The use of biosensors and microfluidics devices is often limited by constraints in terms of volumetric throughput due to the small dimensions of devices in microfluidics and of expensive and complicated sample preparation steps necessary to ensure the operation of biosensing platforms. This can be due to high initial sample volume with low concentration analytes or complex media matrices from which analytes are extracted. While working to analyse Cryptosporidium presence in drinking water a novel technique was developed. The huge advantages from using a label-free, buffer-free hydrodynamic mechanism in terms of cost, coupled with the ease of simply scaling a single design to match any target size and the ability manufacture these quickly and easily using cheap and readily available robust materials (i.e. acrylic sheet) may allow a revolution in the scope of microfluidics applications. Using a cascaded array of hydrodynamic focusing devices uniquely designed for parallelised operation from a single pump or pressure source, the array can be tailored to meet the specific requirements of many applications, in particular high volume and low concentration target analyte enrichment from complex media.

This thesis presents an analysis of the signal-to-noise ratio in liquid core anti-resonant reflecting optical waveguides (ARROWs) and the application of hydrodynamic focusing to the waveguides. These concepts are presented as a method to improve the detection capabilities of the ARROW platform. The improvements are specifically targeted at achieving single molecule detection (SMD) with the devices. To analyze the SNR of the waveguides a test platform was designed and fabricated. This test platform was then used to examine relationship between the SNR and the location of the excitation region. It was determined that the excitation region should be moved closer to the solid-core. By moving the excitation region closer to the solid-core the distance the signal was required to travel in the hollow-core was reduced. This reduction led to a decrease in optical signal loss and resulted in a more than 2x increase in the SNR. Hydrodynamic focusing in the waveguides was developed as a method to increase the consistency of detection of the devices. In hydrodynamic focusing particles in the sample are forced towards the center of the waveguide with a buffer solution. With the particles focused to the center of the channel the percentage that passed through the excitation region can be increased improving the detection consistency of the device. ARROW chips designed for hydrodynamic focusing were simulated, fabricated, and preliminary testing was performed. Initial results have shown a more than 30% increase in particle focusing.

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Microfluidic devices are ideal for analysis of biological systems. The small dimensions result to controlled handling of the flow profile and the cells in suspension. Implementation of additional forces in the system, such as an electric field, promote further manipulation of the cells. In this dissertation, I show novel, unique microfluidic approaches for manipulation and analysis of mammalian cells by the aid of electrical methods or the architecture of the device. Specifically, for the first time, it is shown, that adoption of electrical methods, using surface electrodes, promotes cell concentration in a microchamber due to isoelectric focusing (IEF). In contrast to conventional IEF techniques for protein separation, a matrix is not required in our system, the presence of which would even block the movement of the bulky cells. Electric field is, also, used to breach the cell membrane and gain access to the cell interior by electroporation (irreversible and reversible). Irreversible electroporation is used in a unique, integrated microfluidic device for cell lysis and reagentless extraction of DNA. The genomic material is subsequently analyzed by on-chip PCR, demonstrating the possible elimination of the purification step. On the other hand, reversible electroporation is used for the delivery of exogenous molecules to cells. For the first time, the effect of shear stress on the electroporation efficiency of both attached and suspended cells is examined. On the second part of my dissertation, I explore the capabilities of the architecture of microfluidic devices for cell analysis. A simple, unique method for compartmentalization of a microchamber in an array of picochambers is presented. The main idea of the device lies on the fabrication of solid supports on the main layer of the device. These features may even hold a dual nature (e.g. for cell trapping, and chamber support), in which case, single cell analysis is possible (such as single cell PCR). On the final chapter of my dissertation, a computational analysis of the flow and concentration profiles of a device with hydrodynamic focusing is conducted. I anticipate, that all these novel techniques will be used on integrated microfluidic systems for cell analysis, towards point-of-care diagnostics. / Ph. D.

Cellular growth, development, differentiation, and death are mediated to some degree by the interaction of soluble factors with plasma membrane receptors. Traditionally the cellular response to chemical cues has been studied by exposing entire culture dishes to a desired reagent. While the addition of soluble reagents homogenously to cell culture dishes provides a basis for understanding much of cell biology, greater spatial resolution of reagent delivery is necessary in order to elucidate mechanisms on the subcellular scale. This dissertation explores techniques that may improve the quality and precision of delivering soluble factors to cultured cells in order to better understand the complex processes of cell biology. These advancements were made possible by applying high intensity, focused laser light to soluble materials to achieve microscopic three-dimensional (µ-3D) printing. In combination with a previously developed microfluidic cell dosing platform, microstructures were designed and µ-3D printed to hydrodynamically focus reagent streams for cell dosing. Structures were also µ-3D printed within micrometers of living cells from a solution of gelatin and bovine serum albumin with minimal cytotoxicity. When µ-3D printed, these proteins displayed both temperature and pH-responsive properties. In order to allow for on-the-fly control of reagent stream size and temporal pulse width, microstructures were µ-3D printed from temperature-responsive N- isoproplyacrylamide. To further improve the temporal resolution of the system, a technique for cycling between reagents with millisecond exchange times using laminar flow microfluidics was developed. The utility of these techniques was demonstrated by staining rat Schwann cells and mouse neuroblastoma rat glioma hybrid cells (NG108-15) with focused streams of fluorescent dyes. These advancements may allow future experiments to determine the placement of soluble factors necessary for bacterial quorum sensing or stem cell differentiation. / text

The rise of superbugs, including antibiotic-resistant bacteria, and virus outbreaks, such as the recent coronavirus scare, illustrate the need for rapid detection of disease pathogens. Widespread availability of rapid disease identification would facilitate outbreak prevention and specific treatment. The ARROW biosensor microchip can directly detect single molecules through fluorescence-based optofluidic interrogation. The nature of the microfluidic channels found on optofluidic sensor platforms sets some of the ultimate sensitivity and accuracy limits and can result in false negative test results. Yet higher sensitivity and specificity is desired through hydrodynamic focusing. Novel 3D hydrodynamic focusing designs were developed and implemented on the ARROW platform, an optofluidic lab-on-a-chip single-molecule detector device. Microchannels with cross-section dimensions smaller than 10 μm were formed using sacrificial etching of photoresist layers covered with plasma-enhanced chemical-vapor-deposited silicon dioxide on a silicon wafer. Buffer fluid carried to the focusing junction enveloped an intersecting sample fluid, resulting in 3D focusing of the sample stream. The designs which operate across a wide range of fluid velocities through pressure-driven flow were integrated with optical waveguides in order to interrogate fluorescing particles and confirm 3D focusing, characterize diffusion, and quantify optofluidic detection enhancement of single viruses on chip.