The development of microdevices for applications related to bioanalysis is described. There are two types of microdevices involved in this study: DNA (or RNA) microarrays and bead-based microfluidic devices. First, a new method to fabricate DNA microarrays is developed: replication of DNA microarrays. It was shown that oligonucleotides immobilized on a glass master can hybridize with their biotin-modified complements, and then the complements can be transferred to a streptavidinfunctionalized replica surface. This results in replication of the master DNA array. Several innovative aspects of replication are discussed. First, the zip code approach allows fabrication of replica DNA arrays having any configuration using a single, universal master array. It is demonstrated that this approach can be used to replicate master arrays having three different sequences (spot feature sizes as small as 100 [mu]m) and that master arrays can be used to prepare multiple replicas. Second, it is shown that a surface T4 DNA polymerase reaction improves the DNA microarray replication method by removing the requirement for using presynthesizd oligonucleotides. This in-situ, enzymatic synthesis approach is used to replicate DNA master arrays consisting of 2304 spots and arrays consisting of different oligonucleotide sequences. Importantly, multiple replica arrays prepared from a single master show consistent functionality to hybridization-based application. It is also shown that RNA microarrays can be fabricated utilizing a surface T4 DNA ligase reaction, which eliminates the requirement of modified RNA in conventional fabrication schemes. This aspect of the work shows that the replication approach may be broadly applicable to bioarray technologies. A different but related aspect of this project focuses on biosensors consisting of microfluidic devices packed with microbeads conjugated to DNA capture probes. The focus here is on understanding the parameters affecting the hybridization of DNA onto the probeconjugated microbeads under microfluidic flow conditions. These parameters include the surface concentration of the probe, the flow rate of the solution, and the concentration of the target. The simple microfluidic device packed with probe-conjugated microbeads exhibits efficient target capture resulting from the inherently high surface-area-to-volume ratio of the beads, optimized capture-probe surface density, and good mass-transfer characteristics. Furthermore, the bead-based microchip is integrated with a hydrogel preconcentrator enhancing the local concentration of DNA in a icrochannel. The integration of the preconcentrator into the bead-based capture chip allows significantly lower limit of detection level (~10-fold enhancement in the sensitivity of the microbeadbased DNA detection). / text

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An alternating current electroosmotic micro pumping device has been designed, experimentally tested and theoretically analyzed using an electrohydrodynamic theoretical model applied to a computer simulation model. The device SP-1 is a microelectrode array which uses the principal of AC electroosmosis (EO), ions driven along microelectrode surfaces by coulomb forces produced by tangential electric fields. These ions, when driven, induce a net fluid motion due to viscous drag forces. Three submerged microelectrode wires were deposited on a substrate using microfabrication techniques such that a square spiral geometry was formed. Device SP-1 received asymmetrically applied AC signals creating a travelling wave of potential and resulted in a net fluid flow across the microelectrode array. Microsphere tracer particles were suspended in ethanol to measure the fluid velocity to determine pumping performance and the experimental operating frequency at which maximum fluid velocity is achieved. The experimental results were reviewed and at an AC signal frequency of 125 Hz, device SP-1 was capable of pumping ethanol at a fluid velocity of approximately 270 μm/s. The experimental results were in good agreement with the theoretical predictions produced using the computer simulation model. In addition, the computer simulation model predicted a similar flow profile to those previously predicted and experimentally observed. Overall, novel micropumping device SP-1 was found to produce a net flow comparable to previously tested devices and a computer simulation framework capable of analyzing future micropump design concepts was developed. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2011-04-01 17:12:02.908

No description available.

Bioartificial Liver (BAL) is a term for medical devices designed to replace natural liver functions. The idea behind the use of artificial livers is to either externally support an injured liver to recovery or bridge a patient with a failing liver to transplantation. Central to all BAL systems is a bioreactor for culturing liver cells. The main function of this reactor is to provide a cell adhesion matrix and supply the necessary nutrient solution. A high cellular oxygen uptake rate combined with low solubility in aqueous media makes oxygen supply to the liver cells the most constraining factor in current reactor designs. Devices with parallel-plate channel geometry promise high efficiency for blood detoxification and liver metabolism. However, due to their specific flow regime oxygen depletion in the medium is a major problem in these devices. This thesis explores a unique method of controlling and measuring dissolved oxygen in BAL cell-culture bioreactors and lab-on-a-chip devices. Testing is performed using simulations, prototype bioreactor devices and in-vitro measurement of dissolved oxygen. Several strategies developed to fabricate the bioreactors and integrate oxygen sensing are presented. Emphasis is placed on techniques that provide compatibility with commonly used microfabrication processes, while allowing for laterally-resolved measurement of oxygen in a re-usable, low-cost setup. The most significant contribution presented is the development and assessment of the tapered cell-culture bioreactor with integrated PtOEPK/PS oxygen sensor. The combination adopts a unique approach to oxygen control. Bioreactor shape is used to modulate the oxygen supplied to cells via the resulting shear-stress function. By linearly increasing the shear-stress oxygen concentration can be maintained constant over the length of the reactor. Using the integrated oxygen sensor, the resulting concentration profile can be monitored in real-time with high lateral resolution. The advantage of the device over existing techniques is that no additional oxygenation inside the reactor chamber is required to maintain a certain concentration profile and that oxygen concentration can be mapped in-situ without having to introduce further chemicals into the perfusion medium. This thesis presents a number of other contributions: a grayscale mask process, development of the PtOEPK/PS sensor patterning method and signal optimization regime, demonstration of the multi-stream flow application, an experimental setup for sensor calibration and a process to pattern cell-adhesion proteins simultaneously with the oxygen sensor, a multi-layer BAL prototype and the results of a brief experiment to test an approach using vertically aligned carbon nanotube bundles as fluidic conduits for bile drainage.

Electrospray Ionization (ESI) is a technique that permits the soft ionization of large proteins and biomolecules without fragmenting them, which allows them to be characterized via Mass Spectrometry (MS). It has the potential of permitting the identification of transient intermediate products in biological processes in situ, which would provide great insight to researchers in the growing fields of proteomics and metabolomics. However, this application presents a technical challenge in that most relevant biochemistry occurs in aqueous solutions with high salt content, which makes successful identification of analytes by ESI-MS difficult. This thesis presents the design, fabrication, and characterization of a microfabricated dialysis module that could alleviate this issue by desalting samples inline between sampling and electrospraying interfaces. Its small volume (~10 nL) minimizes sample transit time and, thus, optimizes ESI-MS analysis temporal resolution. A preliminary analytical model of dialysis elucidates the key performance parameters and sets the guidelines for consideration in its design. The device is then microfabricated in a cleanroom environment using techniques that have been well established by the microelectronics industry such as E-beam evaporation and Reactive Ion Etching. The system efficiency is demonstrated experimentally by assessing its salt removal effectiveness as a function of sample residence time. Mass spectrometry analyses of proteins in solutions with high salt content further corroborate its performance.

T-lymphocytes (T-cells) trafficking in the lymph nodes (LNs) is key for T-cells activation and their effector functions in adaptive immune responses. T-cells enter the LNs through high endothelial venules (HEVs) and interact with dendritic cells (DCs) for cognate antigens in the T-cell zone (TCZ). After scanning the TCZ for antigens, T-cells leave the LNs through efferent lymphatic vessel. CCR7 and its ligands, CCL19 and CCL21 are involved in the recruitment and compartmentalization of T-cells in LNs. However, their specific role(s) in mediating T-cells migration in LNs sub-regions remain unclear. In addition, the mechanism behind the passage of T-cells from the TCZ to the abluminal side of medullary sinuses (for their exit through medullary sinuses) is not well understood. Here, I hypothesize that different CCL19 and CCL21 fields in LNs sub-regions, orchestrate T-cells sub-regional migration in LNs.. In this study, I examined the CCL19 and CCL21 distribution profiles in mouse LNs sub-regions by immunofluoroscence staining and confocal microscopy. Using microfluidic devices that can flexibly configure well-defined single and co-existing chemical concentration gradients, I quantitatively analyzed the migration of activated human blood T-cells in LNs relevant CCL19 and CCL21 fields. The results suggested a novel CCL19 and CCL21 based combinatorial guiding mechanism for T-cells migration in different LNs sub-regions. In particular, this mechanism operates in the TCZ periphery region to guide T-cells migration away from the TCZ. Furthermore, the CCL19 and CCL21 fields mimicking the region beyond the TCZ toward the medulla result in disturbed chemotaxis, which prevents T-cells from being attracted back to the TCZ. Taken together, this microfluidics-based in vitro study shows the coordinated T-cells migration in different single and combined CCL19 and CCL21 fields, leading to interesting new insights into the guiding mechanisms for T-cells trafficking in LNs sub-regions.

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Stem cells hold great promise for treating various degenerative diseases. However, the outcomes of preclinical and clinical cell therapy studies are still not close to our expectation. The unsatisfactory outcomes of cell therapy are at least partially due to: 1) insufficient homing of implanted stem cells into target organs and 2) use of heterogeneous cell populations for cell therapy. Therefore, there is a need to develop effective guiding technique for stem cells to migrate to the target organs and to isolate effective stem cell populations. In this project, I developed a microfluidics-based method for selecting chemotactic adipose-derived stem cells (ASCs) to epidermal growth factor (EGF). This method integrates cell patterning, chemotaxis and cell extraction on a single microfluidic device. Post-extraction analysis confirmed the higher chemotactic ability of the extracted cells to EGF. The extracted chemotactic ASCs shows up-regulated surface expression of EGF receptor and its downstream signaling event upon EGF stimulation.

No description available.