On-Chip Nucleic Acid Purification Followed by ddPCR for SARS-CoV-2 Detection.

We developed a microfluidic chip integrated with nucleic acid purification and droplet-based digital polymerase chain reaction (ddPCR) modules to realize a 'sample-in, result-out' infectious virus diagnosis. The whole process involved pulling magnetic beads through drops in an oil-enclosed environment. The purified nucleic acids were dispensed into microdroplets by a concentric-ring, oil-water-mixing, flow-focusing droplets generator driven under negative pressure conditions. Microdroplets were generated with good uniformity (CV = 5.8%), adjustable diameters (50-200 µm), and controllable flow rates (0-0.3 µL/s). Further verification was provided by quantitative detection of plasmids. We observed a linear correlation of R2 = 0.9998 in the concentration range from 10 to 105 copies/µL. Finally, this chip was applied to quantify the nucleic acid concentrations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The measured nucleic acid recovery rate of 75 ± 8.8% and detection limit of 10 copies/µL proved its on-chip purification and accurate detection abilities. This chip can potentially be a valuable tool in point-of-care testing.

Droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction and amplification for the detection of pathogens and tumor mutation sites.

Traditional nucleic acid extraction and detection is based on open operation, which may cause cross-contamination and aerosol formation. This study developed a droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction, purification and amplification. The reagent is sealed in oil to form a droplet, and the nucleic acid is extracted and purified by controlling the movement of the magnetic beads (MBs) through a permanent magnet, ensuring a closed environment. This chip can automatically extract nucleic acid from multiple samples within 20 min, and can be directly placed in the in situ amplification instrument for amplification without further transfer of nucleic acid, characterized by simple, fast, time-saving and labor-saving. The results showed that the chip was able to detect <10 copies/test SARS-CoV-2 RNA, and EGFR exon 21 L858R mutations were detected in H1975 cells as low as 4 cells. In addition, on the basis of the droplet magnetic-controlled microfluidic chip, we further developed a multi-target detection chip, which used MBs to divide the nucleic acid of the sample into three parts. And the macrolides resistance mutations A2063G and A2064G, and the P1 gene of mycoplasma pneumoniae (MP) were successfully detected in clinical samples by the multi-target detection chip, providing the possibility for future application in the detection of multiple pathogens.

An integrated microfluidic chip for nucleic acid extraction and continued cdPCR detection of pathogens.

This paper introduces an enclosed microfluidic chip that integrates sample preparation and the chamber-based digital polymerase chain reaction (cdPCR). The sample preparation of the chip includes nucleic acid extraction and purification based on magnetic beads, which adsorb nucleic acids by moving around the reaction chambers to complete the reactions including lysis, washing, and elution. The cdPCR area of the chip consists of tens of thousands of regularly arranged microchambers. After the sample preparation processes are completed, the purified nucleic acid can be directly introduced into the microchambers for amplification and detection on the chip. The nucleic acid extraction performance and digital quantification performance of the system were examined using synthetic SARS-CoV-2 plasmid templates at concentrations ranging from 101-105 copies per µL. Further on, a simulated clinical sample was used to test the system, and the integrated chip was able to accurately detect SARS-CoV-2 virus particle samples doped with interference (saliva) with a detection limit of 10 copies per µL. This integrated system could provide a promising tool for point-of-care testing of pathogenic infections.

S Trimer Derived from SARS-CoV-2 B.1.351 and B.1.618 Induced Effective Immune Response against Multiple SARS-CoV-2 Variants.

The spread of SARS-CoV-2 and its variants leads to a heavy burden on healthcare and the global economy, highlighting the need for developing vaccines that induce broad immunity against coronavirus. Here, we explored the immunogenicity of monovalent or bivalent spike (S) trimer subunit vaccines derived from SARS-CoV-2 B.1.351 (S1-2P) or/and B.1. 618 (S2-2P) in Balb/c mice. Both S1-2P and S2-2P elicited anti-spike antibody responses, and alum adjuvant induced higher levels of antibodies than Addavax adjuvant. The dose responses of the vaccines on immunogenicity were evaluated in vivo. A low dose of 5 µg monovalent recombinant protein or 2.5 µg bivalent vaccine triggered high-titer antibodies that showed cross-activity to Beta, Delta, and Gamma RBD in mice. The third immunization dose could boost (1.1 to 40.6 times) high levels of cross-binding antibodies and elicit high titers of neutralizing antibodies (64 to 1024) prototype, Beta, Delta, and Omicron variants. Furthermore, the vaccines were able to provoke a Th1-biased cellular immune response. Significantly, at the same antigen dose, S1-2P immune sera induced stronger broadly neutralizing antibodies against prototype, Beta, Delta, and Omicron variants compared to that induced by S2-2P. At the same time, the low dose of bivalent vaccine containing S2-2P and S1-2P (2.5 µg for each antigen) significantly improved the cross-neutralizing antibody responses. In conclusion, our results showed that monovalent S1-2P subunit vaccine or bivalent vaccine (S1-2P and S2-2P) induced potent humoral and cellular responses against multiple SARS-CoV-2 variants and provided valuable information for the development of recombinant protein-based SARS-CoV-2 vaccines that protect against emerging SARS-CoV-2 variants.