Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was classified as a pandemic by the World Health Organization and has caused over 550,000 deaths ...worldwide as of July 2020. Accurate and scalable point-of-care devices would increase screening, diagnosis, and monitoring of COVID-19 patients. Here, we demonstrate rapid label-free electrochemical detection of SARS-CoV-2 antibodies using a commercially available impedance sensing platform. A 16-well plate containing sensing electrodes was pre-coated with receptor binding domain (RBD) of SARS-CoV-2 spike protein, and subsequently tested with samples of anti-SARS-CoV-2 monoclonal antibody CR3022 (0.1 μg/ml, 1.0 μg/ml, 10 μg/ml). Subsequent blinded testing was performed on six serum specimens taken from COVID-19 and non-COVID-19 patients (1:100 dilution factor). The platform was able to differentiate spikes in impedance measurements from a negative control (1% milk solution) for all CR3022 samples. Further, successful differentiation and detection of all positive clinical samples from negative control was achieved. Measured impedance values were consistent when compared to standard ELISA test results showing a strong correlation between them (R2=0.9). Detection occurs in less than five minutes and the well-based platform provides a simplified and familiar testing interface that can be readily adaptable for use in clinical settings.
•Capacitive immunosensing of clinically relevant concentrations of SARS-CoV-2 antibodies.•Antigen/antibody association and dissociation occurs within few seconds.•Rapid, label free detection using commercially available equipment.•Impedance peaks correlated with CR3022 concentration levels and ELISA measurements.
Respiratory droplets emitted during speech can transmit oral bacteria and infectious viruses to others, including COVID-19. Loud speech can generate significantly higher numbers of potentially ...infectious respiratory droplets. This study assessed the effect of speech volume on respiratory emission of oral bacteria as an indicator of potential pathogen transmission risk. Loud speech (average 83 dBA, peak 94 dBA) caused significantly higher emission of oral bacteria (p = 0.004 compared to no speech) within 1 ft from the speaker. N99 respirators and simple cloth masks both significantly reduced emission of oral bacteria. This study demonstrates that loud speech without face coverings increases emission of respiratory droplets that carry oral bacteria and may also carry other pathogens such as COVID-19.
Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Storing ...RBCs in a frozen or dried state is an appealing solution to address storage limitations, but many promising cryoprotectants, including the non-reducing sugar trehalose, are impermeant to mammalian cell membranes and cannot be utilized effectively using currently available compound-loading methods. We found that transient pore formation induced by ultrasound and microbubbles (sonoporation) offers an effective means of loading trehalose into RBCs to facilitate long-term storage in a frozen or desiccated state. The protective potential of trehalose loading was demonstrated by freezing processed RBCs at −1 °C/min to −80 °C, then either storing the cells at −80 °C or lyophilizing them. RBCs were either thawed or rehydrated after 42 days of storage and evaluated for membrane integrity and esterase activity to estimate recovery and cell viability. The intracellular concentration of trehalose reached 40 mM after sonoporation and over 95% of treated RBCs were recovered after loading. Loading of trehalose was sufficient to maintain RBC morphology and esterase activity in most cells during freezing (>90% RBC recovery) and to a lower degree after lyophilization and rehydration (>20% recovery). Combining sonoporation with an integrated fluidics device allowed for rapid loading of up to 70 mM trehalose into RBCs. These results demonstrate the potential of sonoporation-mediated trehalose loading to increase recovery of viable RBCs, which could lead to effective methods for long-term stabilization of RBCs.
Preservation of erythrocytes in a desiccated state for storage at ambient temperature could simplify blood transfusions in austere environments, such as rural clinics, far-forward military ...operations, and during space travel. Currently, storage of erythrocytes is limited by a short shelf-life of 42 days at 4 °C, and long-term preservation requires a complex process that involves the addition and removal of glycerol from erythrocytes before and after storage at −80 °C, respectively. Natural compounds, such as trehalose, can protect cells in a desiccated state if they are present at sufficient levels inside the cell, but mammalian cell membranes lack transporters for this compound. To facilitate compound loading across the plasma membrane via ultrasound and microbubbles (sonoporation), a polydimethylsiloxane-based microfluidic device was developed. Delivery of fluorescein into erythrocytes was tested at various conditions to assess the effects of parameters such as ultrasound pressure, ultrasound pulse interval, microbubble dose, and flow rate. Changes in ultrasound pressure and mean flow rate caused statistically significant increases in fluorescein delivery of up to 73 ± 37% (p < 0.05) and 44 ± 33% (p < 0.01), respectively, compared to control groups, but no statistically significant differences were detected with changes in ultrasound pulse intervals. Following freeze-drying and rehydration, recovery of viable erythrocytes increased by up to 128 ± 32% after ultrasound-mediated loading of trehalose compared to control groups (p < 0.05). These results suggest that ultrasound-mediated molecular delivery in microfluidic channels may be a viable approach to process erythrocytes for long-term storage in a desiccated state at ambient temperatures.
Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging ...technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.
Cell transformation is an important process utilized in a wide variety of research and medical applications. Current methods for cell transformation generally depend on viral delivery or other ...methods which are often limited by inefficiency and/or toxicity. An alternative approach known as sonoporation may avoid these issues. Sonoporation can occur when ultrasound pulses induce microbubble oscillation near cellular membranes causing formation of transient pores. Sonoporation has been shown to enhance molecular delivery to cells. To improve the efficiency and consistency of molecular delivery via sonoporation, we have developed a high-performance ultrasonic flow system which integrates ultrasound and microfluidic technology. One particular application of interest involves loading red blood cells (RBCs) with trehalose for dry preservation. Storage of RBCs is limited by a short shelf-life of 42 days when refrigerated, and frozen storage is limited by the complex process that is required which involves adding and removing glycerol from RBCs before and after freezing them for storage at -80 oC. A technology that enables dry storage of blood at ambient temperature would have significant global impact. A potential solution to achieve this goal is to load RBCs with trehalose which can form a protective barrier around cell membranes during freezing and drying. Trehalose is a sugar molecule found in many organisms that survive freezing and desiccation, but mammalian cells are impermeable to trehalose. Therefore, trehalose must be actively loaded into human cells. In this study we have evaluated the performance of our ultrasonic flow system to load trehalose into RBCs. In addition, this platform technology can potentially be utilized to transform other cell types for a variety of different applications.
PDF
