non-parasitic C. elegans is a widely accepted model in parasitology as most parasitic worms have complex life-cycles and impractical for imaging in their natural environments. These challenges lured us to design a microfluidic platform for growing Arabidopsis plants and imaging live roots within microchannels (Chapter 5). After 7-days period, the roots were inoculated with plant-parasitic worms and imaged for another 10 days. Unlike previous microfluidic platforms designed for short-term experiments, this system showed the possibility of conducting very long-term experiments in microfluidics.
Several aspects of the presented devices and tracking program have been adopted by researchers working on other parasites. Depending on the parasite under study, the original chip dimensions and geometry were altered for specific needs. The tracking program now has a graphic-user-interface for easy video capture, compression, and post-processing. In addition, we are regularly striving to make our system simple and user-friendly so that the developed techniques can be transferred to biology laboratories.
Parashar, Archana, "Microfluidics and real-time behavioral analysis: Tools for screening drugs, toxins, and pathogens" (2013). Graduate Theses and Dissertations. 13527.