BME Master's Defense Announcement for Pankti R. Thakkar (J. Linnes, advisor)

Everyone is invited to attend the public presentation beginning at 12:00 pm.

Thesis Title: Characterization and Development of an Enzymatically Signal Enhanced Lateral Flow Assay Test for HIV using p24 Antigen

Date: April 18^th 2023

Time: 12:00 pm

Location: BRK 2001 and Zoom Link: https://purdue-edu.zoom.us/j/8181492040

Thesis Committee:

     Jacqueline Linnes, PhD, Chair

     Lia Stanciu, PhD

     Scott Bolton, PhD

Abstract:

Authors: Pankti R. Thakkar¹*, Jacqueline Linnes¹

Affiliation: ¹ Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA

In 2021, an estimated 1.5 million people were diagnosed with HIV globally, increasing the total to 38.4 million people. Approximately 16% of this population were unaware of their infected status and required HIV testing, which is a critical first step in HIV prevention, treatment, and care [1]. Hence, there is a need to develop a rapid, user-friendly, and cost-effective point-of-care test for HIV detection. The time between HIV infection and a detectable host HIV antibody concentration can extend up to 90 days. By incorporating more sensitive testing for the HIV p24 antigen on the virus, the diagnosis lag can be reduced to 17 days [2]. This window could be further shortened by using horseradish peroxidase (HRP) enzyme as a signal enhancement technique. The work herein focuses on developing an enzymatically signal-enhanced lateral flow assay test for the p24 antigen to detect HIV during the acute phase of infection. Conjugation chemistry for the sandwich assay was characterized using DLS and UV-Vis. Dot blots were then used to assess and enhance the functionality of the individual components via a visual color gradient formed by the protein coupled with antibody-conjugated gold nanoparticles. A quantitative analysis was performed using ImageJ software through signal pixel intensity analysis. A limit of detection (LoD) of 6 ng/mL was obtained for the detection of p24 antigen in the patient sample. This LoD was improved to 0.2 ng/mL by incorporating HRP signal enhancement with diaminobenzidine substrate [3]. This 30x signal improvement could drive down the LoD even further to improve the sensitivity of the commercial p24 antigen tests. Different fabrication and scalability studies were performed to produce a cost-efficient, fully functional prototype of a paper-based lateral flow device incorporating the signal-enhanced p24 assay. This study serves as a solid foundation to research focused on creating more efficient point-of-care tests that can be used in resource-limited settings to provide early detection of HIV for the 6 million individuals who are currently unaware of their HIV status.

[1] October 25, 2021, “Global HIV/AIDS Organizations.” [2] “Types of HIV tests, HIV basics | HIV/AIDS | CDC.” [3] KM. Byers et al., ACS Omega 5, no. 9 (March 10, 2020): 4673-81