GlycoEVLR: Glycosylated extracellular vesicle-like receptors for targeting and sensing viral antigen

Biosensors are rapid and portable detection devices with great potential for the instant screening of infectious diseases. Receptors are the critical element of biosensors. They determine the specificity, sensitivity and stability. However, current receptors are mainly limited to antibodies and aptamers. Herein, we developed a glycosylated extracellular vesicle-like receptor (GlycoEVLR) for the rapid detection of virus antigens, specifically using SARS-CoV-2 as a model. The human angiotensin-converting enzyme 2 (ACE2)-overexpressed and heparin-functionalized HEK-293T cell membrane-cloaked Fe3O4 nanoparticles (NPs) were prepared as functionalizing GlycoEVLR. They were characterized as spherical core–shell structures with a diameter of around 100 nm, which were perfectly comparable to natural extracellular vesicles. Binding affinities between GlycoEVLR and spike1 (S1) antigen were demonstrated using surface plasmon resonance (SPR). The GlycoEVLR was fixed on magnetic electrodes to construct electrochemical biosensors. Using electrochemical impedance spectroscopy (EIS) as a measurement technique, the S1 antigen was detected down to 1 pg/mL within 20 min and showed a good linearity range from 1 pg/mL to 1 ng/mL. Also, the GlycoEVLR-based electrochemical biosensors showed excellent antifouling performance and stability. Overall, our work provides a useful methodology for developing extracellular vesicle-like receptors for biosensors. Combining the inherit natural receptor proteins and antifouling lipids from the host cells with engineered glycan motifs to target and sense viral antigens will open a newavenue for biosensors.

Micro-PCR chip-based multifunctional ultrafast SARS-CoV-2 detection platform.

When dealing with infectious pathogens, the point-of-care screening and diagnosis strategy should be low-cost, simple, rapid and accurate. Here, we report a multifunctional rapid PCR platform allowing both simultaneous screening of suspected cases and accurate identification and quantification of the virus. Based on the platform, samples suspected of being infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are screened first, after which subsequent precise quantification of the virus (SARS-CoV-2) can be performed if necessary. This fast screening technique offers a detection limit of 10 nucleic acid copies per test during the entire running time of 15 minutes, with a throughput of 9 samples at a time. Besides, depending on a droplet microfluidic chip, this platform could also provide assays of nucleic acids across four orders of magnitude of concentration within less than 15 minutes. Additionally, we successfully use the platform to quickly distinguish between positive and negative cases in clinical samples and rapidly quantify the viral load in each sample, which is consistent with standard RT-qPCR tests. As such, we demonstrate a promising and versatile rapid PCR platform for point-of-care diagnosis of infectious diseases.

Ultrafast multiplexed detection of SARS-CoV-2 RNA using a rapid droplet digital PCR system.

We report the first combination of droplet digital and rapid PCR techniques for efficient, accurate, and quantitative detection of SARS-CoV-2 RNA. The presented rapid digital PCR system simultaneously detects two specific targets (ORF1ab and N genes) and one reference gene (RNase P) with a single PCR thermal cycling period around 7 s and the total running time less than 5 min. A clear positive signal could be identified within 115 s via the rapid digital RT-PCR, suggesting its efficiency for the end-point detection. In addition, benchmark tests with serial diluted reference samples of SARS-CoV-2 RNA reveal the excellent accuracy of our system (R2>0.99). More importantly, the rapid digital PCR system gives consistent and accurate detection of low-concentration reference samples, whereas qPCR yields Ct values with significant variations that could lead to false-negative results. Finally, we apply the rapid digital PCR system to analyze clinical samples with both positive and control cases, where results are consistent with qPCR test outcomes. By providing similar accuracy with qPCR while minimizing the detection time-consuming and the false-negative tendency, the presented rapid digital PCR system represents a promising improvement on the rapid diagnosis of COVID-19.

On-site analysis of COVID-19 on the surfaces in wards.

SARS-Cov-2 has erupted across the globe, and confirmed cases of COVID-19 pose a high infection risk. Infected patients typically receive their treatment in specific isolation wards, where they are confined for at least 14 days. The virus may contaminate any surface of the room, especially frequently touched surfaces. Therefore, surface contamination in wards should be monitored for disease control and hygiene purposes. Herein, surface contamination in the ward was detected on-site using an RNA extraction-free rapid method. The whole detection process, from surface sample collection to readout of the detection results, was finished within 45 min. The nucleic acid extraction-free method requires minimal labor. More importantly, the tests were performed on-site and the results were obtained almost in real-time. The test confirmed that 31 patients contaminated seven individual sites. Among the sampled surfaces, the electrocardiogram fingertip presented a 72.7% positive rate, indicating that this surface is an important hygiene site. Meanwhile, the bedrails showed the highest correlation with other surfaces, so should be detected daily. Another surface with high contamination risk was the door handle in the bathroom. To our knowledge, we present the first on-site analysis of COVID-19 surface contamination in wards. The results and applied technique provide a potential further reference for disease control and hygiene suggestions.