Sample-to-answer viremia diagnostic using thermally responsive alkane partitions.

Introduction: Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has resulted in over 7 million deaths globally since 2019 [1]. Numerous studies demonstrated that SARS-CoV-2 viremia - live virus particles in the bloodstream - is linked to severe health outcomes and more accurately predicts patient outcomes than nasopharyngeal swabs [2-5]. It also has been identified as a predictor for long COVID [6]. Despite the availability of point-of-care (PoC) tests for nasal swabs, developing PoC tests for viremia is challenging due to the lower viral load in blood, requiring RNA purification and amplification in lab settings with trained technicians and expensive equipment. There is a clear need for PoC tests, which must be cost-effective, portable, and sample-to-answer (i.e., no user interventions).
In response, we developed a magnetofluidic approach using Thermally Responsive Alkane Partitions (TRAPs) that enable a fully automated, low-cost, and portable PoC diagnostic for viremia (Figure 1) [7,8]. TRAPs are alkane layers in an assay cassette that, upon heating, liquify. TRAPs thinner than a threshold thickness breach, causing reagents to mix, while thicker ones remain stationary, continuing to partition the reagents but allowing magnetic beads with captured targets to be pulled through [7]. We previously applied TRAPs to detect circulating histones in blood [9] and to create a PoC test for detecting SARS-CoV-2 antibodies in whole blood [10]. The current work refines the TRAP system to be more robust, more user-friendly, and more capable, specifically demonstrating for the first-time sample-to-answer detection of SARS-CoV-2 in blood in a low-cost portable format.

Materials and

Methods: Dual TRAP demonstration: A channel was sequentially loaded with 15 µL of yellow-dyed 0.1% Tween 20 solution (similar to LAMP master mix), 8 µL of melted hexacosane, 10 µL of blue dyed water, 35 µL of melted eicosane, and 25 µL of red dyed streptavidin magnetic beads solution. Each alkane layer solidified before adding the next layer. The assembled cassette was placed on a magnet with a polyimide heater, heating to 65°C.

Sample-to-Answer tests: Cassettes were fabricated from a 24mm thick acrylic sheet into 6.35mm diameter cylinders with 3.2mm wide, 23mm deep channels using CNC. The portable detection device (Figure 2) includes an ArduCAM Camera, a longpass filter, blue LEDs, a DS18B20 digital temperature sensor, polyimide heaters, and neodymium magnets, all in a 3D printed case.
To demonstrate the S2A system with SARS-CoV-2 from blood, 100 fmol biotinylated capture aptamers were attached to 400 µg streptavidin magnetic beads in 8µL PBS. LAMP solution: 50 µL WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix, 2 µL 50X Fluorescent Dye, and 8 µL water. Lysis solution: 4 µL Thermolabile Proteinase K, 10 µL LAMP primer mix for the N15 gene, 2 µL Antarctic Thermolabile UDG, and 24 µL water. These solutions were layered in cassettes, sealed with hexacosane and eicosane. Following the addition of 20 µL of spiked whole blood, the cassettes were heated at 45°C for 6 minutes to melt eicosane, allowing the beads to move through the alkane into the lysis solution. The hexacosane then melted at 65°C, initiating the LAMP reaction.

Results, Conclusions, and Discussions: TRAP Operation: Within the cassette, there are two TRAPs (Figure 1). One TRAP (eicosane, melt temperature 40 °C) remains stationary to continually separate the blood sample from the assay but allows magnetic beads to pull capture virus particles into the assay. The second TRAP (hexacosane, melt temperature 55 °C) breaches when it melts, resulting in the automatic addition of the LAMP reagents. Figure 3 illustrates this process. The red dyed solution, which initially contains magnetic beads, remains separated by the eicosane layer, even after the eicosane melts and the beads are pulled through. The yellow and blue solutions are then mixed when the hexacosane layer melts.
S2A detection in blood: Using our dual-TRAP cassette and portable device, we demonstrated the detection of SARS-CoV-2 virus in blood. Figure 4 shows the average amplification curves for samples with 5000 copies along with blank samples, showcasing rapid and consistent time to positive (TTP). As shown in Figure 5, blood samples with varying concentrations of SARS-CoV-2 were tested with our system. Notably, samples with at least 500 copies of SARS-CoV-2 achieved 100% detecting accuracy. Even with as low as 5 copies, most of the samples tested positive.

Conclusion: This work represents a significant advance in sample-to-answer diagnostics for bloodborne viruses. Notably, our sample-to-answer system utilizes an easy-to-manufacture cassette and an automated portable device that does not require microfluidics, a pump, or precise sample handing steps.

References
[1] WHO COVID-19 Dashboard. (accessed 2024)
[2] Cardenoso D L. et al. J Med Virol 94, 5260 (2022).
[3] Jacobs, J. L. et al. Clinical Infectious Diseases 74, 1525 (2022).
[4] Li, Y. et al. Journal of Clinical Investigation 131 (2021).
[5] Fajnzylber, J. et al. Nat Commun 11, 5493 (2020).
[6] Hagman, K. et al. Diagn Microbiol Infect Dis 102, 115595 (2022).
[7] Boegner, D. J. et al. ACS Appl Mater Interfaces 14, 8865 (2022).
[8] Goertz, J. P. et al. Anal Chem 90, 3708 (2018).
[9] Everitt, M. L., et al. ACS Sens 6, 3006 (2021).
[10] Everitt, M.L., et al. Biosensors (Basel) 12, 1030 (2022).

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