Intermodulation Peak Detection of Branched DNA for Compact-Apparatus Viral Load M

DESCRIPTION (provided by applicant): HIV viral load measurement in resource-limited settings is an area of persistent need. Branched DNA (bDNA), a non-PCR nucleic acid method based on multitiered solution-phase probe structures, is a promising approach to meeting the need for HIV viral load measurement in resource-limited settings. On reference lab instruments, the branched DNA chemistry supports detection of as few as 75 copies of viral RNA in a 1 mL sample without target amplification. Branched DNA is inherently resistant to sample contamination, requires no thermal cycling or extreme temperatures, and is compatible with enclosed-cartridge-format assay instruments. This Phase I project focuses on a specific technical barrier to the development of cartridge-format bDNA systems suitable for use in resource-limited settings: detecting bound, bDNA probe-hybridized viral RNA to determine the assay outcome. While standard bDNA chemiluminescence detection yields excellent results in traditional clinical laboratories, this method presents stability concerns for bDNA systems intended for use outside of tightly controlled environments. To overcome this challenge, a novel method for detecting bound bDNA probe-hybridized viral RNA is proposed: intermodulation peak detection of nonlinear superparamagnetic effects in materials subjected to oscillating fields. Intermodulation peak detection is a recently developed magnetic detection method which shares the robustness of more well established giant magnetoresistance methods, but affords advantages in terms of detecting spatially distributed probes. This method has recently been demonstrated for immunoassays, but the work proposed here is (to our knowledge) the first use of intermodulation peak detection as a tool for solution-phase nucleic acid assays. Preliminary studies indicate that intermodulation peak detection methods can support bDNA assay sensitivities of as few as 10 bDNA-complexed viral RNA molecules within a bDNA-optimized porous structure and an assay dynamic range of at least four orders of magnitude. The project work encompasses bench-level prototype detector construction and extensive experimentation, modeling, and analysis. Reaching these milestones will demonstrate feasibility intermodulation peak detection as a component of the ultimate goal, a compact, low-maintenance, battery-powered, FDA-approved system which analyzes fingerstick samples with a two-hour turnaround time. The proposed research is pioneering, exploring a novel combination of bDNA signal amplification with intermodulation peak detection; it is rigorous, with exhaustive calibration protocols and careful mapping of relevant parameter spaces; and it is of high value, addressing a prominent technical barrier to the deployment of diagnostic devices with the potential to facilitate significant improvement in the level of care afforded HIV patient populations in resource-limited settings across the globe. The central hypothesis of this project is that intermodulation peak detection of bDNA complexes bound within a porous structure supports viral RNA measurement with a limit of detection of 5,000 copies per mL or better, upper bound of dynamic range at least 500,000 copies per mL, and total assay duration less than 120 minutes. PUBLIC HEALTH RELEVANCE: Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project explores new ways of making viral load measurements less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities.