Chlamydia trachomatis accounts for 3.6% of blindness worldwide, and is the leading cause of bacterial-induced blindness in the world. With the subtle initial presentation of the disease and the difficulty in clearing the infection without the aid of antibiotics, C. trachomatis can spread rapidly following introduction into a population. This problem is further compounded in resource limited areas due to the lack of trained personnel (i.e. Medical Doctors, Nurses), equipment, and finances to test and treat large portions of the population. A testing method that is both cheap and easy to interpret is necessary. Lateral flow assays (LFA) have been used for years to evaluate pregnancy status in the developed world, and their low cost, ease of use and disposable nature make them a worthwhile candidate, but the current use of visual reporters (i.e. gold or latex nanoparticles) does not allow for adequate sensitivity for true clinical use. Fluorescent reporters, particularly fluorescent nanoparticles, would lower the limit of detection (LOD) and allow for the detection of acute and subclinical infections, which would allow for an effective and objective screening method for trachoma and many other diseases. An effective, rapid, and disposable test would allow for mass screening to be implemented which, in turn, would allow for rapid and targeted treatment. The results in this study show that the use of fluorescent-based reporters greatly improve the LOD of the LFA, with both FITC and RuSNP reporters showing a reduction in the LOD by 1 and 2.5 logs respectively when compared to traditional colorimetric reporters. This substantial improvement in the LOD of the LFA allows for the tests to be used to detect relevant levels of viral pathogens. A similar improvement in the LOD was seen when using FITC-labeled antibodies which improved the sensitivity of LFAs with regards to the detection of C. trachomatis. The use of fluorescent-based reporters in LFAs greatly improves the LOD for both viruses and bacteria, allowing for their detection at clinically relevant levels.

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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.

abstract: Over the past several decades, there has been a growing interest in the use of fluorescent probes in low-cost diagnostic devices for resource-limited environments. This dissertation details the design, development, and deployment of an inexpensive, multiplexed, and quantitative, fluorescence-based lateral flow immunoassay platform, in light of the specific constraints associated with resource-limited settings. This effort grew out of the need to develop a highly sensitive, field-deployable platform to be used as a primary screening and early detection tool for serologic biomarkers for the high-risk human papillomavirus (hrHPV) infection. A hrHPV infection is a precursor for developing high-grade cervical intraepithelial neoplasia (CIN 2/3+). Early detection requires high sensitivity and a low limit-of-detection (LOD). To this end, the developed platform (DxArray) takes advantage of the specificity of immunoassays and the selectivity of fluorescence for early disease detection. The long term goal is to improve the quality of life for several hundred million women globally, at risk of being infected with hrHPV. The developed platform uses fluorescent labels over the gold-standard colorimetric labels in a compact, high-sensitivity lateral flow assay configuration. It is also compatible with POC settings as it substitutes expensive and bulky light sources for LEDs, low-light CMOS cameras, and photomultiplier tubes for photodiodes, in a transillumination architecture, and eliminates the need for expensive focusing/transfer optics. The platform uses high-quality interference filters at less than $1 each, enabling a rugged and robust design suitable for field use. The limit of detection (LOD) of the developed platform is within an order of magnitude of centralized laboratory diagnostic instruments. It enhances the LOD of absorbance or reflectometric and visual readout lateral flow assays by 2 - 3 orders of magnitude. This system could be applied toward any chemical or bioanalytical procedure that requires a high performance at low-cost. The knowledge and techniques developed in this effort is relevant to the community of researchers and industry developers looking to deploy inexpensive, quantitative, and highly sensitive diagnostic devices to resource-limited settings. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2018

A low cost compact diagnostic has many implications in today’s society. Smart phone technology has exponentially grown and with it the imaging capabilities associated with smart phones. The goals of this research are i) to determine the feasibility of combining in the field smart phone images with color dependent assay results, ii) to develop a MatLab® image analysis code to analyze these results, and iii) compare limits of detection between the un-aided eye and MatLab® image analysis software. Orange G dye is used to create a stock solution and subsequent titers for analysis. Autocad is used to design an assay platform of 10x10 wells that are printed via a Xerox® Phaser printer with wax ink onto nitrocellulose paper. Dilutions are performed and pipetted into the wells. The image analysis code is used to determine hue, saturation, and value (HSV) values of wells. A limit of detection study using the dye is performed. HSV values are used to form calibration curves. The resulting curve fit equations are then integrated into the image analysis code to determine dye concentration. Finally, the complete capability is demonstrated by using an analogous 10x10 well experimental nitrocellulose sheet, which included a follow-up experiment via a spot check analysis. This study illustrates the feasibility of a low cost image analysis as a tool for lateral flow assay diagnostic versus the unaided eye. Future work includes using this protocol in conjunction with a lateral flow immunoassay and developing an application for the analysis.