Hybrid Microfluidic Devices For On-Demand Manipulation and Screening of Neurons and Organs of Small Model Organisms

Caenorhabditis elegans and Drosophila melanogaster are widely used model organisms for neurological and cardiac studies due to their simple neuronal and cardiac systems, genome similarity to humans, and ease of maintenance in laboratories. However, their 50m-1mm sizes and continuous mobility impede their precise spatiotemporal manipulation, thereby, reducing the throughput of biological assays. By integrating glass capillaries into microfluidic devices and using 3D-printed fixtures for precise control, we have developed hybrid lab-on-a-chip devices to facilitate the processes of animal manipulation and stimuli control, using modules for single-organism selection, orientation, imaging and chemical stimulation. These microdevices enabled us to manipulate organisms individually and to orient them at any desired direction for imaging purposes. The applications of these hybrid microdevices were demonstrated in the optical and fluorescent imaging of C. elegans cells as well as cardiac screening of Drosophila larvae. This technique can be applied in fundamental biology, toxicology, and drug discovery

High-throughput microfluidic assay devices for culturing of soybean and microalgae and microfluidic electrophoretic ion nutrient sensor

In the past decade, there are significant challenges in agriculture because of the rapidly growing global population. Meanwhile, microfluidic devices or lab-on-a-chip devices, which are a set of micro-structure etch or molded into glass, silicon wafer, PDMS, or other materials, have been rapidly developed to achieve features, such as mix, separate, sort, sense, and control biochemical environment. The advantages of microfluidic technologies include high-throughput, low cost, precision control, and highly sensitive. In particular, they have offered promising potential for applications in medical diagnosis, drug discovery, and gene sequencing. However, the potential of microfluidic technologies for application in agriculture is far from being developed. This thesis focuses on the application of microfluidic technologies in agriculture. In this thesis, three different types of microfluidic systems were developed to present three approaches in agriculture investigation. Firstly, this report a high throughput approach to build a steady-state discrete relative humidity gradient using a modified multi-well plate. The customized device was applied to generate a set of humidity conditions to study the plant-pathogen interaction for two types of soybean beans, Williams and Williams 82. Next, a microfluidic microalgal bioreactor is presented to culture and screen microalgae strains growth under a set of CO2 concentration conditions. C. reinhardtii strains CC620 were cultured and screened in the customized bioreactor to validate the workability of the system. Growth rates of the cultured strain cells were analyzed under different CO2 concentrations. In addition, a multi-well-plate-based microalgal bioreactor array was also developed to do long-term culturing and screening. This work showed a promising microfluidic bioreactor for in-line screening based on microalgal culture under different CO2 concentrations. Finally, this report presents a microchip sensor system for ions separation and detection basing electrophoresis. It is a system owning high potential in various ions concentration analysis with high specificity and sensitivity. In addition, a solution sampling system was developed to extract solution from the soil. All those presented technologies not only have advantages including high-throughput, low cost, and highly sensitive but also have good extensibility and robustness. With a simple modification, those technologies can be expanded to different application areas due to experimental purposes. Thus, those presented microfluidic technologies provide new approaches and powerful tools in agriculture investigation. Furthermore, they have great potential to accelerate the development of agriculture

Advanced Microfluidic Assays for Caenorhabditis elegans

The in vivo analysis of a model organism, such as the nematode Caenorhabditis elegans, enables fundamental biomedical studies, including development, genetics, and neurobiology. In recent years, microfluidics technology has emerged as an attractive and enabling tool for the study of the multicellular organism. Advances in the application of microfluidics to C. elegans assays facilitate the manipulation of nematodes in high-throughput format and allow for the precise spatial and temporal control of their environment. In this chapter, we aim to illustrate the current microfluidic approaches for the investigation of behavior and neurobiology in C. elegans and discuss the trends of future development

Master of Science

thesisTwo microfluidics devices are presented, which are used to further study of the Caenorhabditis elegans worm (C. elegans). The first device is a tool for ranking the muscle force between mutant and wild type strains of worms. The second device is a screening chip, which is designed to decrease the amount of time needed to screen the C. elegans worm, and does not require the use of anesthetics to immobilize them for imaging. The muscle force tool operates by compressing a worm in a microchannel with a flexible Polydimethylsiloxane (PDMS) membrane. The force the membrane exerts on the worm is determined by air pressure controlled using a sensitive regulator. The method mimics the natural environment of the worm, where it must move through soil. Worms are tested by loading them into the chip and air pressure against the membrane is increased in increments until the worm becomes immobilized. To rank strains of worms according to muscle force, the pressures at which the worms become immobilized are compared. The chip operates on the hypothesis that a higher pressure indicates a greater muscle contraction force. The chip was tested using three strains of worms: a wild type and two strains with genetic knockouts of specific ion channels at the neuromuscular junction. Immobilization pressures are given for each strain. The screening chip is designed to be operated on a confocal microscope, and is used for taking high magnification images and videos of the worms. To perform the screening process, the chip separates a single worm form a solution containing many worms using a tapered channel and filter. Second, the worm is immobilized for imaging using a flexible PDMS membrane, which compresses the worm against a cover slip. Third, the worm is transported to one of two holding containers. Worm movement through the device is controlled on screen through a custom computer program. This work discusses the design, fabrication and testing results of the microfluidic chips

Monitoring single heart cell biology using lab-on-a- chip technologies

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states

Microfluidic in vivo laser microsurgery screen for identification of compounds enhancing neural regeneration

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 41-42).Discovery of small molecules and novel mechanisms for enhancing neurite regeneration in animal models is significant for therapeutics of central nervous system injuries and neurodegenerative disorders. C. elegans is a widely studied model organisms due to their fully mapped neural network of 302 neurons and amenable genetics. Their small size and short life cycle allows for rapid studies to be conducted; however, after decades of use the manual methods of manipulation have still remained unchanged. This thesis details the development of automated, high-throughput optical and microfluidic technologies for screening C. elegans and demonstrates the production of a reliable system for screening over ten thousand animals. Using the screening system, femtosecond laser microsurgery was performed on thousands of animals followed by incubation in compounds from a chemical library. The screens revealed several high-scoring drug candidates that enhance regeneration after laser microsurgery of C. elegans mechanosensory neurons.by Cody Lee Gilleland.S.M

Microfuidic Devices and Open Access Tool for Localized Microinjection and Heart Monitoring of Drosophila Melanogaster

This thesis aims to address the current research gaps associated with the use of Drosophila larvae as an in-vivo model for cardiac toxicity and cardiac gene screening. In objective 1, we have developed a hybrid multi-tasking microfluidic platform that enables desired orientation, reversible immobilization, and localized microinjection of intact Drosophila larvae for recording heart activities upon injection of controlled dosages of different chemicals. In objective 2. we have developed software for in-vivo quantification of essential heartbeat parameters on intact Drosophila larvae. Several image segmentation and signal processing algorithms were developed to detect the heart, extract the heartbeat signal, and quantify heart rate and arrhythmicity index automatically, while other heartbeat parameters were quantified semi-automatically using the M-mode. In objective 3a, we demonstrated the application of our microfluidic device and heartbeat quantification software for investigating the effect of different chemicals (e.g., serotonin and heavy metals) on Drosophila larval heart function. Also, we applied our technology to genetically modified Drosophila larvae to investigate the effect of metal responsive transcription factor (MTF-1) against heavy metals cardiac toxicity (objective 3b)

Microfuidic Devices and Open Access Tool for Localized Microinjection and Heart Monitoring of Drosophila Melanogaster

This thesis aims to address the current research gaps associated with the use of Drosophila larvae as an in-vivo model for cardiac toxicity and cardiac gene screening. In objective 1, we have developed a hybrid multi-tasking microfluidic platform that enables desired orientation, reversible immobilization, and localized microinjection of intact Drosophila larvae for recording heart activities upon injection of controlled dosages of different chemicals. In objective 2. we have developed software for in-vivo quantification of essential heartbeat parameters on intact Drosophila larvae. Several image segmentation and signal processing algorithms were developed to detect the heart, extract the heartbeat signal, and quantify heart rate and arrhythmicity index automatically, while other heartbeat parameters were quantified semi-automatically using the M-mode. In objective 3a, we demonstrated the application of our microfluidic device and heartbeat quantification software for investigating the effect of different chemicals (e.g., serotonin and heavy metals) on Drosophila larval heart function. Also, we applied our technology to genetically modified Drosophila larvae to investigate the effect of metal responsive transcription factor (MTF-1) against heavy metals cardiac toxicity (objective 3b)