UCLA

Providing effective healthcare to underrepresented populations is a major challenge worldwide due to environmental, economic, and social disparities. Both developing countries and low income rural/urban areas in the US tend to have reduced healthcare access which limits the frequency and quality of medical screenings as well as treatment options. This has led to an increased effort in the development of point-of-care devices which emphasize, immediate and onsite results. One device that has the potential to fulfill these requirements is the lateral-flow immunoassay (LFA). While the LFA has been successful in the form of the over-the-counter pregnancy test, it still does have some disadvantages that limit its ability to fully replace sophisticated laboratory-based assays. Throughout this thesis, I will be discussing the development of cutting-edge technologies to address the two major disadvantages of the LFA which are its poor quantitative characteristics and low sensitivity.Cerebrospinal fluid (CSF) leaks are a common complication of numerous procedures in Otolaryngology. These leaks are relatively difficult to diagnose and the current laboratory-based tests to do so have a turn-around time that is unacceptable for rapid clinical decision making. We addressed this problem by developing the first rapid diagnostic test kit for the detection of CSF leaks. In Chapter 2, we describe the development of a barcode-style LFA for the semi-quantitative detection of beta-trace protein, which is present in CSF at high concentrations. This work was expanded upon in Chapter 3, where we created a complete, standalone rapid diagnostic test kit composed of our barcode-style LFA, a collection swab, dilution buffers, disposable pipettes, and instructions. Clinical studies demonstrated excellent predictive capabilities of this kit in distinguishing patient samples containing CSF from those that did not. In some cases, a more quantitative readout than what the barcode-style LFA can achieve is required. In Chapter 4, we developed a new technique to introduce an even more quantitative readout to the LFA without the need of electronic readers. This technology was applied to the therapeutic drug monitoring of the cardiac glycoside digoxin. Digoxin capture and binding occurs on a modified LFA test strip which utilizes platinum nanozyme probes that possess catalase-like activity. This test strip is combined with a reaction that controls the anisotropic etching of gold nanorods to produce a wide range of visible color hues dependent on the initial digoxin concentration in the sample which allowed for naked-eye quantification. We then shifted to the development of technologies to improve the detection limit and sensitivity of the LFA. Our lab previously discovered the phenomenon of an aqueous two-phase system (ATPS) separating on paper, which allowed for the seamless integration of biomarker preconcentration and detection on the LFA. In Chapter 5, we have extended the functionality of an ATPS separating on paper to automate the sequential delivery of signal enhancement reagents in addition to concentrating biomarkers. The timing of reagent delivery was controlled by changing the initial composition of the ATPS and a mathematical model was developed to help predict the ATPS flow behavior. We applied this technology to automate biomarker preconcentration and nanozyme signal enhancement on the LFA, resulting in a 30-fold improvement in detection limit over the conventional LFA when detecting Escherichia coli, all while maintaining a single application step. In late 2019, a new virus known as SARS-CoV-2 began to sweep across the globe, leading to the COVID-19 pandemic. The development of highly sensitive and easily deployable rapid diagnostic tests is a vital aspect of managing this pandemic. In Chapter 6, we discuss ongoing efforts to develop a highly sensitive, nanozyme signal enhanced LFA for the rapid detection of the SARS-CoV-2 nucleocapsid protein. A 3D-printed casing was designed to store the test strip and all reagents in a way that maintains user friendly operation by eliminating liquid handling steps. Besides developing highly sensitive diagnostics, the ability to deploy these tests quickly and in a highly scalable manner is crucial for being able to manage a pandemic. The work described in Chapter 7 highlights the integration of novel fibronectin-based antibody mimetics, called monobodies, into the LFA for the detection of the nucleocapsid protein of SARS-CoV-2. These monobodies are advantageous over monoclonal antibodies in that they are rapidly developed and produced recombinantly in Escherichia coli which is more affordable and scalable than monoclonal antibody production.