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<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>

Antibiotic resistance is now a recognized problem in global health. In attempts to find solutions to detect bacteria causing antibiotic resistance we turn to technological solutions that are miniaturized, portable and cheap. The current diagnostic procedures cannot provide correct information outside laboratory settings, at the point-of-care, within necessary time. This has led to ineffective treatment of urinary tract infections causing recurrent infections and multi-drug resistant bacteria to spread. The bacteria genes show which antibiotic that is required to eliminate disease and spread of resistance. Hence, the solution would be to perform nucleic acid testing at the point-of-care. By using new DNA amplification methods it is possible to miniaturize the diagnostic test to a so-called Lab-on-a-chip. These solutions would enable sample-in-results-out capability of the system at the point-of-care. For this to work one of the most important factors is fluorescent signal read-out from DNA amplification products. In this project the design parameters of such a read-out device was investigated with focus on image sensor sensitivity and device integration. During the project it was found that a low-cost commercial image sensor could be used to record images of a (3.76 x 2.74 mm2) micro well array of nanoliter sized PCR chambers. Different imaging artifacts appearing during sample partitioning were observed, distance dependency between sensor surface well array was investigate, and finally the image sensor function was compared to a fluorescent microscope.

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Antimicrobial resistance in bacteria is a global health challenge with a projected fallout of 10 million deaths annually and cumulative costs of over 1 trillion dollars by 2050. The currently available tools exploited in the detection of bacteria or their DNA can be expensive, time inefficient, or lack multiplex capabilities among others. The research work highlighted in this dissertation advances techniques employed in the phenotypic or genotypic detection of bacteria and their DNA. In this dissertation, I present polymethyl methacrylate-pressure sensitive adhesive microfluidic platforms developed using a time-efficient, inexpensive fabrication technique. Microfluidic devices were then equipped with functionalized monoliths and utilized for sequence-specific capture and detection of picomolar concentrations of bacterial plasmid DNA harvested from cultured bacteria. I then showed multiplex detection of multiple bacteria gene targets in these devices with an improved monolith column. Finally, I demonstrated a genotypic approach to studying single bacteria growth in water-in-oil droplets with nanomolar concentrations of a fluorescence reporter, and detection via laser-induced fluorescence after convenient room temperature 2-h incubation conditions. The systems and methods described herein show potential to advance tools needed to address the surging problems and effects of drug-resistant bacteria.

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<p>Swift recognition of disease-causing pathogens at the point-of-care enables life-saving treatment and infection control. However, current rapid diagnostic devices often fail to detect the low concentrations of pathogens present in the early stages of infection, causing delayed and even incorrect treatments. Rapid diagnostics that require multiple steps and/or elevated temperatures to perform have a number of barriers to use at the point-of-care and in the field, and despite efforts to simplify these platforms for ease of use, many still require diagnostic-specific training for the healthcare professionals who use them. Most nucleic acid amplification assays require hours to perform in a sterile laboratory setting that may be still more hours from a patient’s bedside or not at all feasible for transport in remote or low-resourced areas. The cold-chain storage of reagents, multistep sample preparation, and costly instrumentation required to analyze samples has prohibited many nucleic acid detection and antibody-based assays from reaching the point-of-care. There remains a critical need to bring rapid and accessible pathogen identification technologies that determine disease status and ensure effective treatment out of the laboratory.</p> <p>Paper-based diagnostics have emerged as a portable platform for antigen and nucleic acid detection of pathogens but are often limited by their imperfect control of reagent incubation, multiple complex steps, and inconsistent false positive results. Here, I have developed mechanisms to economically improve thermal incubations, automate dried reagent flow for multistep assays, and specifically detect pathogenic antigens while improving final output sensitivity on paper-based devices. First, I characterize miniaturized inkjet printed joule-heaters (microheaters) that enable thermal control for pathogen lysis and nucleic acid amplification incubation on a low-cost paper-based device. Next, I explore 2-Dimensional Paper Networks as a means to automate multistep visual enhancement reactions with dried reagents to increase the sensitivity and readability of nucleic acid detection with paper-based devices. Lastly, I aim to create a novel Reverse-Transcription Recombinase Polymerase Reaction mechanism to amplify and detect a specific region of the Spike protein domain of SARS-CoV-2. This will allow the rapid detection of SARS-CoV-2 infections to aid in managing the current COVID-19 pandemic. In the future, these tools could be integrated into a rapid diagnostic test for SARS-CoV-2 and other pathogens, ultimately improving the accessibility and sensitivity of rapid diagnostics on multiple fronts.</p>

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.

Despite the increasing number of electrochemical and photoelectrochemical biosensors reported in the research literature, few have achieved success outside of a laboratory setting. This can partly be attributed to accessibility issues with commercially available readout instruments. Consequently, low-cost and portable readout instruments have been developed by researchers, but these devices fail to address other key compatibility and accessibility challenges. Much like the commercial systems, these devices are not natively compatible with multiplexed signal assays consisting of two or more working electrodes, cannot control optical excitation sources for photoelectrochemical biosensing, nor can they interface with auxiliary instruments such as heaters and electromagnets. To this end, we have developed a low-cost smartphone-operated electrochemical and photoelectrochemical readout system for point-of-care biosensing. Our readout system can perform standard voltammetric techniques and is capable of synchronously controlling an optical excitation source to support photoelectrochemical biosensing. This device is compatible with standard three-electrode assays as well as dual signal assays with two working electrodes. We have also created a portable sample heater that can be controlled by this readout system to facilitate on-site sample heating and have also integrated a portable electromagnet to perform away-from-lab magnetic manipulation. / Thesis / Master of Applied Science (MASc) / Early and prompt detection of disease biomarkers is crucial in order to develop effective disease management strategies. Unfortunately, many gold-standard diagnostic techniques for infectious diseases, cancers, heart diseases, among other conditions prove to be time-consuming, costly, and reliant on trained professionals in a laboratory setting. Electrochemical and photoelectrochemical detection are two sensing modalities that show promising potential for point-of-care applications, as they are easily miniaturized, inexpensive, and can be used to detect both the presence of and the amount of analyte present. However, up until now, these sensing modalities have mostly been confined to research settings. To expedite the commercialization of such sensors and to facilitate their translation to point-of-care diagnostics, we have developed a low-cost smartphone-operated electrochemical and photoelectrochemical readout system. Through the integration of peripheral instruments including a sample heater, electromagnet, and optical excitation source, this system is compatible with a number of different biosensors.

No description available.