Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 98-101).

Every environment of the biosphere supports a particular mix of microorganisms called a microbiome. These diverse microbial communities play critical roles in the health of ecosystems and in higher organisms, including humans. Disruption or translocation of microbiomes may cause lethal infections, contaminate food and drug supplies, and adversely impact industrial activities. Microbiome detection and molecular characterization have emerged as priorities in many fields. Available methods cannot quickly and efficiently extract rare microorganisms in real specimens. Therefore, microbial detection and analysis require long incubation periods or the use of technically challenging molecular biotechnologies. These strategies are impractical in situations requiring immediate intervention. The intrinsic electric and dielectric properties of microbes permit their isolation by the phenomenon of dielectrophoresis in microfluidic devices. These microsystems have the potential to enhance microbial analysis but are plagued by low processing rates and the inability to interface with biological specimens containing high levels of interfering cells and debris. In this study, a method was created to discriminate between target microbes and undesired cells on the basis of their differential susceptibility to permeabilizing agents that altered cell dielectrophoretic responses. Fabrication techniques were developed to manufacture high-aspect ratio microfluidic channels that allowed the physical forces of gravity, diffusion and dielectrophoresis to be exploited to control cell positions over microscale distances normal to a Poiseuille flow gradient. Because the positioning effects were exploited in only one dimension, the other two dimensions of the channels could be scaled up to create large channel cross-sectional areas that supported rapid specimen processing rates while maintaining high separation efficiencies expected for the microscale effects. These strategies were applied in various ways to isolate microbes from whole blood, platelets, stool, saliva, and skin specimens. The dielectrophoretic extraction of microbes enabled by this approach was used to enable electrical impedance detection of ~100 bacteria in less than five hours. As a result, important technological barriers that have limited the applicability of dielectrophoresis in clinical and industrial settings were overcome by increasing throughput and addressing sample preparation requirements. These proof-of-concept data demonstrate the potential for accelerating microbial isolation and detection in diagnostics, screening, and microbiome research. / text

Early work in electrowetting on dielectric (EWOD) devices has demonstrated their great potential in microfluidics; however, further work is needed to integrate EWOD technology into a system deployable for point-of-care (POC) diagnostics. This research is aimed at providing enabling technologies that foster a development path of EWOD devices using a process similar to the development of application-specific integrated circuits (ASICs). A field-programmable lab-on-a-chip (FPLOC), which allows designers to electrically program the prefabricated chip into EWOD applications, was fabricated and demonstrated based on novel microelectrode dot array (MEDA) architecture. The MEDA architecture proposes a standard EWOD component called “microelectrode cell”, which can be dynamically configured into microfluidic components to perform microfluidic operations of the biochip. The FPLOC is the first EWOD biochip fabricated by the standard low-voltage complementary metal-oxide-semiconductor (CMOS) technology, which allows smooth on-chip integration of microfluidics and microelectronics. A total of 900 droplet detection electrical circuits were integrated into the chip and a real-time droplet location map could show shapes and locations of all droplets on the chip. The daisy-chained control structure of the MEDA architecture allowed individual control of 900 microelectrodes by only using three control pads. This control structure was also leveraged to add the built-in self-test (BIST), which was proven to be very useful in diagnosing the chip, of the FPLOC. The FPLOC successfully demonstrated seamless hierarchical field-programmability. Compared to conventional bottom-up and full-custom design approaches, the FPLOC brings microfluidic technology closer to POC diagnostics by providing biochip designers with CAD support at a level similar to that of the semiconductor industry, without the time-consuming and costly process of hardware design, testing, and maintenance.

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Herein is presented the collective experimental record of research performed in the Laboratory for Biological Surface Science. These investigations are generally classified under the category of bioanalytical surface science and include the following projects. Chapters III and IV describe the creation of a microfluidic device capable of generating fixed arrays of concentration gradients. Experimental results were matched with computational fluid dynamics simulations to predict analyte distributions in these systems. Chapters V and VI demonstrate the discovery and utility of photobleaching fluorophores for micropatterning applications. Bleached fluorophores were found to rapidly attach to electron rich surfaces and this property was used to pattern enzymes inside microfluidic channels in situ. Finally, Chapter VII exhibits a method by which solid supported lipid bilayers can be dried and preserved by specifically bound proteins. The intrinsic property of lateral lipid mobility was maintained during this process and a mechanism by which the protein protects the bilayer was suggested.

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Lab‐On‐a‐Chip (LOC) or Micro Total Analysis System (μTAS) technology requires precise control of minute amounts of liquid. Moving liquids in small capillaries requires bulky expensive external pumps that defy the purpose of microfabrication. By integrating a micropump into the device, it allows the system to be transportable, reliable, energy efficient and inexpensive. Such a microsystem built on a chip has been designed to study separation by dielectrophoretic chromatography. Nanobeads were successfully separated and used separately to measure fluid velocity and study the electroosmosis effect. Cell or beads of different type can be trapped in this system. This system encompasses a solid‐state AC electroosmotic pump for the manipulation of liquid‐containing cells or molecules. AC Electroosmosis is the movement of induced charges over polarised electrodes created by a non‐uniform electric field. The charges undergo Coulomb forces and drag the fluid with their motion. This results in bulk flow over the electrodes. This micro pump is used in a LOC by fabricating the pump on two sides of a microfluidic channel. The transport of material from what can be an analyte to a cell is of critical interest. The described system in the second part of this thesis presents the advantage of having a defined number of droplets, each of which is a lab on chip. The paradigm is the droplet and therefore the vessel that carries the information. Surfaces are then the place of interaction with the vessel which carries the second aspect of this thesis. Several approaches have been investigated, in particular by enclosing the droplet between two slides in order to increase the change of contact angle under the presence of polarised electrodes. This system is known as EWOD (ElectroWetting On Dielectric). It follows the approach of modified Lippmann laws and the modification of the apparent contact angle and therefore the motion of the droplet. The lid is somewhat a problem and the possibility of using liquid dielectrophoresis to create a multitude of droplets of calibrated volume is an advantage, as it is harder to create fixed‐volume droplets with an open geometry by EWOD due to contact angle hysteresis.