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To enable inexpensive molecular detection at the point-of-care and at home with minimal or no instrumentation, it is necessary to streamline unit operations and store reagents refrigeration-free. To address this need, a multifunctional enzymatic amplification reactor that combines solid-phase nucleic acid extraction, concentration, and purification; refrigeration-free storage of reagents with just-in-time release; and enzymatic amplification is designed, prototyped, and tested. A nucleic acid isolation membrane is placed at the reactor's inlet, and paraffin-encapsulated reagents are prestored within the reactor. When a sample mixed with chaotropic agents is filtered through the nucleic acid isolation membrane, the membrane binds nucleic acids from the sample. Importantly, the sample volume is decoupled from the reaction volume, enabling the use of relatively large sample volumes for high sensitivity. When the amplification reactor's temperature increases to its operating level, the paraffin encapsulating the reagents melts and moves out of the way. The reagents are hydrated, just-in-time, and the polymerase reaction proceeds. The amplification process can be monitored, in real-time. We demonstrate our reactors' ability to amplify both DNA and RNA targets using polymerase with both reverse-transcriptase and strand displacement activities to obtain sensitivities on-par with benchtop equipment and a shelf life exceeding 6 months.

The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors’ offices, clinics, and at home.

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Rapid and quantitative molecular diagnostics in the field, at home, and at remote clinics is essential for evidence-based disease management, control, and prevention. Conventional molecular diagnostics requires extensive sample preparation, relatively sophisticated instruments, and trained personnel, restricting its use to centralized laboratories. To overcome these limitations, we designed a simple, inexpensive, hand-held, smartphone-based mobile detection platform, dubbed “smart-connected cup” (SCC), for rapid, connected, and quantitative molecular diagnostics. Our platform combines bioluminescent assay in real-time and loop-mediated isothermal amplification (BART-LAMP) technology with smartphone-based detection, eliminating the need for an excitation source and optical filters that are essential in fluorescent-based detection. The incubation heating for the isothermal amplification is provided, electricity-free, with an exothermic chemical reaction, and incubation temperature is regulated with a phase change material. A custom Android App was developed for bioluminescent signal monitoring and analysis, target quantification, data sharing, and spatiotemporal mapping of disease. SCC’s utility is demonstrated by quantitative detection of Zika virus (ZIKV) in urine and saliva and HIV in blood within 45 min. We demonstrate SCC’s connectivity for disease spatiotemporal mapping with a custom-designed website. Such a smart- and connected-diagnostic system does not require any lab facilities and is suitable for use at home, in the field, in the clinic, and particularly in resource-limited settings in the context of Internet of Medical Things (IoMT).

Often, high-sensitivity, point-of-care (POC) clinical tests, such as HIV viral load, require large volumes of plasma. Although centrifuges are ubiquitously used in clinical laboratories to separate plasma from whole blood, centrifugation is generally inappropriate for on-site testing. Suitable alternatives are not readily available to separate the relatively large volumes of plasma from milliliters of blood that may be needed to meet stringent limit-of-detection specifications for low-abundance target molecules. We report on a simple-to-use, low-cost, pump-free, membrane-based, sedimentation-assisted plasma separator capable of separating a relatively large volume of plasma from undiluted whole blood within minutes. This plasma separator consists of an asymmetric, porous, polysulfone membrane housed in a disposable chamber. The separation process takes advantage of both gravitational sedimentation of blood cells and size exclusion-based filtration. The plasma separator demonstrated a "blood in-plasma out" capability, consistently extracting 275 ± 33.5 μL of plasma from 1.8 mL of undiluted whole blood within less than 7 min. The device was used to separate plasma laden with HIV viruses from HIV virus-spiked whole blood with recovery efficiencies of 95.5% ± 3.5%, 88.0% ± 9.5%, and 81.5% ± 12.1% for viral loads of 35,000, 3500, and 350 copies/mL, respectively. The separation process is self-terminating to prevent excessive hemolysis. The HIV-laden plasma was then injected into our custom-made microfluidic chip for nucleic acid testing and was successfully subjected to reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP), demonstrating that the plasma is sufficiently pure to support high-efficiency nucleic acid amplification.

The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors' offices, clinics, and at home.