POWER-FREE HIV RNA EXTRACTION FROM WHOLE BLOOD FOR SMARTPHONE-BASED DETECTION

Introduction:: Of the 38.4 million people were living with HIV/AIDS in 2021, 32% of those individuals had not achieved viral suppression ¹. The reduction of viral load to undetectable levels via antiretroviral therapy typically translates to very low transmission risk and prolonged life expectancy yet monitoring of viral load to inform proper treatment can be difficult ². Here, we present progress towards a modular platform to perform on-site, sample-to-answer HIV viral load quantification from patient’s whole blood sample. Our device couples an upstream syringe-based sample preparation module for extraction of the viral RNA, to a downstream detection module for on-chip reverse-transcription loop-mediated isothermal amplification (RT-LAMP) of both HIV and an internal amplification control (IAC) to ensure clinical accuracy. The detection of HIV amplicons is achieved via a real-time smartphone-based particle diffusometry (PD) measurement which performs ensemble detection of the motion of particles within a sample over time.


Materials and Methods:: We have developed a PD-RT-LAMP assay, including an IAC that is specific to HIV integrase gene. The IAC incorporates replication deficient HIV lyophilized into the RT-LAMP reaction components, thereby removing cold-chain storage requirements. To extract HIV from blood, we devised a power-free sample processing technique by modifying a standard QIAamp Viral RNA spin-column to incorporate a 3D printable (9mm ID, 20mm length) luer-locked adaptor component (Figure 1A) and connecting reagent storage tubing to the adaptor (Figure 1B). Viral RNA is released in the cell lysis step, the lysate is transferred to the column, and a negative pressure then forces the sequential flow of reagents through the capture membrane. Bound HIV RNA is washed and eluted directly into the detection chip (Figure 1C). Direct imaging of particle motion during PD-RT-LAMP is performed on a microfluidic chip heated using a thermally resistive ITO heater³.


Results, Conclusions, and Discussions:: RESULTS AND DISCUSSION

HIV RNA was purified from fresh human blood and detected via RT-LAMP off-chip, down to 70 viral particles (vp)/uL. Our chip design spatially multiplexes real-time imaging chambers for the test, IAC (positive control), and the negative (contamination control) reactions to ensure clinically relevant sample evaluation. Using our robust real-time detection of PD-RT-LAMP in a partially integrated prototype, we have performed semi-quantitative detection of HIV spiked in water at concentrations from 1E5 (25000 vp/uL) to 1E2 vp/reaction (25 vp/uL) (Figure 1D).



CONCLUSION

We demonstrated a promising tool to aid patient monitoring of their HIV viral load status. Future work will reduce the limit of detection in the microfluidic chip. Further, we will integrate heating, imaging, and on-device PD algorithm, enabling real-time detection on our smartphone-based platform. Finally, we will perform clinical testing of the integrated platform, including the extraction module.


Acknowledgements (Optional): :

References (Optional): : < ![if !supportLists] >1. < ![endif] >Global HIV & AIDS statistics — Fact sheetat https://www.unaids.org/en/resources/fact-sheet

< ![if !supportLists] >2. < ![endif] >The science is clear: with HIV, undetectable equals untransmittable. ,2019.at https://www.nih.gov/news-events/news-releases/science-clear-hiv-undetectable-equals-untransmittable

< ![if !supportLists] >3. < ![endif] >Colbert, A. J., D. H. Lee, K. N. Clayton, S. T. Wereley, J. C. Linnes, and T. L. Kinzer-Ursem. PD-LAMP smartphone detection of SARS-CoV-2 on chip. Analytica Chimica Acta 1203:339702, 2022.