RESUMO
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-of-detection 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 × 1011 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.
RESUMO
As the capabilities of low-resource field testing have begun to expand to incorporate more complex diagnostic technologies, many of these devices remain tethered to large heaters requiring relatively high-power inputs. Highly efficient microheaters would enable miniaturization of devices for more economic and effective heating with high temperatures and sustained incubation. This work reports the development and application of resistive microheaters printed with nanosilver ink for improved methods of automated sample heating in paper-based point-of-care (POC) and in-field diagnostics. Resistance is easily predicted, and shapes can be altered to fit space and heat-transfer needs, sustained and discrete heating of precise regions are possible. Here, we demonstrate both isothermal nucleic acid amplification at 65 °C and bacterial culture at 37 °C using our microheaters. Printed nanosilver microheaters are easily integrated into reactions that require low-power battery heating, can sustain heating for 16-hour incubations, and cost between 0.17 and 0.58 US dollars each. Further, the microheaters are reusable, stable over 6 months, and can be wetted without degradation or reduction in conductivity. These versatile printed microheaters enable thermal control for a variety of low power heating applications.
RESUMO
While identifying acute HIV infection is critical to providing prompt treatment to HIV-positive individuals and preventing transmission, existing laboratory-based testing methods are too complex to perform at the point of care. Specifically, molecular techniques can detect HIV RNA within 8-10 days of transmission but require laboratory infrastructure for cold-chain reagent storage and extensive sample preparation performed by trained personnel. Here, we demonstrate our point-of-care microfluidic rapid and autonomous analysis device (microRAAD) that automatically detects HIV RNA from whole blood. Inside microRAAD, we incorporate vitrified amplification reagents, thermally-actuated valves for fluidic control, and a temperature control circuit for low-power heating. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) products are visualized using a lateral flow immunoassay (LFIA), resulting in an assay limit of detection of 100 HIV-1 RNA copies when performed as a standard tube reaction. Even after three weeks of room-temperature reagent storage, microRAAD automatically isolates the virus from whole blood, amplifies HIV-1 RNA, and transports amplification products to the internal LFIA, detecting as few as 3 × 105 HIV-1 viral particles, or 2.3 × 107 virus copies per mL of whole blood, within 90 minutes. This integrated microRAAD is a low-cost and portable platform to enable automated detection of HIV and other pathogens at the point of care.
