Development of a Fully Automated Desktop Analyzer and Ultrahigh Sensitivity Digital Immunoassay for SARS-CoV-2 Nucleocapsid Antigen Detection.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. METHODS: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. RESULTS: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. CONCLUSIONS: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.

Regeneration of Escherichia coli Giant Protoplasts to Their Original Form.

The spheroplasts and protoplasts of cell wall-deficient (CWD) bacteria are able to revert to their original cellular morphologies through the regeneration of their cell walls. However, whether this is true for giant protoplasts (GPs), which can be as large as 10 µm in diameter, is unknown. GPs can be prepared from various bacteria, including Escherichia coli and Bacillus subtilis, and also from fungi, through culture in the presence of inhibitors for cell wall synthesis or mitosis. In this report, we prepared GPs from E. coli and showed that they can return to rod-shaped bacterium, and that they are capable of colony formation. Microscopic investigation revealed that the regeneration process took place through a variety of morphological pathways. We also report the relationship between GP division and GP volume. Finally, we show that FtsZ is crucial for GP division. These results indicate that E. coli is a highly robust organism that can regenerate its original form from an irregular state, such as GP.

Antibody-free digital influenza virus counting based on neuraminidase activity.

There is large demand for a quantitative method for rapid and ultra-sensitive detection of the influenza virus. Here, we established a digital influenza virus counting (DIViC) method that can detect a single virion without antibody. In the assay, a virion is stochastically entrapped inside a femtoliter reactor array device for the fluorogenic assay of neuraminidase, and incubated for minutes. By analyzing 600,000 reactors, the practical limit of detection reached the order of 103 (PFU)/mL, only 10-times less sensitive than RT-PCR and more than 1000-times sensitive than commercial rapid test kits (RIDTs). Interestingly, neuraminidase activity differed among virions. The coefficient of variance was 30-40%, evidently broader than that of alkaline phosphatase measured as a model enzyme for comparison, suggesting the heterogeneity in size and integrity among influenza virus particles. Sensitivity to oseltamivir also differed between virions. We also tested DIViC using clinical gargle samples that imposes less burden for sampling while with less virus titre. The comparison with RIDTs showed that DIViC was largely superior to RIDTs in the sensitivity with the clinical samples although a few false-positive signals were observed in some clinical samples that remains as a technical challenge.

Osmolyte-Enhanced Protein Synthesis Activity of a Reconstituted Translation System.

Molecular crowding is receiving great attention in cell-free synthetic biology because molecular crowding is a critical feature of natural cell discrimination from artificial cells. Further, it has significant and generic influences on biomolecular functions. Although there are reports on how the macromolecular crowder reagents affect cell-free systems such as transcription and translation, the second class of molecular crowder reagents with low molecular weight, osmolyte, was much less studied in cell-free systems. In the present study, we focused on trimethylamine- N-oxide (TMAO) and betaine, methylamine osmolytes, and investigated the effectiveness of these osmolytes on gene expression activity of reconstituted cell-free protein synthesis. The gene expression activity of the fluorescent proteins Venus and tdTomato and the enzymes ß-galactosidase and dihydrofolate reductase were tested. At 37 °C, 0.4 M TMAO showed the highest enhancement of translational activity by a factor of 1.6-3.8, regardless of protein type. In contrast, betaine showed only a moderate effect that was limited to fluorescent proteins. Excess amounts of osmolytes suppressed gene expression activity. An mRNA-start assay and SDS-PAGE quantitative analysis provided firm evidence that TMAO enhances the translation process, instead of transcription, folding, or the maturation of fluorescent proteins. Interestingly, at 26 °C, TMAO and betaine showed the highest enhancement of protein synthesis activity at lower concentrations than at 37 °C. These findings provide implications on how osmolytes assist translation in natural cells. Further, they provide guidelines for modulation of protein synthesis activity in artificial cells through osmolyte addition.

Hybrid cell reactor system from Escherichia coli protoplast cells and arrayed lipid bilayer chamber device.

We developed a novel hybrid cell reactor system via functional fusion of single Escherichia coli protoplast cells, that are deficient in cell wall and expose plasma membrane, with arrayed lipid bilayer chambers on a device in order to incorporate the full set of cytosolic and membrane constituents into the artificial chambers. We investigated gene expression activity to represent the viability of the hybrid cell reactors: over 20% of hybrid cells showed gene expression activity from plasmid or mRNA. This suggests that the hybrid cell reactors retained fundamental activity of genetic information transduction. To expand the applicability of the hybrid cell reactors, we also developed the E. coli-in-E. coli cytoplasm system as an artificial parasitism system. Over 30% of encapsulated E. coli cells exhibited normal cell division, showing that hybrid cells can accommodate and cultivate living cells. This novel artificial cell reactor technology would enable unique approaches for synthetic cell researches such as reconstruction of living cell, artificial parasitism/symbiosis system, or physical simulation to test functionality of synthetic genome.