Evaluation of the analytical performance of the 15-minute point-of-care DASH™ SARS-CoV-2 RT-qPCR test.

Accurate and timely diagnosis for COVID-19 diagnosis allows highly effective antiviral medications to be prescribed. The DASH™ Rapid PCR System is a sample-to-answer point-of-care platform combining state-of-the-art PCR kinetics with sequence specific hybridization. The platform's first assay, the DASH™ SARS-CoV-2/S test for anterior nares direct swab specimens, received FDA Emergency Use Authorization in March 2022 for point-of-care use. Here we report the analytical characteristics of the assay including limit of detection, dynamic range, and robustness of SARS-CoV-2 variant detection. The limit of detection was determined by testing swabs contrived with one hundred copies of wild type or Omicron BA.5 virus and detecting 20/20 and 19/20, respectively. The dynamic range was assessed with contrived swabs containing 102-106 copies; the log-linear relationship between Cq and copy input was plotted, and the qPCR efficiency calculated from the slope of the line was 101.4%. Detection of seven SARS-CoV-2 variants was demonstrated.

Point-of-care p24 antigen detection for early infant diagnosis of HIV infection: cross-sectional and longitudinal studies in Zambia.

BACKGROUND: Early infant diagnosis of HIV infection is challenging in sub-Saharan Africa, particularly in rural areas, leading to delays in diagnosis and treatment. Use of a point-of-care test would overcome many challenges. This study evaluated the validity of a novel point-of-care p24 antigen detection test (LYNX) in rural and urban settings in southern Zambia. METHODS: Two studies were conducted: a cross-sectional study from 2014 to 2015 at Macha Hospital (LYNX Hospital study) and a longitudinal study from 2016 to 2018 at 12 health facilities in Southern Province, Zambia (NSEBA study). In both studies, children attending the facilities for early infant diagnosis were enrolled and a blood sample was collected for routine testing at the central lab and immediate on-site testing with the LYNX test. The performance of the LYNX test was measured in comparison to nucleic acid-based testing at the central lab. RESULTS: In the LYNX Hospital study, 210 tests were performed at a median age of 23.5 weeks (IQR: 8.9, 29.0). The sensitivity and specificity of the test were 70.0 and 100.0%, respectively. In the NSEBA study, 2608 tests were performed, including 1305 at birth and 1222 on children ≥4 weeks of age. For samples tested at birth, sensitivity was 13.6% (95% CI: 2.9, 34.9) and specificity was 99.6% (95% CI: 99.1, 99.9). While specificity was high for all ages, sensitivity increased with age and was higher for participants tested at ≥4 weeks of age (80.6%; 95% CI: 67.4, 93.7). Children with positive nucleic acid tests were more likely to be negative by the LYNX test if their mother received antiretroviral therapy during pregnancy (60.7% vs. 24.2%; p = 004). CONCLUSIONS: Considering the high specificity and moderate sensitivity that increased with age, the LYNX test could be of value for early infant diagnosis for infants ≥4 weeks of age, particularly in rural areas where centralized testing leads to long delays. Point-of-care tests with moderate sensitivity and high specificity that are affordable, easy-to-use, and easily implemented and maintained should be developed to expand access to testing and deliver same-day results to infants in areas where it is not feasible to implement nucleic acid-based point-of-care assays.

A point-of-care PCR test for HIV-1 detection in resource-limited settings.

A low-cost, fully integrated sample-to-answer, quantitative PCR (qPCR) system that can be used for detection of HIV-1 proviral DNA in infants at the point-of-care in resource-limited settings has been developed and tested. The system is based on a novel DNA extraction method, which uses a glass fiber membrane, a disposable assay card that includes on-board reagent storage, provisions for thermal cycling and fluorescence detection, and a battery-operated portable analyzer. The system is capable of automated PCR mix assembly using a novel reagent delivery system and performing qPCR. HIV-1 and internal control targets are detected using two spectrally separated fluorophores, FAM and Quasar 670. In this report, a proof-of-concept of the platform is demonstrated. Initial results with whole blood demonstrate that the test is capable of detecting HIV-1 in blood samples containing greater than 5000 copies of HIV-1. In resource-limited settings, a point-of-care HIV-1 qPCR test would greatly increase the number of test results that reach the infants caregivers, allowing them to pursue anti-retroviral therapy.

Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device.

In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers.

Autonomously-triggered microfluidic cooling using thermo-responsive hydrogels.

We present autonomously-triggered on-chip microfluidic cooling devices that utilize thermo-responsive hydrogels to adapt to local environmental temperatures. An external rotating magnetic stirrer couples with an in situ fabricated nickel impeller in these centrifugal-based microfluidic cooling devices to recirculate cooler water. Temperature-responsive hydrogels, which exhibit volumetric expansion and contraction, are integrated at the axle of the impeller. In this design, the hydrogels behave similar to an automotive clutch, to autonomously control the impeller's rotation as a function of the local environmental temperature. Therefore, the hydrogels act as both sensors and actuators and help take away the necessity for additional temperature sensing, feedback, and/or control units here. Cooling devices capable of on-chip thermal management at multiple predetermined onset operation points are realized by changes to the composition of hydrogel to alter its lowest critical solution temperature (LCST). Furthermore, the effect of magnetic stirrer frequency on the fluid cooling and flowrates for different two-blade nickel impeller designs are presented.

Polarity effect in electrovibration for tactile display.

Electrovibration is the tactile sensation of an alternating potential between the human body and a smooth conducing surface when the skin slides over the surface and where the current is too small to stimulate sensory nerves directly. It has been proposed as a high-density tactile display method, for example to display pictographic information to persons who are blind. Previous models for the electrovibration transduction mechanism are based on a parallel-plate capacitor in which the electrostatic force is insensitive to polarity. We present experimental data showing that electrovibratory perceptual sensitivity to positive pulses is less than that for negative or biphasic pulses and propose that this disparity may be due to the asymmetric electrical properties of human skin. We furthermore propose using negative pulses for insulated tactile displays based on electrovibration because their sensory thresholds were found to be more stable than for waveforms incorporating positive pulses.

Adaptive liquid microlenses activated by stimuli-responsive hydrogels.

Despite its compactness, the human eye can easily focus on different distances by adjusting the shape of its lens with the help of ciliary muscles. In contrast, traditional man-made optical systems achieve focusing by physical displacement of the lenses used. But in recent years, advances in miniaturization technology have led to optical systems that no longer require complicated mechanical systems to tune and adjust optical performance. These systems have found wide use in photonics, displays and biomedical systems. They are either based on arrays of microlenses with fixed focal lengths, or use external control to adjust the microlens focal length. An intriguing example is the tunable liquid lens, where electrowetting or external pressure manipulates the shape of a liquid droplet and thereby adjusts its optical properties. Here we demonstrate a liquid lens system that allows for autonomous focusing. The central component is a stimuli-responsive hydrogel integrated into a microfluidic system and serving as the container for a liquid droplet, with the hydrogel simultaneously sensing the presence of stimuli and actuating adjustments to the shape--and hence focal length--of the droplet. By working at the micrometre scale where ionic diffusion and surface tension scale favourably, we can use pinned liquid-liquid interfaces to obtain stable devices and realize response times of ten to a few tens of seconds. The microlenses, which can have a focal length ranging from -infinity to +infinity (divergent and convergent), are also readily integrated into arrays that may find use in applications such as sensing, medical diagnostics and lab-on-a-chip technologies.

Dissolvable membranes as sensing elements for microfluidics based biological/chemical sensors.

We demonstrate a chemical and biological sensing mechanism in microfluidics that transduces chemical and biological signals to electrical signals with large intrinsic amplification without need for complex electronics. The sensing mechanism involves a dissolvable membrane separating a liquid sample chamber from an interdigitated electrode. Dissolution of the membrane (here, a disulfide cross-linked poly(acrylamide) hydrogel) in the presence of a specific target (here, a reducing agent-dithiothreitol) allows the target solution to flow into contact with the electrode. The liquid movement displaces the air dielectric with a liquid, leading to a change (open circuit to approximately 1 kOmega) in the resistance between the electrodes. Thus, a biochemical event is transduced into an electrical signal via fluid movement. The concentration of the target is estimated by monitoring the difference in dissolution times of two juxtaposed sensing membranes having different dissolution characteristics. No dc power is consumed by the sensor until detection of the target. A range of targets could be sensed by defining membranes specific to the target. This sensing mechanism might find applications in sensing targets such as toxins, which exhibit enzymatic activity.