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Lightning Talk Presentation 8

4:05 PM to 4:55 PM
Multiplexed Enzymatic Assay for the Rapid Measurement of Antiretroviral Drug Levels
Presenter
  • Katherine Ye Zhang, Senior, Bioengineering CoMotion Mary Gates Innovation Scholar
Mentors
  • Jonathan Posner, Mechanical Engineering
  • Ayokunle Ayokunle Olanrewaju, Mechanical Engineering
Session

Multiplexed Enzymatic Assay for the Rapid Measurement of Antiretroviral Drug Levelsclose

Antiretroviral therapy (ART) and pre-exposure prophylaxis (PrEP) can treat and prevent Human Immunodeficiency Virus (HIV), respectively. However, good medication adherence (≥4 doses/week) is crucial for these treatments to work effectively. Our research group recently developed the REverse TranscrIptase Chain Termination (RESTRICT) assay, a rapid enzymatic assay that measures the amount of tenofovir diphosphate (TFV-DP) present in blood, a drug that is a good indicator of long-term (1-3 month) ART/PrEP adherence. The goal of this project is to modify RESTRICT to additionally measure the amount of emtricitabine triphosphate (FTC-TP) in blood, which serves as a good indicator of short-term (1 week) adherence. RESTRICT provides the HIV reverse transcriptase (HIV RT) enzyme all the required reagents for synthesizing double-stranded DNA (dsDNA) and measures the concentration of TFV-DP and FTC-TP based on their inhibition of HIV RT activity. We designed custom DNA templates that bind preferentially to either TFV-DP or FTC-TP based on the nucleotides that they mimic. We also designed molecular beacon probes with different fluorescence dyes that bind to each DNA template and can provide fluorescence output corresponding to the amount of complementary DNA (cDNA) synthesized by HIV RT. High drug levels will lead to less cDNA synthesis and lower fluorescence intensities, indicating good adherence to treatment. Preliminary results indicate that molecular beacon probes can distinguish between the cDNA associated with each drug, which would allow for the simultaneous detection of TFV-DP and FTC-TP in a single reaction tube through the fluorescence measurements of the two types of dyes. Ongoing work is focused on optimizing RESTRICT reactions to measure clinically relevant concentrations of TFV-DP and FTC-TP. Gaining information about long- and short-term adherence to treatment through the detection of TFV-DP and FTC-TP respectively, would allow for more informed interventions by healthcare professionals to help patients improve adherence, leading to better health outcomes.