Pancreatic islets are heavily vascularized micro-organs containing insulin secreting beta-cells coupled with endothelial cells (EC). These EC slowly deteriorate in static culture, precluding long term study of beta-cell-EC interaction, and likely limiting tissue revascularization post-transplantation. We postulate this EC deterioration is due to an absence of hemodynamics, blood movement. We created a microfluidic device to mimic aspects of hemodynamics, delivering a range of media flow to ex vivo islets. With our resulting desk-top system, we have conducted long term incubations (72 hrs), fixed tissue treatments (maintaining endothelial cell morphology) and real-time live tissue imaging (glucose-stimulated Ca2+-response). Our data show that flow in a microfluidic device maintains EC morphology in ex vivo islets better than non-flowing culture, providing an improved platform to study ex vivo islets and to examine the interaction between beta-cells and EC. Our data also suggest an opportunity to prime islet EC for revascularization using microfluidic flow prior to transplantation.

The specific membrane capacitance (SMC) of biological cell membranes correlates with cells’ electrical activity and morphology, which are physiological markers for cellular phenotype and health. Conventionally, SMC measurements are conducted using electro-rotation and Patch-clamping, which entail long time training and stringent operation skills. Both techniques also suffer from limited throughput and lengthy measurement time. In this study, a microfluidic device, which enables impedance spectroscopy measurements, was developed to quantify the SMC of single biological cells. The device has a testing speed of approximately one cell per minute and is relatively easy to operate. Three-dimensional finite element simulations of the microfluidic device confirm the feasibility of this approach. SMC measurement of two AML (Acute Myeloid Leukemia) subtypes and two UCC (Urothelium Cell Carcinoma) subtypes were conducted. Measured SMC results were found to lie in the comparable range with previously reported publications.

We have investigated and implemented several general strategies in the development of microfluidics-based chemical/biochemical sensing systems. The research in this dissertation covers the immobilization of biological reagents inside microfluidic channels using polystyrene (PS) microbeads and photopolymerizable hydrogel, electrochemical sensing via electrochemiluminescence (ECL) reporting with bipolar and two-electrode configurations, and integration of these general functions to realize multiplexing and networking on-chip. Photopolymerizable hydrogel based on Poly(ethylene glycol) (PEG) and streptavidin-coated polystyrene (PS) microbeads were employed as building blocks as well as functional components in microfluidic system. PEG hydrogels can be used to define local microenvironments at different locations in the same microchannel, which enables the introduction of multiple sensing events on the same device. Monitoring of DNA hybridization and enzyme/substrate interaction were realized thereafter by using either fluorescence or electrochemistry as the detection method. Electrogenerated chemiluminescence based on Ru(bpy)32+ (bpy = 2,2??-bipyridine) and tripropylamine (TPA) was used to photonically report various redox events in microfluidic systems. By using microfluidic electrochemical cells based on either two-electrode or bipolar electrode (one-electrode), electroactive species that undergo reduction can be electrically linked to this anodic ECL process and thus be reported by the latter. This ECL sensing scheme essentially broadens the spectrum of redox compounds that can be detected by ECL since the analytes are not required to directly participate into the light-generating processes. Microfluidics offers some unique technical advantages of performing electrochemistry over conventional methods. In particular, laminar flow allows multiple analyte streams to be brought together in parallel with little mixing. Moreover, electrochemical signals can be generally utilized as a convenient means to link individual microchannels together hence to realize microfluidic networking and cross-communication. Electrochemical microfluidic devices can be used to mimic general functions of microelectronic devices such as diodes, transistors, and logic gates. These novel functions rendered by electrochemistry are believed to bring us closer to the final goals of micro total analysis systems and lab-on-a-chip.

Novel microscale velocity and temperature measurement techniques were studied based on confocal laser scanning microscopy (CLSM) and optical serial sectioning microscopy (OSSM). Two microscopic measurement systems were developed, 1) a CLSM micro particle image velocimetry (PIV) system with a dual Nipkow disk confocal unit (CSU-10), a CW argon-ion laser and an upright microscope, and 2) an OSSM micro- particle tracking velocimetry (PTV) system with an epi-fluorescence microscope and a non-designed specimen to make a three-dimensional (3-D) diffraction particle image. The CLSM micro-PIV system shows a unique optical slicing capability allowing true depth-wise resolved vector field mapping. A comparative study is presented between the CLSM micro-PIV and a conventional epi-fluorescence micro-PIV. Both have been applied to the creeping Poiseuille flows in two different microtubes of 99-µm (Re = 0.00275) and 516-µm ID diameters (Re = 0.021). The CLSM micro-PIV consistently shows significantly improved particle image contrasts, the definition of "optical slicing" and measured flow vector fields more accurately agreeing with predictions based on the Poiseuille flow fields, compared to the conventional micro-PIV. The OSSM micro-PTV technique is applied for a 3-D vector field mapping in a microscopic flow and a Brownian motion tracking of nanoparticles. This technique modifies OSSM system for a micro-fluidic experiment, and the imaging system captures a diffracted particle image having numerous circular fringes instead of an in-focus particle image. The 3-D particle tracking is based on a correlation between the 3-D diffraction pattern of a particle and the defocus distance from a focal plane. A computational program is invented for the OSSM micro-PTV, and provides a 3-D velocity vector field with a spatial resolution of 5.16 µm. In addition, a concept of nonintrusive thermometry is presented based on the correlation of the Brownian motion of suspended nanoparticles with the surrounding fluid temperature. Detection of fully three-dimensional Brownian motion is possible by the use of the OSSM, and the measured value of mean square displacement (MSD) is compared fairly well with Einstein's predictions.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

No description available.

The impedance response of a quadrupolar microelectrode array was studied over a wide frequency range to determine whether particles captured at the center of the array could be detected impedimetrically. The microelectrode array (denoted as DEP chip) uses dielectrophoretic forces to concentrate particles at its center. Initial results showed that there was a large electrode-silicon-electrode (ESE) capacitance which dominated at high frequencies. This capacitance was reduced by decreasing the electrode area and increasing the insulating layer thickness. These measures however proved fruitless as this capacitance was still significantly greater then the dielectric capacitance of the chip. This ESE capacitance can be eliminated through the use of a glass substrate so that the dielectric response of the chip dominates at higher frequencies. Since the ESE capacitance prevented experimental validation of impedance spectroscopy as a signal transduction method, computer simulations were performed. These simulations indicated that capture with the current DEP chips would not have a significant impact on the impedance of the chip. Decreasing the electrode gap distance and reducing the area of the electrodes, which is recommended for future work, can remedy this. As measureable changes in the dielectric capacitance of the chip are not possible, a reaction scheme was developed to translate the capture of viral particles into a change in medium conductivity. An ELISA type system was proposed where the viral particles would be functionalized with urease. This uease would then be used to degrade non-ionic urea into ionic products thereby increasing the medium conductivity. A model was formulated to predict the conductivity increase expected for low concentrations, and validated using higher concentrations of biotinylated-urease. Urease from commercial sources proved not to be a viable option as it does not possess a high enough activity to produce a significant conductivity change given the low concentrations of viral particles expected after collection. Urease with suitable activity is produced by the organism Ureaplasma urealyticum which has an activity of 180 000 µmol urea catalyzed min-1 mg urease-1. It is not recommended that this method be pursued further due to technical challenges that would be encountered. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-05-20 02:35:15.678

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 68-71). Also available on the World Wide Web.

Thesis (doctoral)--Helsinki University of Technology, 2007. / Includes bibliographical references. Also available on the World Wide Web.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references. Also available on the World Wide Web.