This handheld sample-to-answer platform combines blood cell separation, viral lysis, and isothermal nucleic acid amplification with capillary fluidics and heating controls to automatically detect HIV from blood samples within 90 minutes.
Two-dimensional paper networks (2DPNs) have enabled the use of paper-based platforms to perform multistep immunoassays for detection of pathogenic diseases at the point-of-care. To date, however, detection has required the user to provide multiple signal enhancement solutions and been limited to protein targets. We solve these challenges by using mathematical equations to guide the device design of a novel 2DPN, which leverages multiple fluidic inputs to apply fully dried solutions of hydrogen peroxide, diaminobenzidine, and horseradish peroxidase signal enhancement reagents to enhance the limit-ofdetection of numerous nucleic acid products. Upon rehydration in our unique 2DPN design, the dried signal enhancement solution reduces the limit-of-detection (LOD) of the device to 5 × 10 11 nucleic acid copies/mL without increasing false positive detection. Our easy-to-use device retains activity after 28 days of dry storage and produces reliable signal enhancement 40 min after sample application. The fully integrated device demonstrated versatility in its ability to detect double-stranded and single-stranded DNA samples, as well as peptide nucleic acids.
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.
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