Thesis (Ph. D.)--Auburn University, 2009. / Abstract. Vita. Includes bibliographical references (p. 168-174).

Thesis--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 78-81).

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 61-63). Also available on the World Wide Web.

Biological systems of bacteria were investigated at the single-cell and single-molecule level. Additionally, aspects of the techniques employed were studied. A unifying theme in each project is the reliance on optical imaging techniques coupled to microfluidic devices. Hypo-osmotic shock experiments with an Escherichia coli mechanosensitive channel deletion mutant were carried out at the single-cell level. E. coli MJF465 cells in which the three major mechanosensitive channel genes are deleted (∆mscL, ∆mscS, ∆mscK) show only 10% cell viability upon hypo-osmotic shock (from LB + 0.5 M NaCl into distilled water), compared to 90% viability of the wild-type strain. Bacterial cells were trapped with optical tweezers in microfluidic devices, enabling the first direct observation of single-cell behaviour upon hypo-osmotic shock. Phase-contrast microscopy revealed intra-population diversity in the cells response: Different features of lysis included cells bursting rapidly and leakage of ribosomes, DNA and protein from the cytoplasm. Fluorescence microscopy of hypo-osmotically-shocked GFP-expressing MJF465 cells showed either bursting of cells, which was a rare event, or fast leakage of GFP, indicating cell membrane ruptures. Data were analysed in terms of their kinetic behaviour and showed that lysis occurs on a timescale of milliseconds to seconds. The implications of these findings for the bacterial cell wall and cell membranes are discussed. Enzymes involved in homologous recombination and repair of double-stranded DNA (dsDNA) breaks are essential for maintaining genomic integrity in both eukaryotes and prokaryotes. RecBCD of E. coli and AddAB, found widely in bacteria, are involved in these processes, carrying out the same function. Both enzymes were studied kinetically with single-molecule total internal reflection fluorescence microscopy (TIRFM). Surface-tethered, hydrodynamically stretched lambda-DNA molecules, stained with YOYO-1, were imaged with TIRFM in a microfluidic flowcell. The RecBCD enzyme is a well characterised DNA helicase and was introduced to this system for method validation purposes. The AddAB enzyme of Bacteroides fragilis was then characterised as a helicase acting on lambda-DNA. It was found that AddAB helicase unwinds dsDNA with high processivity of on average 14,000 bp and up to 40,000 bp for individual enzyme complexes at an ATP-dependent rate ranging from 50-250 bp s−1 (for Mg2+-ATP concentrations larger or equal than 0.1 mM). This activity was detected by DNA binding dye (YOYO-1) displacement from the dsDNA and studied for different Mg2+-ATP concentrations, flow (shear) rates and different YOYO-1 staining ratios of DNA. Aspects of this last experimental setup were investigated. A kinetic analysis of intercalation of YOYO-1 into lambda-DNA is presented, occurring on a timescale of minutes. Different flow rates and staining ratios that influence the apparent (stretched) DNA molecule length were also examined. Several image analysis techniques were employed to enhance the data quality in images showing stretched lambda-DNA molecules. The Singular Value Decomposition was found to be the most effective technique which strongly reduces the noise in the obtained kymograph images.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Microfluidic devices have a wide variety of biological applications. My Ph.D. dissertation focuses on three major projects. A) culturing a non-adherent immortal cell line within a microfluidic device under static and dynamic media flow conditions; B) designing and fabricating novel microfluidic devices for electrokinetic injecting analytes from a hydrodynamic fluid; and C) using this novel injection method to lyse single non-adherent cells by applying a high electric field across the cell at a microfluidic channel intersection. There are several potential advantages to the use of microfluidic devices for the analysis of single cells: First, cells can be handled with care and precision while being transported in the microfluidic channels. Second, cell culturing, handling, and analysis can be integrated together in a single, compact microfluidic device. Third, cell culturing and analysis in microfluidic devices uses only extremely small volumes of culturing media and analysis buffer. In this dissertation a non-adherent immortal cell line was studied under static media flow conditions inside a CO[subscript]2 incubator and under dynamic media flow conditions in a novel portable cell culture chamber. To culture cells they must first be trapped on a microfluidic device. To attempt to successfully trap cells, three different types of cellular traps were designed, fabricated and tested in polydimethylsiloxane (PDMS)-based microfluidic devices. In the first generation device, cubic-shaped traps were used. After 48 h of culturing in these devices the cell viability of 79 [plus or minus] 6 % (n = 3). In the second generation device, circular wells with narrow connecting channels were employed. However, after 12 h of culturing, no viable cells were found. While the second generation device was not capable of successfully culturing cells, it did demonstrate the importance of culturing under dynamic conditions which lead to next design. The third generation microfluidic device consisted of hydrodynamic shaped traps that were used to culture the cells in a less confined environment. The cell viability after 12 h in this design was 29 [plus or minus] 41% (n = 3). In addition to cell trapping, a novel electrokinetic injection method was developed for injecting analytes from a hydrodynamic flow into a separation channel that was followed by an electrokinetic separation. As the hydrodynamic flow could introduce some excess band broadening in the separation, the actual band broadening of an analyte was measured for different channel depths and hydrodynamic fluid flow rates. The results consistently showed that the separations performed on these devices were diffusion limited. Finally, using this novel injection method, single cell lysis was performed by applying a high voltage at the microfluidic channel intersection. The results of these studies may eventually be applied to help answer some fundamental questions in the areas of biochemistry and pharmaceutical science.

Point-of-care (POC) tests can improve the management of infectious diseases and clinical outcomes, through prompt diagnosis and appropriate delivery of treatments for preventable and treatable diseases, especially in resource-limited settings where health care infrastructure is weak, and access to quality and timely medical care is challenging. Microfluidics or lab-on-chip technology is appropriate for POC tests when general design constraints such as integration, portability, low power consumption, automation, and ruggedness are met. Although many POC tests have been designed for use in developed countries, they might not be readily transferable to resource-limited settings. These new technologies need to be accessible, affordable and practical to be implemented at resource-limited settings to save lives in developing countries. The overall goal of this dissertation is to develop microfluidic diagnostic devices which are practical and reliable for global health. We first focused on immunoassays, an important class of diagnostic tests which utilize antibodies to quantify host immunity or pathogen protein markers. We developed and evaluated a rapid, accurate, multiplexed, and portable microfluidic immunoassay for diagnosis of HIV and syphilis on hundreds of archived specimens (whole blood, plasma, and sera). Our assay exhibited performance equal to lab-based immunoassays in less than 20 minutes. In addition, our technique quantified signals using a handheld instrument, allowing for objective measurements as opposed to current rapid HIV tests which require subjective interpretation of band intensities. We further integrated three important off-chip processes in a diagnostic test - liquid handling, optical signal detection, and data communication – in a low-cost, versatile, handheld instrument capable of performing immunoassays on reagent-loaded (i.e. “ready-to-run”) cassettes at high analytical performance characteristic of ELISA but with the speed, portability and ease-of-use of a rapid test. We also evaluated this immunoassay device in Rwanda on archived samples and achieved analytical performance comparable to that of benchtop standards. To simplify the user interface and reduce the cost of the diagnostic device, we integrated our microfluidic immunoassay with a smartphone to replace computers or high-cost processors for diagnostic devices in low-resource settings. Our low-cost ($34), smartphone-supported device for a multiplexed immunoassay detected three antibody markers from HIV, treponemal- and non-treponemal syphilis from fingerstick whole blood simultaneously in 15 minutes. This device was designed to eliminate the number of manual steps, through the use of lyophilized secondary antibodies and anti-coagulant, preloaded reagents on cassette, and an automatic result readout. A step-by-step user guide was included on the smartphone to make the device simple enough to be used by an untrained operator. The analytical performance of the device was evaluated in Rwanda by local health care workers. We also accessed user experiences for improvement of the device in future. While immunoassays offer rapid and accurate diagnosis for infectious diseases, various infections cannot be confirmed using protein markers. Due to increasing clinical demand for detection of DNA and RNA signatures for diagnosis and monitoring of patients in resource-limited settings, we also explored how microfluidic and nanoparticle technologies can improve nucleic acid amplification test at the point of care. Nucleic acid tests are arguably some of the most challenging assays to develop due to additional steps required for sample pre-treatment (e.g. cell sorting, isolation, and lysis, as well as nucleic acid extraction), signal amplification (due to low physiological concentrations, target contamination, and instability) and product detection. Here we developed a sputum processor to isolate and lyse mycobacteria (M.smegmatis) from a more complex sample matrix, using magnetic beads-based target isolation to replace the need of a centrifuge or other complicated sample preparation technique. We also investigated a technique to detect Mycobacterium tuberculosis using multiplex polymerase chain reaction (PCR) and silver-gold amplification detection.

Precision medicine represents a shift in medicine where large datasets are gathered for massive patient groups to draw correlations between disease cohorts. An individual patient can then be compared to these large datasets to determine the best treatment strategy. While electronic health records and next generation sequencing techniques have enabled much of the early applications for precision medicine, the human genome only represents a fraction of the information present and important to a person’s health. A person’s proteome (peptides and proteins) and glycome (glycans and glycosylation patterns) contain biomarkers that indicate health and disease; however, tools to detect and analyze such biomarkers remain scarce. Thus, precision medicine databases are lacking a major source of phenotypic data due to the absence of available methods to explore these domains, despite the potential of such data to allow further stratification of patients and individualized therapeutic strategies. Available methods to detect non-nucleic acid biomarkers are currently not well suited to address the needs of precision medicine. Mass spectrometry techniques, while capable of generating high throughput data, lack standardization, require extensive preparative steps, and have many sources of errors. Immunoassays rely on antibodies which are time consuming and expensive to produce for newly discovered biomarkers. Aptamers, analogous to antibodies but composed of nucleotides and isolated through in vitro methods, have potential to identify non-nucleic acid biomarkers but methods to isolate aptamers remain labor and resource intensive and time consuming. Recently, microfluidic technology has been applied to the aptamer discovery process to reduce the aptamer development time, while consuming smaller amounts of reagents. Methods have been demonstrated that employ capillary electrophoresis, magnetic mixers, and integrated functional chambers to select aptamers. However, these methods are not yet able to fully integrate the entire aptamer discovery process on a single chip and must rely on off-chip processes to identify aptamers. In this thesis, new approaches for aptamer selection are developed that aim to integrate the entire process for aptamer discovery on a single chip. These approaches are capable of performing efficient aptamer selection and polymerase chain reaction based amplification while utilizing highly efficient bead-based reactions. The approaches use pressure driven flow, electrokinetic flow or a combination of both to transfer aptamer candidates through multiple rounds of affinity selection and PCR amplification within a single microfluidic device. As such, the approaches are capable of isolating aptamer candidates within a day while consuming <500 µg of a target molecule. The utility of the aptamer discovery approach is then demonstrated with examples in precision medicine over a broad spectrum (small molecule to protein) of molecular targets. Seeking to demonstrate the potential of the device to generate probes capable of accessing the human glycome (an emerging source of precision medicine biomarkers), aptamers are isolated against gangliosides GM1, GM3, and GD3, and a glycosylated peptide. Finally, personalized, patient specific aptamers are isolated against a multiple myeloma patient serum sample. The aptamers have high affinity only for the patient derived antibody.

Chau, Long Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 73-82). / Abstract also in Chinese. / ABSTRACT --- p.III / 摘要 --- p.IV / PUBLICATIONS CORRESPOND TO THIS THESIS --- p.V / ACKNOWLEDGEMENTS --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.XI / ABBREVIATIONS AND NOTATIONS --- p.XII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.1 / Chapter 1.1.1 --- Types of Biofuel Cells --- p.1 / Chapter 1.1.2 --- Properties of Using Enzymes in Bio-energy Generation Systems --- p.2 / Chapter 1.1.3 --- Application of Bio-energy Generation Systems --- p.3 / Chapter 1.2 --- Objectives of the Project --- p.4 / Chapter 1.3 --- Organization of the Thesis --- p.5 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.7 / Chapter 2.1 --- Working Principle of a Typical Fuel Cell --- p.7 / Chapter 2.2 --- Introduction of Enzymes and Co-enzymes --- p.9 / Chapter 2.3 --- Functions and Activities of Glucose Dehydrogenase (GDH) --- p.10 / Chapter 2.4 --- Functions and Activities of Laccase --- p.11 / Chapter 2.5 --- Introduction of Carbon Nanotubes (CNTs) --- p.12 / Chapter 2.6 --- Introduction of Gold Nanoparticles (AuNPs) --- p.13 / Chapter 2.7 --- Introduction of PdNPs --- p.14 / Chapter 2.8 --- Summary of Literature Review --- p.15 / Chapter CHAPTER 3 --- WORKING PRINCIPLE OF AN ENZYMATIC BIOFUEL CELL --- p.16 / Chapter 3.1 --- Enzymatic Biofuel Cell Using Glucose as a Fuel --- p.16 / Chapter 3.2 --- Deterministic Factors of the Fuel Cell´ةs Performance --- p.19 / Chapter 3.3 --- Energy --- p.22 / Chapter 3.3 --- Chapter Conclusion --- p.23 / Chapter CHAPTER 4 --- ENZYMATIC BIOFUEL CELL DESIGN --- p.24 / Chapter 4.1 --- Engineering Structure of the EBFC --- p.24 / Chapter 4.2 --- Chemical Structures of the EBFCs --- p.25 / Chapter 4.2.1 --- 1st Structure of EBFC - Au-Ll-CNTs-Ll-AuNPs-L2-{(GDH-NAD)/Laccase} --- p.26 / Chapter 4.2.2 --- 2nd Structure of EBFC - Au-Ll-CNTs-Ll-AuNPs-L2-{GDH/Laccase} --- p.28 / Chapter 4.2.3 --- 3rd Structure of EBFC- Pd-Ll-CNTs-Ll-AuNPs-L2-{(GDH-NAD)/Laccase} --- p.28 / Chapter 4.2.4 --- 4th Structure of EBFC - Pd-Ll -A uNPs-L2-{(GDH~NAD)/Laccase} --- p.29 / Chapter 4.2.5 --- 5th Structure of EBFC- Au-Ll-CNTs~L4'{(GDH-NAD)/Laccase} --- p.30 / Chapter 4.2.6 --- 6th Structure ofEBFC 一 Au-Ll-CNTs-{L3- NAD-GDH/L4-Laccase} --- p.31 / Chapter 4.3 --- Chapter Conclusion --- p.33 / Chapter CHAPTER 5 --- FABRICATION AND CHARACTERIZATION OF EBFCS --- p.34 / Chapter 5.1 --- Materials Preparation --- p.34 / Chapter 5.1.1 --- Preparation of Linker 1 --- p.34 / Chapter 5.1.2 --- Preparation of Linker 2 --- p.35 / Chapter 5.1.3 --- Preparation of Linker 4 --- p.35 / Chapter 5.1.4 --- Purification of Linkers --- p.35 / Chapter 5.1.5 --- Verification of Linkers --- p.36 / Chapter 5.2 --- 3-D Micro Electrode Fabrication --- p.37 / Chapter 5.3 --- Electrode Modification --- p.40 / Chapter 5.3.1 --- 1st Structure of EBFC --- p.40 / Chapter 5.3.2 --- 2nd Structure of EBFC --- p.41 / Chapter 5.3.3 --- 3rd Structure of EBFC --- p.41 / Chapter 5.3.4 --- 4th Structure of EBFC --- p.42 / Chapter 5.3.5 --- 5th Structure of EBFC --- p.42 / Chapter 5.3.6 --- 6th Structure of EBFC --- p.42 / Chapter 5.4 --- Characterization --- p.43 / Chapter 5.4.1 --- Atomic Force Microscopy (AFM) --- p.43 / Chapter 5.4.2 --- Scanning Electron Microscopy (SEM) & Energy-Disperse X-ray Spectroscopy (EDX) --- p.46 / Chapter 5.4.3 --- Cyclic Voltammetry (CV) --- p.47 / Chapter 5.5 --- Chapter Conclusion --- p.52 / Chapter CHAPTER 6 --- RESULTS OF EBFCS --- p.53 / Chapter 6.1 --- Experimental Setup --- p.53 / Chapter 6.2 --- Results --- p.55 / Chapter 6.2.1 --- Results of 1st EBFC --- p.55 / Chapter 6.2.2 --- Results of 2nd EBFC --- p.57 / Chapter 6.2.3 --- Results of 3rd EBFC --- p.58 / Chapter 6.2.4 --- Results of 4th EBFC --- p.60 / Chapter 6.2.5 --- Results of 5th EBFC --- p.60 / Chapter 6.2.6 --- Results of 6th EBFC --- p.65 / Chapter 6.3 --- Chapter Conclusion --- p.67 / Chapter CHAPTER 7 --- CONCLUSION --- p.69 / Chapter 7.1 --- Conclusion --- p.69 / Chapter 7.2 --- Future Work for the Biofuel Cell Project --- p.70 / Chapter 7.2.1 --- Study the Effect of Temperature Change --- p.70 / Chapter 7.2.2 --- Study the Effect of the Change of pH in Substrates --- p.70 / Chapter 7.2.3 --- Further Modified the Electrodes to Enhance the Output Power --- p.70 / APPENDIX --- p.71 / BIBLIOGRAPHY --- p.73

Han, Zuoyan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 43-46). / Abstracts in English and Chinese. / Abstract (Chinese) --- p.i / Abstract (English) --- p.ii / Acknowledgements --- p.iv / Glossary --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Physics parameter viscosity --- p.1 / Chapter 1.2 --- PDMS microfluidics device --- p.4 / Chapter Chapter 2 --- PDMS viscometer for microliter Newtonian fluid / Chapter 2.1 --- Introduction --- p.5 / Chapter 2.2 --- Configuration of the PDMS Viscometer --- p.8 / Chapter 2.3 --- Mechanism of passive pumping --- p.10 / Chapter 2.4 --- Theory of the PDMS viscometer --- p.11 / Chapter 2.5 --- Viscosity Measurement in PDMS Viscometer --- p.15 / Chapter 2.5.1 --- Preparation of Blood Plasma --- p.16 / Chapter 2.5.2 --- Measurements of Glycerol Solutions --- p.16 / Chapter 2.5.3 --- Measurements of Protein Solution and Blood Plasma --- p.19 / Chapter 2.5.4 --- Measurements of Organic Solvents --- p.19 / Chapter 2.6 --- Data Analysis --- p.21 / Chapter 2.7 --- Dynamic Contact Angle --- p.22 / Chapter 2.8 --- Conclusions --- p.23 / Chapter Chapter 3 --- PDMS viscometer for microliter Non-Newtonian fluid / Chapter 3.1 --- Introduction --- p.25 / Chapter 3.2 --- Configuration of the PDMS viscometer --- p.29 / Chapter 3.3 --- Theory for non-Newtonian fluid --- p.31 / Chapter 3.4 --- Viscosity Measurement of non-Newtonian fluids --- p.35 / Chapter 3.4.1 --- Preparation of Blood Plasma --- p.36 / Chapter 3.4.2 --- Measurement of starch solutions --- p.36 / Chapter 3.5 --- Data analysis --- p.37 / Chapter 3.6 --- Conclusion --- p.41 / References --- p.43

Han, Zuoyan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 75-81). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.