Automatic system for high-throughput and high-sensitivity diagnosis of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had severe consequences for health and the global economy. To control the transmission, there is an urgent demand for early diagnosis and treatment in the general population. In the present study, an automatic system for SARS-CoV-2 diagnosis is designed and built to deliver high specification, high sensitivity, and high throughput with minimal workforce involvement. The system, set up with cross-priming amplification (CPA) rather than conventional reverse transcription-polymerase chain reaction (RT-PCR), was evaluated using more than 1000 real-world samples for direct comparison. This fully automated robotic system performed SARS-CoV-2 nucleic acid-based diagnosis with 192 samples in under 180 min at 100 copies per reaction in a "specimen in data out" manner. This throughput translates to a daily screening capacity of 800-1000 in an assembly-line manner with limited workforce involvement. The sensitivity of this device could be further improved using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based assay, which opens the door to mixed samples, potentially include SARS-CoV-2 variants screening in extensively scaled testing for fighting COVID-19.

A proof of concept study for the differentiation of SARS-CoV-2, hCoV-NL63, and IAV-H1N1 in vitro cultures using ion mobility spectrometry.

Rapid, high-throughput diagnostic tests are essential to decelerate the spread of the novel coronavirus disease 2019 (COVID-19) pandemic. While RT-PCR tests performed in centralized laboratories remain the gold standard, rapid point-of-care antigen tests might provide faster results. However, they are associated with markedly reduced sensitivity. Bedside breath gas analysis of volatile organic compounds detected by ion mobility spectrometry (IMS) may enable a quick and sensitive point-of-care testing alternative. In this proof-of-concept study, we investigated whether gas analysis by IMS can discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from other respiratory viruses in an experimental set-up. Repeated gas analyses of air samples collected from the headspace of virus-infected in vitro cultures were performed for 5 days. A three-step decision tree using the intensities of four spectrometry peaks correlating to unidentified volatile organic compounds allowed the correct classification of SARS-CoV-2, human coronavirus-NL63, and influenza A virus H1N1 without misassignment when the calculation was performed with data 3 days post infection. The forward selection assignment model allowed the identification of SARS-CoV-2 with high sensitivity and specificity, with only one of 231 measurements (0.43%) being misclassified. Thus, volatile organic compound analysis by IMS allows highly accurate differentiation of SARS-CoV-2 from other respiratory viruses in an experimental set-up, supporting further research and evaluation in clinical studies.

Fully automated fast-flow synthesis of antisense phosphorodiamidate morpholino oligomers.

Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases.

High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

Future perspective: high-throughput construction of new ultrasensitive cytokine and virion liquid chips for high-throughput screening (HTS) of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases.

Pathogen-host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane-protein interactions were found to mediate E. coli invasion of the blood-brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)-host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract.