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DEVELOPMENT OF VIRAL MOLECULAR DETECTION PLATFORMS FOR POINT-OF-CARE DIAGNOSTICS

thesis
posted on 2024-04-22, 19:10 authored by Navaporn SritongNavaporn Sritong

The emergence of infectious diseases like HIV, influenza, and COVID-19 highlights the urgent need for highly scalable testing methods that can be deployed outside traditional laboratory settings. Despite decades of research in point-of-care (POC) diagnostics, the main challenge remains the limited performance of assays, especially in terms of sensitivity. Furthermore, most POC assays originating from academic research struggle to transition beyond the laboratory due to manufacturability issues. This dissertation aims to enhance the effectiveness of viral molecular detection platforms for POC diagnostics by improving analytical and clinical sensitivity and facilitating the practical adaptation of academic-developed POC devices for use outside laboratory settings.

Each aim addresses a separate aspect of device development. The first aim addresses the need for clinical accuracy during test interpretation, especially in POC or at-home diagnostic tests, by developing an internal amplification control (IAC). Here, I develop a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detecting SARS-CoV- 2 along that incorporates a housekeeping gene as an IAC to ensure the quality of collected samples and the validity of assay reagents. The valid results can be easily visualized in triple-line lateral flow immunoassay (LFIA). The second aim makes progress towards overcoming the limited analytical sensitivity of existing rapid diagnostic tests for acute HIV infection screening. Here, I introduce a novel antibody-initiated LAMP assay targeting the HIV p24 capsid protein that combines LAMP sensitivity with the specificity of HIV p24 and its antibody. There are 3000 p24 capsid proteins present in the virion compared to only 2 viral RNA copies. In the assay, two DNA- conjugated antibody probes will each bind to p24 and their proximity will allow the DNA overlaps to generate a complete DNA target that acts as a trigger for the LAMP reaction. An LFIA is integrated into this design to enable simple result visualization. The third aim improves manufacturability and assembly of our existing nucleic acid detection platform by simplifying the platform components while maintaining the user-friendly sample-to-answer concept. Here, I validate material compatibility testing, assess chamber fabrication methods amenable to large-scale manufacturing, evaluate alternative heating units, and examine fluid flow control mechanisms of the redesigned wax valve. These combined aims demonstrate promising outcomes for practical implementation of molecular diagnostics to the POC.

Funding

NIH NIDA DP2DA051910

Moore Inventor Fellowship# 9687

History

Degree Type

  • Doctor of Philosophy

Department

  • Biomedical Engineering

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Jacqueline C. Linnes

Additional Committee Member 2

Tamara Kinzer-Ursem

Additional Committee Member 3

Natalia Rodriguez

Additional Committee Member 4

Karin F. K. Ejendal

Additional Committee Member 5

Mohit Verma

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