Microfluidic Rapid and Autonomous Analytical Device (microRAAD) to Detect HIV from Whole Blood Samples

Abstract

Early Human Immunodeficiency Virus (HIV) testing is critical to preventing transmission and providing treatment to HIV-positive individuals, yet an estimated 30% of HIV-positive individuals do not know their status because of barriers to early diagnosis. Readily accessible, highly sensitive, and rapid diagnostic tests would enable patients' prompt treatment with antiretroviral therapies and reduce transmission. However, existing HIV diagnostic technologies either do not detect early stages of infection or require multiple days of laboratory processing, delaying notification of patients' status.Molecular techniques that amplify HIV RNA can detect the earliest stages of infection, within 8-10 days after transmission. However, most of these molecular assays require cold-chain storage of reagents, significant sample preparation, and extensive laboratory infrastructure. To achieve early detection, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with a limit of detection of 10 HIV-1 RNA copies visualized by eye using a lateral flow immunoassay. To demonstrate automated sample-to-answer detection of HIV, we incorporate dried amplification reagents and wax valves in low-cost substrates with resistive heating elements and circuitry. By combining controlled heating with paper's capillary flow, our assembled device automatically isolates viral particles from human blood samples, amplifies HIV-1 RNA, and transports products to a detection zone. We determine that as few as 10 5 HIV-1 viral particles can be separated from whole blood, amplified, and visually detected within 90 minutes of sample addition into our Microfluidic Rapid and Autonomous Analysis Device (microRAAD). The low-cost and automated attributes of microRAAD demonstrate its utility as a point-of-care testing platform.

Towards the Use of a Smartphone Imaging-Based Tool for Point-of-Care Detection of Asymptomatic Low-Density Malaria Parasitemia

“…The diffusion coefficient values were significantly different from NTC for 12.6 (****p<0.0001), 1. 26 significantly different from NTC (uninfected blood) but followed the same trend of having lower diffusivity than the NTC. Thus, PD-LAMP is sensitive enough to detect amplicons even when they cannot be ascertained via agarose gel electrophoresis and a LOD of 0.126 parasites/µL was obtained with the smartphone device from patient samples.…”

“…Ultimately, a smartphone-enabled hardware device could integrate a portable heating element for a standalone, sample-to-answer, portable diagnostic. (26) Total reaction volumes can also be increased to allow for higher input of blood sample while keeping the overall concentration of blood at 10%. Additionlly, malaria PD-LAMP applications could also be extended for use with alternative sample matrices, such as dried blood spots and urine, which would provide users to have alternative sample storage and/or perform non-invasive screening.…”

“…(21)(22)(23)(24) Isothermal amplification methods eliminate the thermal cycling that is needed for highly accurate methods such as PCR and have the ability to robustly amplify nucleic acids in complex matrices while maintaining sensitivity and specificity. (25,26) This simplifies the process and decreases the time from sample to answer. (25,26) The use of one such isothermal technique, loop mediated isothermal amplification (LAMP) is an attractive nucleic acid amplification technique for field use due to its simplicity and robustness in complex matrices in comparison to other isothermal methods.…”

“…(25,26) This simplifies the process and decreases the time from sample to answer. (25,26) The use of one such isothermal technique, loop mediated isothermal amplification (LAMP) is an attractive nucleic acid amplification technique for field use due to its simplicity and robustness in complex matrices in comparison to other isothermal methods. (27) LAMP-based assays have also been deemed appropriate for detection of low-level parasitemia with the commercialized Loopamp TM malaria Pan/Pf kit by having an excellent limit of detection of 2 parasites/L.…”

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

“…Researchers have shown that inhibitors found in common sample matrices such as blood, urine, saliva, and environmental water ( Figure 1) do not completely suppress LAMP amplification [15][16][17][18][19] and that thermal lysis of microorganisms is possible at standard LAMP conditions [20,21]. This allows users to potentially skip tedious lysis, extraction, and/or purification steps traditionally required prior to nucleic acid amplification.…”