No description available.

As demand for food increases, rapid testing methods are becoming increasingly important. In the past few years, yogurt has become popular. Yeast species are the most common spoilage organism, costing consumers and food companies money. A novel lateral flow assay has been developed to detect yeast oligonucleotide sequences. Gold nanoparticles were used as the standard reporter and fluorescent nanoparticles were developed as the novel reporter. The fluorescent nanoparticles were ruthenium-doped silica nanoparticles synthesized using the modified Stöber method. Visual analysis of assays using standard reporters showed the limit of detection to be 10 femtomoles of target sequence. Analysis of the fluorescent nanoparticles using a plate reader showed the limit of detection to be 0.027 femtomoles. The fluorescent reporter’s limit of detection is 1000 fold lower due to a sophisticated, more sensitive analysis method. Gold nanoparticles are appropriate for presence or absence testing, but fluorescent nanoparticles are best for obtaining quantitative data with low detection limits. Pathogens have been used as biological warfare for centuries. A brief review of common biowarfare agents is included. Yersinia pestis, the causative agent of the Plague, and Bacillus anthracis, the causative agent of Anthrax, are the focus. Additional work using gold nanoparticles as reporter in a sandwich assay is also included. The novel dye covered reporter was compared to the control, which was a single dye molecule linked to the reporter sequence. Repeated testing showed the novel reporter had a lower limit of detection and higher sensitivity due to increased ability to bind target.

No description available.

Iron is an essential nutrient for energy and DNA replication. Its homeostasis is commonly perturbed by chronic inflammatory mechanisms. Chronic inflammation upregulates a cytokine, hepcidin, that degrades the iron export protein ferroportin. Without a way to export iron into the bloodstream iron availability in blood becomes depleted. Iron depletion in the blood stream hinders erythropoiesis and is termed anemia. Herein I investigate and inhibit the mechanism of hepcidin activation. Inhibition of hepcidin activation has released iron from tissues and alleviated anemic conditions in a cancer model. I have laid the foundation to investigate this pathway in a 3D spheroid model. The results show that hepcidin-25 inhibition is a promising treatment for anemia of cancer. More work needs to be done to confirm efficacy in an in vivo model. In addition to anemia of cancer I have also worked with diabetic rats and investigated their anemic state using common anemia diagnostic methods. I found that in this high fat high sugar diet Wistar rat model anemia was not induced. In addition to my studies on anemia I have investigated the use of portable x-ray fluorescence (pXRF) as an accessible and affordable elemental analysis technique for lateral flow immunoassays and biological samples such as cell lysates and animal tissue. While pXRF shows promising results more work needs to be done to increase its sensitivity and pixel size.

The purpose of this thesis project is to demonstrate and evaluate an enzyme-linked immunosorbent assay (ELISA) on a paper microfluidic device platform. The integration of ELISA technology onto paper microfluidic chips allows for a quantitative detection of stroke biomarkers, such as glial fibrillary acidic protein (GFAP). Dye experiments were performed to confirm fluid connectivity throughout the 3D chips. Several chip and housing designs were fabricated to determine an optimal design for the microfluidic device. Once this design was finalized, development time testing was performed. The results confirmed that the paper microfluidic device could successfully route fluid throughout its channels at a reasonable rate. For the biochemistry portion of this thesis project, antibodies were selected to target the intended stroke biomarker: GFAP. However, due to antibody pairing complications, the protein chosen for this project was natural human cardiac troponin T, which is elevated in the bloodstream of patients who have suffered a stroke. Several antibody experiments were performed to help finalize the procedure for performing an ELISA on the paper chip. The final antibody experiment was able to demonstrate that a paper microfluidic device utilizing ELISA techniques can successfully detect a stroke biomarker at physiologically relevant concentrations. Overall, this project supported the ability to accurately and effectively diagnose stroke in a timely manner through the use of a paper microfluidic device.

No description available.

Asthma is a chronic disease affecting over 300 million people worldwide. Airway inflammation is a central feature of asthma. Quantitative sputum cytometry is the most validated method to assess this and to adjust anti-inflammatory therapy, yet it is underutilized due to rigorous processing that requires expensive specialized technicians. To address these limitations, this thesis focuses on the development of several point of need biosensors that rapidly quantify respiratory biomarkers as alternatives to traditional laboratory tests. The project began by developing a paper based sensor for detection of myeloperoxidase (MPO), a neutrophil biomarker. A test was developed using commercially available antibodies, showing direct correlation between the test line colour intensity and total neutrophils. This work was expanded to include a second protein target, eosinophil peroxidase (EPX), for identification of eosinophils. Although the test performed well using neat samples, it failed to identify EPX in clinical sputum samples. Analyzing pre-treatment methods identified that a quick immunoprecipitation technique using protein A/G beads followed by syringe filtration allowed for the device to successfully quantify EPX, eliminating the need for a centrifuge. However, the limited supply of commercial anti-EPX antibodies combined with the need for sample pre-treatment prompted investigation into alternative detection avenues. Nucleic acid aptamers were explored, with aptamer selection for EPX producing several aptamer candidates. Binding affinity and specificity tests were performed, with the T1-5 aptamer emerging. T1-5 was capable of selectively binding EPX over MPO with high affinity. This aptamer was integrated into a simple pull-down assay, capable of detecting EPX with an order of magnitude lower limit of detection than the antibody test. Overall this work has developed multiple sensors with the potential to overcome the limitations of accessibility to sputum cytometry, rapidly identify the presence and type of airway inflammation, and deliver personalized treatment strategies that not only reduce the global healthcare burden, but also greatly improve a patient’s quality of life. / Thesis / Doctor of Philosophy (PhD)

Lateral flow assay (LFA) has been demonstrated as a promising point-of-care biosensor due to its facile use and low cost. These immunoassays utilize nanoparticles as a colorimetric label to conjugate with the antibody and create a colored signal for antigen detection. Typically, gold or silver nanoparticles are used for this procedure. However, the sensitivity of these materials is not high enough to detect certain biomarkers such as the prostate specific antigen (PSA) which is a biomarker for prostate cancer. Replacing the nanoparticles with dual metal nanocages with a hollow interior has potential to improve the state of the flow test. Dual metal nanocages generated through galvanic replacement have been studied for their unique plasmonic and catalytic properties. In this study, silver-palladium nanocages were synthesized using a galvanic replacement reaction to create dual-metal, hollow nanocages. The particles were characterized for their bimetallic nature with x-ray photoelectron spectroscopy, their hollow structure with transmission electron microscopy, and their plasmonic properties with UV-Vis spectroscopy. Particles of three different sizes were created to investigate a size effect on antigen detection. The nanocages were used to as the label for immunoassay which produced a black, colored signal, and the medium and large AgPd NPs improved the tests’ naked eye limit of detection against standard 40 nm gold nanoparticles by tenfold and twenty-fivefold respectively. The medium and large AgPd NPs also had a considerable increase in calibration sensitivity when converting qualitative measurement into quantitative signal. With this work, it is our hope to improve the sensitivity of lateral flow assay and sustain the procedure as a reliable form of point-of-care testing.

Im Rahmen dieser Arbeit wurde die Möglichkeit untersucht, eine bestimmte Klasse von Pestiziden mit einem Lateral-Flow-Test als Plattform und einem Smartphone-Setup für die Signalerkennung nachzuweisen. Konkret handelt es sich bei Typ-I-Pyrethroiden um Insektizide, die zur Bekämpfung von Stechmücken eingesetzt werden, die z.B. Überträger von Viruserkrankungen sein können. Permethrin und Phenothrin, die bekanntesten Pyrethroid-Verbindungen, werden seit einiger Zeit zur Desinfektion von Flugzeugen auf Flügen aus und in tropischen Gebieten verwendet, um die Ausbreitung gefährlicher Krankheiten wie Malaria, Zika oder Dengue zu verhindern. Da es immer noch keine Kontrolle über die Menge der an Bord versprühten Insektizide gibt, wird eine neue effektive und schnelle Methode zur Analyse von Pyrethroiden direkt im Flugzeug benötigt. Zu diesem Zweck wurde ein tragbarer Test im Lateral-Flow-Assay- (LFA-) Format realisiert, bei dem gegatterte Antikörper-Indikator-Freisetzungssysteme zum Nachweis eingesetzt werden. In der ersten Phase der Arbeit wurden verschiedene Träger aus mesoporösem Siliziumdioxid (von Nano- und Mikropartikeln in Form von Plättchen und kurzen Fasern), unterschiedliche Funktionalisierungswege sowie Beladungssequenzen untersucht und bewertet. Da sich diese Materialien gut für LFAs eignen, wurde die Leistungsfähigkeit nicht nur mit herkömmlichen Assays in Suspension, sondern auch im Teststreifenformat untersucht. Die Kombination von Farbstoffen als Indikatoren und Smartphones zum Auslesen konnten einfache analytische Tests entwickelt werden, die in Zukunft von ungeschultem Personal direkt am Ort des Geschehens, z. B. in einer Flugzeugkabine, eingesetzt werden können und Ergebnisse in weniger als 5 Minuten erforderlichen Selektivität liefern. / Across this work, it was investigated the possibility of detecting a specific class of pesticides with a lateral flow test as a platform and a smartphone set-up for signal detection. Specifically, type-I pyrethroids are insecticides employed eventually to kill mosquitos that can be vectors for e.g., viral diseases. Permethrin and phenothrin, the most well-known pyrethroid compounds, are recently used for disinfection purposes on airplanes from and to tropical areas to avoid the spread of dangerous illnesses as malaria, zika or dengue. As there is still no control on the amount of insecticide sprayed onboard, a new effective and rapid method for pyrethroids analysis directly in the plane is needed. For this purpose, a portable test in lateral flow assay format was realized involving gated antibody indicator delivery systems for sensing. In the first stage of the work, different mesoporous silica supports (from nano and microparticles to platelets and short fibers), different functionalisation routes, and different loading sequences were assessed. Then, the materials’ performances were evaluated by studying their temporal response behaviour and detection sensitivity, including the tightness of pore closure (through the amount of blank release in the absence of an analyte) and the release kinetics. Finally, because such materials are well-suited for LFAs, performance assessment included a test-strip format besides conventional assays in suspension. The combination of dyes as indicators and smartphones for read-out, simple analytical tests for use by untrained personnel directly at a point-of-need such as an airplane cabin, can be devised, allowing for results in less than 5 min with the-required selectivity.

<div>Pathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples.</div><div><br></div><div>Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use.</div>