Signalling states of cells are heterogeneous even within clonally derived populations due to cell cycle status and their local microenvironment. As a result multiplexed single-cell signal transduction measurements represent a powerful tool which could potentiate a much greater understanding of subcellular processes. However, multiplexed single-cell analysis remains challenging due to several factors, most notably the low copy number of analytes present, difficulties in cellular manipulation and the availability of well characterized and stable probes for use in intact cells. In order to address these issues, a capillary electrophoresis system with laser induced fluorescence (CE-LIF) suitable for screening methods to facilitate single cell analysis was designed and assembled. This system has the requisite sensitivity for single-cell analysis, with the capability of detecting down to 10000 molecules of fluorescein, and has been designed to reduce the time required for analyte separations compared to similar systems by integrating a compact detection module which allows for shorter electrophoretic separation distances. This system has been employed to develop a method to determine the sampling efficiency of laser-based cell lysis of single cells allowing more accurate quantitative measurements of fluorescent peptides from single cells. Furthermore, a fluorescent probe based on amyloid precursor protein (β-APP peptide) has been designed and conditions were found which allowed resolution of enzyme-modified versions within single cells. To identify the enzymatic conversion products produced, a novel method was developed employing bulk cell samples in conjunction with LC-MS. As a testament to the resolution afforded by this technique, the peptide fragments identified from single cells represented peptides which differed by single uncharged amino acids. Together, the methods here developed are able to provide higher quality quantitative data and more informative analysis of fluorescent signal transduction reporters in single cells and represents progress towards being able to obtain highly multiplexed data needed for accurate cellular models.

Signalling states of cells are heterogeneous even within clonally derived populations due to cell cycle status and their local microenvironment. As a result multiplexed single-cell signal transduction measurements represent a powerful tool which could potentiate a much greater understanding of subcellular processes. However, multiplexed single-cell analysis remains challenging due to several factors, most notably the low copy number of analytes present, difficulties in cellular manipulation and the availability of well characterized and stable probes for use in intact cells. In order to address these issues, a capillary electrophoresis system with laser induced fluorescence (CE-LIF) suitable for screening methods to facilitate single cell analysis was designed and assembled. This system has the requisite sensitivity for single-cell analysis, with the capability of detecting down to 10000 molecules of fluorescein, and has been designed to reduce the time required for analyte separations compared to similar systems by integrating a compact detection module which allows for shorter electrophoretic separation distances. This system has been employed to develop a method to determine the sampling efficiency of laser-based cell lysis of single cells allowing more accurate quantitative measurements of fluorescent peptides from single cells. Furthermore, a fluorescent probe based on amyloid precursor protein (β-APP peptide) has been designed and conditions were found which allowed resolution of enzyme-modified versions within single cells. To identify the enzymatic conversion products produced, a novel method was developed employing bulk cell samples in conjunction with LC-MS. As a testament to the resolution afforded by this technique, the peptide fragments identified from single cells represented peptides which differed by single uncharged amino acids. Together, the methods here developed are able to provide higher quality quantitative data and more informative analysis of fluorescent signal transduction reporters in single cells and represents progress towards being able to obtain highly multiplexed data needed for accurate cellular models.

A microfluidic device, based on electrophoretic transport and electrostatic trapping of charged particles, has been developed for continuous capture and concentration of microorganisms from water. A generic design, utilizing mobility and zeta potential measurements of various microorganisms exposed to different environmental conditions and physiological states, was employed. Water and buffer samples at pH values ranging from 5.2–7.0 were seeded with bacteria (E. coli, Salmonella, and Pseudomonas) and viruses (MS-2 and Echovirus). Negative control and capture experiments were performed simultaneously using two identical devices. Both culture based methods and real-time PCR analysis were utilized to characterize the capture efficiency as a function of time, flowrate, and applied electric field. Based on differences between the capture and negative control data, capture efficiencies of 90% to 99% are reported for E. coli, Salmonella, Pseudomonas, and MS-2, while the capture efficiency for Echovirus was around 75%. Overall, the device exhibits 16.67 fold sample volume reduction within an hour at 6 mL/hr. This results in a concentration factor of 15 at 90% capture efficiency. Direct quantification of capture on the anode of the prototype microfluidic device was also performed by particle tracking using fluorescent microscopy. Based on image processing, the capture data at different locations on the electrode surface is quantified as a function of the wall shear stress at these locations, which is calculated using CFD simulations. Finally, the Faradaic processes in the microchannel due to electrochemical reactions are studied to predict the amount of electrophoresis in the system. Scaling of the device to sample 5 L/hr can be achieved by stacking 835 identical microchannels. Power and wetted volume for the prototype and scaled devices are presented. The device can thus function either as a filtration unit or as a sample concentrator to enable the application of real-time detection sensor technologies. The ability to continuously sample water without chemical additives facilitates the use of this device in drinking water distribution systems. This work constitutes the first step in our development of a continuous, microbial capture and concentration system from large volumes of potable water.

Isoelectric trapping (IET), has proven to be one of the most successful electrophoretic techniques used for separations of ampholytic compounds. IET is carried out in multicompartment electrolyzers (MCEs) in which adjacent compartments are joined through buffering membranes whose pH values bracket the pI of the ampholytic component to be trapped in the compartment. The present small-scale instruments use plastics as their structural materials, which causes poor Joule heat dissipation. The separation compartments have cylindrical or pear-shaped interiors with large internal diameters, which create long heat transfer paths. The long electrode distances yield low field strengths that lead to low electrophoretic velocities for the analytes. These factors interrelatedly limit the electric power that can be applied to the system, contributing to long separation times. Furthermore, these devices do not offer a realistic solution to the problems associated with the detection of low abundance proteins. To address these problems, two novel IET devices have been developed for small-scale IET separations. The first device, named MSWIFT, was constructed using thermally conductive, high-purity alumina as the structural material of the separation compartments. By creating narrow, 0.1- or 0.2-mL channels in thin alumina blocks, the heat transfer path from the center of the compartment to the wall was significantly decreased; and the distance between electrodes was greatly shortened. MSWIFT achieved 6 to 50 times faster IET separations compared to other MCEs. The second device, named ConFrac, was developed to simultaneously fractionate and concentrate ampholytic components from a complex sample into 0.1-mL collection compartments. By designing a system with a 2-dimensional pH gradient and allowing recirculation of the sample feed, the ConFrac demonstrated enrichment of analytes by a factor of 100 and greater.

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Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 127-134).